i

Biofertilizers for Sustainable

Soil Management

The alkaline calcareous nature, high pH, salinity, heavy metals pollution, and low organic matter
content of soils in many parts of the world have diminished the soil fertility and made essential
nutrients unavailable to crops. To cope with the poor availability of soil nutrients, improve soil
health, and feed the fast-​growing global population, the farming community is using millions of tons
of expensive chemical fertilizers in their fields to maintain an adequate level of nutrients for crop
sustainability as well as to ensure food security. In this scenario, the exploitation of biofertilizers
has become of paramount importance in the agricultural sector for their potential role in food safety
and sustainable crop production. Bearing in mind the key importance of biofertilizers, this book
examines the role of biofertilizers in sustainable management of soil and plant health under different
conditions of the changing climate. Finally, it provides a platform for scientists and academicians all
over the world to promote, share, and discuss various new issues, developments, and limitations in
biofertilizers, crops, and beneficial microbes.

Salient Features:

• Mainly focuses on the role of biofertilizers in managing soils for improving crop and vege-
table yields as a substitute for chemical fertilizers.
• Highlights the valuable information for the mechanism of action, factors affecting, and
limitations of biofertilizers in the wider ecosystem.
• Presents a diversity of techniques used across plant science.
• Designed to cater to the needs of researchers, technologists, policy makers, and undergraduates
and postgraduates studying in the fields of organic agriculture, soil microbiology, soil biology,
soil fertility, and fertilizers.
• Addresses plant responses to biofertilizers.
ii
iii

Biofertilizers for Sustainable

Soil Management

Edited by
Shah Fahad, Shah Saud, Fazli Wahid,
and Muhammad Adnan
iv

First edition published 2024

by CRC Press
6000 Broken Sound Parkway NW, Suite 300, Boca Raton, FL 33487-​2742
and by CRC Press
4 Park Square, Milton Park, Abingdon, Oxon, OX14 4RN
CRC Press is an imprint of Taylor & Francis Group, LLC
© 2024 selection and editorial matter, Shah Fahad, Shah Saud, Fazli Wahid, and Muhammad Adnan; individual chapters,
the contributors
Reasonable efforts have been made to publish reliable data and information, but the authors and publisher cannot assume
responsibility for the validity of all materials or the consequences of their use. The authors and publishers have attempted to
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Trademark notice: Product or corporate names may be trademarks or registered trademarks and are used only for identification
and explanation without intent to infringe.
Library of Congress Cataloging‑in‑Publication Data
Names: Fahad, Shah (Assistant professor of agriculture), editor. | Saud, Shah, editor. |
Wahid, Fazli (Soil scientist), editor. | Adnan, Muhammad (Lecturer in agriculture), editor.
Title: Biofertilizers for sustainable soil management / edited by Shah Fahad, Shah Saud,
Fazli Wahid, and Muhammad Adnan.
Description: First edition. | Boca Raton, FL : CRC Press, 2024. |
Includes bibliographical references and index.
Identifiers: LCCN 2023012718 (print) | LCCN 2023012719 (ebook) | ISBN 9781032260419 (hardback) |
ISBN 9781032260433 (paperback) | ISBN 9781003286233 (ebook)
Subjects: LCSH: Biofertilizers. | Sustainable agriculture. | Soil management.
Classification: LCC S654.5 .B567 2024 (print) | LCC S654.5 (ebook) |
DDC 631.8/6–dc23/eng/20230530
LC record available at https://lccn.loc.gov/2023012718
LC ebook record available at https://lccn.loc.gov/2023012719
ISBN: 978-​1-​032-​26041-​9 (hbk)
ISBN: 978-​1-​032-​26043-​3 (pbk)
ISBN: 978-​1-​003-​28623-​3 (ebk)
DOI: 10.1201/​9781003286233
Typeset in Times
by Newgen Publishing UK
v

Contents
Editor Biographies............................................................................................................................vii
List of Contributors............................................................................................................................ix
Acknowledgements........................................................................................................................... xv

Chapter 1 Endophytic Microbes: Deciphering Their Role and Mechanism to Mitigate

Abiotic Stresses in Plants as an Eco-​friendly Tool........................................................ 1
Sammina Mahmood, Adeel Sattar, Umair Ashraf, Adnan Hassan Tahir,
and Muhammad Zia-​Ur-​Rehman

Chapter 2 Legacy Phosphorus: Tracking Budget, Vulnerability, and Mobility to Ensure

Sustainable Management in the Agricultural System.................................................25
Sammina Mahmood and Umair Ashraf

Chapter 3 Bio-​fertilizers for Improving Micronutrient Availability............................................ 43

Nazish Huma Khan, Muhammad Sufyan Khan, Tooba Saeed, Fazli Zuljalal,
and Muhammad Adnan

Chapter 4 Role of Plant Bio-​stimulants and Their Classification................................................ 65

Waseem Ahmed, Adnan Noor Shah, Asad Abbas, Muhammad Nawaz,
Abdul Qayyum, Muhammad Umair Hassan, Muther Mansoor Qaisrani,
and Jallat Khan

Chapter 5 Approaches for Using Bio-fertilizers as a Substitute for Chemical Fertilizers to

Improve Soil Health and Crop Yields in Pakistan....................................................... 89
Anas Iqbal, Muhammad Izhar Shafi, Mazhar Rafique, Maid Zaman,
Izhar Ali, Waqar-​un-​Nisa, Ayesha Jabeen, Sofia Asif, Bushra Gul,
Muhammad Adnan, Rafi Ullah, Nazeer Ahmed, Muhammad Haroon,
Muhammad Romman, and Ligeng Jiang

Chapter 6 Phosphate-​mobilizing Mycorrhizal-​based Bio-​fertilizers......................................... 119

Muhammad Jawad, Ayesha Jabeen, Ali Raza Gurmani, and Mazhar Rafique

Chapter 7 Quality Standards for Production and Marketing of Biofertilizers........................... 137

Adeel Ahmad, Amir Aziz, Muhammad Irfan, Mukkram Ali Tahir,
Faiza Rawish, and Noman Younas

Chapter 8 Limitations of Using Biofertilizers as an Alternative to Chemical Fertilizers.......... 163

Muhammad Haroon, Fazli Wahid, Rafi Ullah, Muhammad Adnan,
Mukhtar Alam, Hidayat Ullah, Muhammad Saeed, Muhammad Saeed,

v
vi

vi Contents

Shah Fahad, Muhammad Romman, Nazeer Ahmed, Taufiq Nawaz,

Anas Iqbal, Zia Ur Rehman, Ayman El Sabagh, Shah Saud, and Shah Hassan

Chapter 9 Phosphorus-​solubilizing Bio-​fertilizers..................................................................... 173

Kinza Iqbal, Sajid Masood, Qaiser Hussain, Rabia Khalid, Khalid
Saifullah Khan, Muhammad Akmal, Shahzada Sohail Ijaz, Muhammad
Jamil, Muhammad Azeem, and Servat Jehan

Chapter 10 Potential Applications of Algae-​based Bio-​fertilizers............................................... 185

Hafiz Muhammad Rashad Javeed, Mazhar Ali, Rafi Qamar,
Fahim Nawaz, Humaira Yasmin, Koushik Chakraborty, Zainul Abideen,
Muhammad Zahid Ihsan, and Muhammad Adnan Bukhari

Chapter 11 Ectomycorrhizal Fungi: Role as Bio-​fertilizers in Forestry...................................... 197

Hafiz Muhammad Rashad Javeed, Mazhar Ali, Muhammad Shahid Ibni Zamir,
Rafi Qamar, Muhammad Mubeen, Atique-​ur-​Rehman, Muhammad Shahzad,
Samina Khalid, and Ayman EL Sabagh

Chapter 12 Plant Growth-​promoting Rhizobacteria/​Pseudomonas as a Biofertilizer................. 209

Rafiq Ahmad, Sohail Khan, Muhammad Hayat, Syed Muhammad Afzal, and
Javed Muhammad

Chapter 13 Nitrogen-​fixing Biofertilizers.................................................................................... 233

Muhammad Romman, Rainaz Parvez, Muhammad Adnan, Farhana Gul,
Muhammad Haroon, Rafi Ullah, Shah Saud, Nazeer Ahmed, Ishfaq Hameed,
Taufiq Nawaz, Muhammad Hamzah Saleem, Sahar Mumtaz, Amanullah,
Muhammad Arif, Maid Zaman, Abdel Rahman Altawaha, and Shah Hassan

Chapter 14 Status of Research and Applications of Bio-​fertilizers: Global Scenario................. 255

Rafi Ullah, Muhammad Junaid, Mehwish Kanwal, Muhammad Adnan,
Taufiq Nawaz, Nazeer Ahmed, Fazli Subhan, Muhammad Romman,
Shah Saud, Shakeel Ahmad, Anas Iqbal, Fazli Wahid, Muhammad Haroon,
Muhammad Zamin, Nazish Huma Khan, Jamal Nasar, and Shah Hassan

Chapter 15 Bio-​fertilizer Effects on Plant-​parasitic Nematodes.................................................. 279

Taufiq Nawaz, Muhammad Junaid, Mehwish Kanwal, Saeed Ahmed,
Nazeer Ahmed, Rafi Ullah, Muhammad Adnan, Muhammad Saeed,
Muhammad Romman, Shah Fahad, Maid Zaman, Muhammad Haroon,
Muhammad Shahab, Shah Saud, and Shah Hassan

Index............................................................................................................................................... 290
vii

Editor Biographies
Shah Fahad works as Assistant Professor in the Department of Agronomy, Abdul Wali Khan
University Mardan, Khyber Pakhtunkhwa, Pakistan. He obtained his PhD in Agronomy from
Huazhong Agriculture University, China, in 2015. After completing his postdoctoral research in
Agronomy at the Huazhong Agriculture University (2015–​17), he accepted the position of Assistant
Professor at the University of Haripur. He has published over 430 peer-​reviewed papers (impact
factor 1834.76) with more than 370 research and 60 review articles, on important aspects of cli-
mate change, plant physiology and breeding, plant nutrition, plant stress responses and toler-
ance mechanisms, and exogenous chemical priming-​induced abiotic stress tolerance. He has also
contributed 80 book chapters to various book editions published by Springer, Wiley-​Blackwell, and
Elsevier. He has edited 17 book volumes, including this one, published by CRC Press, Springer,
and Intech Open. He won the Young Rice International Scientist award and Distinguished Scholar
award in 2014 and 2015, respectively. He has won 17 projects from international and national donor
agencies. Dr Shah Fahad figured in the Clarivate’s 2021 and 2022 lists of Highly Cited Researchers
in the field of Plant and Agriculture sciences. His name is included among the top two percent of
scientists in a global list compiled by Stanford University, USA. He has worked and is presently
continuing to work on a wide range of topics, including climate change, greenhouse gas emissions,
abiotic stresses tolerance, roles of phytohormones and their interactions in abiotic stress responses,
heavy metals, and the regulation of nutrient transport processes.

Shah Saud received his PhD and Post Doctorate in Turf grasses (Horticulture) from Northeast
Agricultural University, Harbin, China. He is currently working as Assistant Professor in the College
of Life Science, Linyi University, Linyi, Shandong, 276000, China. Dr. Shah Saud has published
over 200 research publications in peer-​reviewed journals. He has edited six books and written 40
book chapters on important aspects of plant physiology, plant stress responses, and environmental
problems in relation to agricultural plants. According to Scopus®, Dr. Shah Saud’s publications
have received roughly 8566 citations with an h-​index of 44.

Fazli Wahid completed his PhD in the field of soil microbiology and plant nutrition at the University
of Agriculture Peshawar and University of Natural Resources and Life Sciences, Vienna, Austria. He
also completed his postdoc at Nigde University, Turkey, in plant nutrition. Currently, he is working
as Assistant Professor in the Department of Agriculuture, University of Swabi. His research work
is mainly focused on the isolation and purification of beneficial microbes, and the production of
biofertilizers to improve soil and plant health. He has isolated several bacterial strains for solubiliza-
tion of P from rock phosphate in high-​pH soils. He has published 70 research articles in well-​reputed
journals with 10 book chapters, while he has two books in progress as editor. He is also working as a
potential reviewer for Chemosphere and Pedospher. He has won one international and two national
research projects and organized several international and national conferences and seminars at the
University of Swabi.

Muhammad Adnan is a lecturer at the Department of Agriculture at the University of Swabi

(UOS), Pakistan. He completed his PhD (soil fertility and microbiology) at the Department of
Soil and Environmental Sciences (SES), the University of Agriculture Peshawar, Pakistan, and the
Department of Plant, Soil and Microbial Sciences, Michigan State University, USA. He received
his MSc and BSc (Hons) in Soil and Environmental Sciences, from the Department of SES at the
University of Agriculture, Peshawar, Pakistan.

vii
viii
ix

Contributors
Asad Abbas Muhammad Akmal
School of Horticulture, Anhui Agricultural Institute of Soil & Environmental Sciences,
University, Hefei, China PMAS-​Arid Agriculture University,
Rawalpindi, Pakistan
Zainul Abideen
Institute of Sustainable Halophyte Utilization, Mukhtar Alam
University of Karachi, Sindh, Pakistan Department of Agriculture, University of
Swabi, Swabi, Pakistan
Muhamad Adnan
Department of Agriculture, University of Izhar Ali
Swabi, Pakistan College of Agriculture, Guangxi University,
Nanning, China
Syed Muhammad Afzal
Center of Biotechnology and Microbiology, Mazhar Ali
University of Peshawar, KP, Pakistan Department of Environmental Sciences,
COMSATS University, Islamabad, Punjab,
Rafiq Ahmad Pakistan
Department of Microbiology, The University of
Haripur, KP, Pakistan Abdel Rahman Altawaha
Department of Biological Sciences Al Hussein
Shakeel Ahmad Bin Talal University, Ma’an, Jordan
Guangxi Colleges and Universities, key
laboratory of Crop Cultivation and Tillage, Amanullah
National Demonstration Center for Department of Agronomy, the University of
Experimental Plant Science Education, Agriculture Peshawar, Khyber Pakhtunkhwa,
Agricultural College of Guangxi University, Pakistan
Nanning, China
Muhammad Arif
Adeel Ahmad Department of Agronomy, the University of
Institute of Soil and Environmental Sciences, Agriculture Peshawar, Khyber Pakhtunkhwa,
University of Agriculture, Faisalabad, Pakistan
Pakistan
Umair Ashraf
Nazeer Ahmed Department of Botany, Division of Science
Department of Agriculture, University of and Technology, University of Education,
Swabi, Khyber Pakhtunkhwa, Pakistan Lahore, Punjab, Pakistan

Saeed Ahmed Sofia Asif

Agricultural Research Center, Londrina State Department of Food Science and Technology,
University, Londrina, Brazil The University of Haripur, Khyber
Pakhtunkhwa, Pakistan
Waseem Ahmed
Department of Horticulture, The University of Atique-​ur-​Rehman
Haripur, Khyber Pakhtunkhwa, Pakistan Department of Agronomy, Bahauddin Zakariya
University, Multan, Punjab, Pakistan

ix
x

x Contributors

Muhammad Azeem Muhammad Haroon

Institute of Soil & Environmental Sciences, Department of Agriculture, University of
PMAS-​Arid Agriculture University, Swabi, Khyber Pakhtunkhwa, Pakistan
Rawalpindi, Pakistan
Shah Hassan
Amir Aziz Department of Agricultural Extension
Department of Soil and Environmental Education and Communication, The
Sciences, College of Agriculture, University University of Agriculture, Peshawar, Pakistan
of Sargodha, Sargodha, Pakistan
Muhammad Umair Hassan
Muhammad Adnan Bukhari Research Center on Ecological Sciences,
Department of Agronomy, Faculty of Jiangxi Agricultural University,
Agriculture and Environment, The Islamia Nanchang, China
University of Bahawalpur, Bahawalpur,
Punjab, Pakistan Muhammad Hayat
State Key Laboratory of Microbial Technology,
Koushik Chakraborty Institute of Microbial Technology, Shandong
Department of Crop Physiology & University Qingdao, China
Biochemistry, ICAR-​National Rice
Research Institute Bidyadharpur, Cuttack, Qaiser Hussain
Odisha, India Institute of Soil & Environmental Sciences,
PMAS-​Arid Agriculture University,
Ayman EL Sabagh Rawalpindi, Pakistan
Department of Agronomy, Faculty of
Agriculture, University of Kafrel-​Sheikh, Muhammad Zahid Ihsan
Kafrel-​Sheikh, Egypt The Cholistan Institute of Desert Studies,
Faculty of Agriculture and Environment
Shah Fahad Sciences, The Islamia University of
Department of Agronomy, Abdul Wali Khan Bahawalpur, Bahawalpur, Punjab, Pakistan
University Mardan, Khyber Pakhtunkhwa,
Pakistan Shahzada Sohail Ijaz
Institute of Soil & Environmental Sciences,
Bushra Gul PMAS-​Arid Agriculture University,
Department of Biosciences, University of Wah, Rawalpindi, Pakistan
Punjab, Pakistan
Anas Iqbal
Farhana Gul College of Agriculture, Guangxi University,
Department of Botany, Government Girls Nanning, China
Degree College Dargai, Malakand, Khyber
Pakhtunkhwa, Pakistan Kinza Iqbal
Institute of Soil & Environmental Sciences,
Ali Raza Gurmani PMAS-​Arid Agriculture University,
Department of Soil & Climate Sciences, The Rawalpindi, Pakistan
University of Haripur, Khyber Pakhtunkhwa,
Pakistan Muhammad Irfan
School of Environmental Science and
Ishfaq Hameed Engineering, Tianjin University, Tianjin,
Department of Botany, University of Chitral, PR China
Khyber Pakhtunkhwa, Pakistan
xi

Contributors xi

Ayesha Jabeen Sohail Khan

Department of Soil & Climate Sciences, The College of Life Science and Technology,
University of Haripur, Khyber Pakhtunkhwa, State Key Laboratory for Conservation
Pakistan and Utilization of Subtropical Agro-​
bioresources, Guangxi University, Nanning,
Muhammad Jamil Guangxi, China
Soil and Water Testing Laboratory, Sahiwal,
Pakistan Jallat Khan
Department of Chemistry, Khwaja Fareed
Hafiz Muhammad Rashad Javeed University of Engineering & Information
Department of Environmental Sciences, Technology, Rahim Yar Khan, Punjab,
COMSATS University, Islamabad, Punjab, Pakistan
Pakistan
Nazish Huma Khan
Muhammad Jawad Department of Environmental Sciences,
Department of Soil & Climate Sciences, The University of Swabi, Pakistan
University of Haripur, Khyber Pakhtunkhwa,
Pakistan Muhammad Sufyan Khan
Department of Environmental Sciences,
Servat Jehan University of Swabi, Pakistan
Institute of Soil & Environmental Sciences,
PMAS-​Arid Agriculture University, Khalid Saifullah Khan
Rawalpindi, Pakistan Institute of Soil & Environmental Sciences,
PMAS-​Arid Agriculture University,
Ligeng Jiang Rawalpindi, Pakistan
Department of Botany, University of Chitral,
Khyber Pakhtunkhwa, Pakistan Sammina Mahmood
Department of Botany, Division of Science
Muhammad Junaid and Technology, University of Education,
Graduate School of Life and Environmental Lahore, Punjab, Pakistan
Sciences, University of Tsukuba, Tsukuba,
Ibaraki, Japan Sajid Masood
Institute of Soil & Environmental Sciences,
Mehwish Kanwal PMAS-​Arid Agriculture University,
Ministry of National Food Security & Rawalpindi, Pakistan
Research, Pakistan Tobacco Board,
Peshawar, Khyber Pakhtunkhwa, Muhammad Mubeen
Pakistan Department of Environmental Sciences,
COMSATS University Islamabad, Punjab,
Samina Khalid Pakistan
Department of Environmental Sciences,
COMSATS University Islamabad, Punjab, Javed Muhammad
Pakistan Department of Microbiology, University of
Haripur, Khyber Pakhtunkhwa, Pakistan
Rabia Khalid
Institute of Soil & Environmental Sciences, Sahar Mumtaz
PMAS-​Arid Agriculture University, Division of Science and Technology,
Rawalpindi, Pakistan Department of Botany University of
Education, Lahore, Pakistan
xii

xii Contributors

Jamal Nasar Faiza Rawish

Guangxi Colleges and Universities Key Superior College of Commerce Sat Sira
Laboratory of Crop Cultivation and Tillage, Chowk, Mandi Bahauddin, Punjab, Pakistan
Agricultural College of Guangxi University,
Nanning, China Muhammad Romman
College of Agriculture, Guangxi University,
Muhammad Nawaz Nanning, China
Department of Agricultural Engineering,
Khwaja Fareed University of Engineering Muhammad Saeed
and Information Technology, Rahim Yar Department of Agriculture, University of
Khan, Punjab, Pakistan Swabi, Anbar, Swabi, Khyber Pakhtunkhwa,
Pakistan
Fahim Nawaz
Department of Agronomy, Muhammad Nawaz Muhammad Saeed
Shareef University of Agriculture, Multan, Department of Weed Science and Botany,
Punjab, Pakistan University of Agriculture, Peshawar, Pakistan

Taufiq Nawaz Tooba Saeed

Department of Food Science and Technology, National Centre of Excellence in Physical
The University of Agriculture, Peshawar, Chemistry, University of Peshawar, Pakistan
Pakistan
Muhammad Hamzah Saleem
Rainaz Parvez MOA Key Laboratory of Crop Ecophysiology
Department of Botany, Government Girls and Farming System in the Middle Reaches
Degree College Dargai, Malakand, Khyber of the Yangtze River, College of Plant
Pakhtunkhwa, Pakistan Science and Technology, Huazhong, China

Muther Mansoor Qaisrani Adeel Sattar

Department of Bioscience & Technology Department of Pharmacology and Toxicology,
Khwaja Fareed University of Engineering University of Veterinary and Animal
and Information Technology, Rohm Yar Sciences, Lahore, Pakistan
Khan, Punjab, Pakistan
Shah Saud
Rafi Qamar College of Life Science, Linyi University,
Department of Agronomy, College of Linyi, Shandong, 276000, China
Agriculture, University of Sargodha,
Sargodha, Punjab, Pakistan Muhammad Izhar Shafi
Department of Soil and Environmental
Abdul Qayyum Sciences, Faculty of Crop Production
Department of Agronomy, The University of Sciences, The University of Agriculture,
Haripur, Khyber Pakhtunkhwa, Pakistan Peshawar, Pakistan

Mazhar Rafique Adnan Noor Shah

Department of Soil & Climate Sciences, The Department of Agricultural Engineering,
University of Haripur, Khyber Pakhtunkhwa, Khwaja Fareed University of Engineering
Pakistan and Information Technology, Rahim Yar
Khan, Punjab, Pakistan
xii

Contributors xiii

Muhammad Shahab Waqar-​un-​Nisa

Department of Agriculture, University of Department of Soil & Climate Sciences, The
Swabi, Anbar, Swabi, Khyber Pakhtunkhwa, University of Haripur, Khyber Pakhtunkhwa,
Pakistan Pakistan

Muhammad Shahzad Humaira Yasmin

Department of Agronomy, University of Department of Biosciences, COMSATS
Poonch Rawalakot, AJK, Pakistan University Islamabad, Islamabad, Pakistan

Fazli Subhan Noman Younas

Department of Agriculture, University of Institute of Soil and Environmental Sciences,
Swabi, Anbar, Swabi, Khyber Pakhtunkhwa, University of Agriculture, Faisalabad
Pakistan
Maid Zaman
Adnan Hassan Tahir Department of Entomology, The University of
Department of Clinical Science, Faculty of Haripur, Khyber Pakhtunkhwa, Pakistan
Veterinary and Animal Sciences, PMAS-​Arid
Agriculture University, Rawalpindi, Pakistan Muhammad Zamin
Department of Agriculture, University of
Mukkram Ali Tahir Swabi, Anbar, Swabi, Khyber Pakhtunkhwa,
Department of Soil and Environmental Pakistan
Sciences, College of Agriculture, University
of Sargodha, Sargodha, Pakistan Muhammad Shahid Ibni Zamir
Department of Agronomy, University of
Rafi Ullah Agriculture, Faisalabad, Punjab, Pakistan
Department of Agriculture, University of
Swabi, Khyber Pakhtunkhwa, Pakistan Muhammad Zia-​Ur-​Rehman
Institute of Soil and Environmental Sciences,
Hidayat Ullah University of Agriculture, Faisalabad,
Department of Agriculture, University of Punjab, Pakistan
Swabi, Swabi, Pakistan
Fazli Zuljalal
Zia Ur Rehman Department of Environmental Sciences,
Department of Agriculture, University of University of Swabi, Pakistan
Swabi, Swabi, Pakistan

Fazli Wahid
Department of Agriculture, University of
Swabi, Anbar, Swabi, Khyber Pakhtunkhwa,
Pakistan
xiv
newgenprepdf

xv

Acknowledgements
Words are bounded and knowledge is limited in praising ALLAH, the Instant and Sustaining Source
of all Mercy and Kindness, and the Sustainer of the Worlds. My greatest and ultimate gratitude
is due to ALLAH (Subhanahu wa Taqadus). I thank ALLAH with all my humility for everything
that I can think of. His generous blessing and exaltation succeeded my thoughts and caused my
ambitions to thrive and to bare the cherished fruit of my modest efforts in the form of this piece of
literature from the blooming spring of blossoming knowledge. May ALLAH forgive my failings and
weaknesses, strengthen and enliven my faith in HIM, and endow me with knowledge and wisdom.
All praise and respect are for Holy Prophet Muhammad Salle Allah Alleh Wassalam, the greatest
educator, the everlasting source of guidance and knowledge for humanity. He taught the principles
of morality and eternal values and enabled us to recognize our Creator. I have a deep sense of obli-
gation to my parents, brothers, sisters, and son. Their unconditional love, care, and confidence in my
abilities helped me achieve this milestone in my life. For this and much more, I am forever in their
debt. It is to them that I dedicate this book. In this arduous time, I also appreciate the patience and
serenity of my wife, who brought joy to my life in so many different ways. It is indeed on account
of her affections and prayers that I was able to achieve something in my life.

Shah Fahad

xv
xvi
1

1 Endophytic Microbes
Deciphering Their Role and
Mechanism to Mitigate Abiotic
Stresses in Plants as an
Eco-​friendly Tool
Sammina Mahmood,*1 Adeel Sattar,2 Umair Ashraf,1
Adnan Hassan Tahir,3 and Muhammad Zia-​Ur-​Rehman4
1
Department of Botany, Division of Science and Technology,
University of Education, Lahore, Punjab, Pakistan
2
Department of Pharmacology and Toxicology, University of Veterinary
and Animal Sciences, Lahore, Pakistan
3
Department of Clinical Science, Faculty of Veterinary and Animal
Sciences, PMAS-​Arid Agriculture University, Rawalpindi, Pakistan
4
Institute of Soil and Environmental Sciences, University of Agriculture,
Faisalabad, Punjab, Pakistan
* Correspondence: [email protected]

CONTENTS
1.1 Introduction............................................................................................................................... 1
1.2 Ecology of the Microbiome....................................................................................................... 2
1.3 Reference Environment for Plant–​microbe Interactions........................................................... 3
1.3.1 Plant–​microbe Interactions in the Rhizosphere............................................................ 3
1.4 Role of Bacteria in Salinity Tolerance....................................................................................... 4
1.5 Drought Stress........................................................................................................................... 5
1.6 Heavy Metal Stress.................................................................................................................... 6
1.7 Conclusion................................................................................................................................. 8
References........................................................................................................................................... 8

1.1 INTRODUCTION
Under natural conditions, plants are continuously exposed to a number of biotic and abiotic
challenges which pose a threat to plant productivity. Particularly, abiotic stresses like drought, sal-
inity, heavy metal stress etc. are more severe than biotic threats as these change the physiochemical
properties of soil and hence the above-​and belowground ecology of the plant. The severity of and
long exposure to stress induces toxicity at the physiological, biochemical, and molecular levels of
the plant (Ventura-​Lima et al. 2011; Al-​Zahrani et al. 2022; Rajesh et al. 2022; Anam et al. 2021;
Deepranjan et al. 2021; Haider et al. 2021; Amjad et al. 2021). Mostly, physiochemical strategies
are applied to combat abiotic stresses, but these contribute to ecological toxicity. Several bacterial
and fungal strains have been discovered which upon exposure to stress situations undergo struc-
tural alterations and secrete certain compounds such as organic acid, humic acid, polysaccharides,

DOI: 10.1201/9781003286233-1 1
2

2 Biofertilizers for Sustainable Soil Management

proteins, etc., which in turn interact with the soil conditions and contaminants in an eco-​friendly
manner. This interaction greatly affects the physical and chemical properties of soil and helps to
combat the toxic effects by adopting differential strategies based on the types of stresses imposed
on the plant (Bruins et al. 2000; Etesami 2018; Sajjad et al. 2021a,b; Fakhre et al. 2021; Khatun
et al. 2021; Ibrar et al. 2021; Bukhari et al. 2021; Haoliang et al. (2022); Sana et al. 2022; Abid et al.
2021). Under drought stress, plants adopt a strategy to produce ACC deaminase, IAA, phosphate
solubilization, exopolysaccharide synthesis (EPS), growth on N-​free culture, osmolyte accumula-
tion, e.g. HCN, in multiple crop species (Kumar et al. 2016; Mariotti et al. 2021; Niu et al. 2018;
Vettori et al. 2010; Zaman et al. 2021; Sajjad et al. 2021a,b; Rehana et al. 2021; Yang et al. 2022;
Ahmad et al. 2022; Shah et al. 2022; Muhammad et al. 2022; Wiqar et al. 2022). The bacterial
cells and their metabolites promote plant growth under water stress conditions. Plants infected with
inoculants were found to contain higher amounts of chlorophyll a, b, total chlorophyll contents,
carotenoids, and stress-​related phytohormones like ABA, JA, and SA (Heidari et al. 2011; Mariotti
et al. 2021). The modified level of enzymes under salinity stress has also been found to have a posi-
tive modulating effect on plant growth and development. However, the rate of this effect depends
upon the severity of the stress, inoculated bacteria used, growth stage of the plant, and the type and
concentration of enzyme produced (Ilangumaran & Smith 2017; Khan et al. 2017; Sun et al. 2020;
Farhat et al. 2022; Niaz et al. 2022; Ihsan et al. 2022; Chao et al. 2022; Qin et al. 2022; Xue et al.
2022; Ali et al. 2022; Mehmood et al. 2022; El Sabagh et al. 2022; Ibad et al. 2022). Under a heavy
metal stress situation, microbes use strategies to adsorb, physical entrapment on the cell surface,
and a reduction of the uptake of metals by plants by altering the cellular architecture. Cellular archi-
tecture modulations include reduced uptake of metal ions, chelation with other organic compounds,
and efflux pump activation in the case of huge uptake (Mazhar et al. 2020; Wei et al. 2018; Zhang
et al. 2019; Deepranjan et al. 2021; Haider et al. 2021; Huang Li et al.2021; Ikram et al. 2021;
Jabborova et al. 2021; Khadim et al. 2021a,b; Manzer et al. 2021; Muzammal et al. 2021; Abdul
et al. 2021a,b; Ashfaq et al. 2021; Amjad et al. 2021; Atif et al. 2021; Athar et al. 2021). Each
microbe has its own unique evolutionary route of emergence, adaptation, and significance under a
range of environmental conditions. Local ecological conditions also influence the establishment and
development of particular microbial communities in an ecological zone. This ecology dominance
not only intensifies the range of microbiota but also diversifies their role in sustainable agriculture
(Compant et al. 2019; Lawson et al. 2019; Adnan et al. 2018a,b; Adnan et al. 2019; Akram et al.
2018a,b; Aziz et al. 2017a,b; Chang et al. 2021; Chen et al. 2021; Emre et al. 2021; Habib et al.
2017; Hafiz et al. 2016; Hafiz et al. 2019; Ghulam et al. 2021; Guofu et al. 2021; Hafeez et al. 2021;
Khan et al. 2021; Kamaran et al. 2017; Muhmmad et al. 2020; Safi et al. 2021).
In this chapter, we aim to disentangle the comprehensive details of microbiota, their mechanistic
relationship with soil ecology and plant growth, and their potential use under the most pronounced
abiotic stresses, i.e. drought, salinity, and heavy metal stress situations as plant growth-​promoting/​
modulating tools. In this respect, microbe community architecture and their interactions in the root
are also discussed in this chapter.

1.2 ECOLOGY OF THE MICROBIOME

An inherently enriched gene pool of microbes called the microbiome, which includes mycobiomes
and bacteria, has been described by the several researchers and reviewers. Their enormous richness
has been attributed to the natural actions and habitats of microbes as they rarely occur as a single
species in a single environment. Multiple sites, as well as a huge range of experimental areas (soil,
plants, animals, sediments, etc.), have shown that great variation exists, even in a small sample,
which is attributed to their hypermutability that accelerates their evolution. Their co-​occurrence in
an environment leads to co-​evolution and adaptation, resulting in the establishment of a large var-
iety of relationships. Untapping this relationship has revealed two major levels of interaction: (1)
3

Endophytic Microbes 3

interactions among the organisms involved, e.g. bacteria–​bacteria, bacteria–​fungus, bacteria–​plant,

bacteria–​fungus–​plant, fungus–​fungus, and fungus–​plant (van Elsas et al. 2012), and (2) based on
the microbial interaction, the type of relationship/​mechanism (e.g. epi/​endo symbiosis, mutualistic,
pathogenic, parasitic, competitive, antagonistic, etc.) that facilitates the cohabitation and coevolution
(Faust & Raes 2012).
Each interaction and mechanism has its own unique evolutionary route of emergence, adapta-
tion, and significance under a range of environmental conditions (Sajjad et al. 2019; Saud et al.
2013; Saud et al. 2014; Saud et al. 2017; Saud et al. 2016; Shah et al. 2013; Saud et al. 2020; Saud
et al. 2022a,b; Qamar et al. 2017; Hamza et al. 2021; Irfan et al. 2021;Wajid et al. 2017; Yang et al.
2017; Zahida et al. 2017; Depeng et al. 2018). The establishment of new species, called “invaders,”
clearly shows the development and adaptation pattern in a new environment, that could affect the
local microbial community of a particular niche either positively or negatively. This dominance
of ecology not only intensifies the range of microbiota but also diversifies their role in sustainable
agriculture (Compant et al. 2019; Lawson et al. 2019). The recruitment of plant microbiota, either
bacteria or fungi, to the plant host needs extensive physiological, metabolic, and genome repro-
gramming that shapes the natural evolution of microbiota. The particular level of reprogramming
recapitulates the shift between free living and symbiotic lives of an organism under the selection
pressure of the host plant (Phelan et al. 2012; Nadal Jimenez et al. 2012; Semchenko et al. 2018;
Rodriguez et al. 2009; Genre et al. 2020).
Based on the mode of occurrence and association, plant-​associated microbiota are mainly
categorized into two classes: rhizospheric (present in soil) and endophytic (present inside the host).
However, bacteria (bacteriota) have many more applications and success stories relevant to plant
stress mitigation compared to fungi (mycobiota), owing to the size and complexity of genomes.
Indeed, the lack of a reference genome, complexity of life cycle and somatic structures, and
limited genetic transformation ability hampered its potential research and put mycobiota far behind
bacteriota ((Rodriguez et al. 2009; Naranjo-​Ortiz & Gabaldón 2020).

1.3 REFERENCE ENVIRONMENT FOR PLANT–​MICROBE INTERACTIONS

Plants, being biologically intact organisms, are directly dependent on the soil composition for their
growth and development (Hussain et al. 2020; Hafiz et al. 2020 a,b; Shafi et al. 2020; Wahid et al.
2020; Subhan et al. 2020; Zafar-​ul-​Hye et al. 2020a,b; Zafar et al. 2021; Adnan et al. 2020; Ilyas
et al. 2020; Saleem et al. 2020a,b,c; Rehman 2020; Farhat et al. 2020; Wu et al. 2020; Mubeen
et al.2020; Farhana 2020; Jan et al. 2019; Wu et al.2019; Ahmad et al,. 2019). Ground and aerial
parts are exposed to different types of environments and hence face diverse challenges (Baseer
et al. 2019; Hafiz et al. 2018; Tariq et al. 2018; Fahad and Bano 2012; Fahad et al. 2017; Fahad
et al. 2013; Fahad et al. 2014a,b). In reference to microbial interactions with plants, these environ-
ments are categorized into rhizosphere, endosphere, and phyllosphere. Each environment presents
different microbial loads and variants, which further depend upon the ecology of the particular
niche. However, by all means, the microbial genome is considered as the second genome of plants,
which directly or indirectly determines the plant fitness and development and hence its productivity
under multiple biotic and abiotic circumstances (Mendes & Raaijmakers 2015; Haldar & Sengupta
2015; Lakshmanan et al. 2014; Fahad et al. 2016a,b,c,d; Fahad et al. 2015a,b; Fahad et al. 2018a,b;
Fahad et al. 2019a,b; Fahad et al. 2020; Fahad et al. 2021a,b,c,d,e,f; Fahad et al. 2022a,b; Hesham &
Fahad 2020).

1.3.1 Plant–​microbe Interactions in the Rhizosphere

The roots are the anchoring appendages of plants, which exude carbohydrates, amino acids, and
organic acids depending upon the type of plant species and the physical and chemical condition
4

4 Biofertilizers for Sustainable Soil Management

of the soil, which determine the biotic and abiotic conditions of the rhizosphere (Iqra et al. 2020;
Akbar et al. 2020; Mahar et al. 2020; Noor et al. 2020; Bayram et al. 2020; Amanullah 2018a,b;
Amanullah 2017; Amanullah et al.2020; Amanullah et al.2021; Rashid et al.2020; Arif et al.2020;
Amir et al.2020; Saman et al. 2020; Muhammad Tahir ul Qamar et al. 2020; Jakirand Allah 2020;
Mahmood et al. 2021; Farah et al. 2020; Sadam et al. 2020; Unsar et al. 2020; Fazli et al. 2020).
Exudates do not only act as signaling molecules, which are recognized by the microbial community,
but also as screening agents for the recruitment of specific microorganisms to specific plant species
(Berg et al. 2016; Haldar & Sengupta 2015). In bulk soil, the microbial community presents a great
diversity and is influenced by soil type and environmental factors. Microbe colonization and devel-
opment are strongly and directly influenced by the prevailing soil chemical and physical conditions.
Further complexity arises when the plant root type is taken into consideration, as each plant species
has specific types of roots, which further dependsupon the prevailingd soil type for its establish-
ment and development. Only specialized communityiesof microbes present in the area close to the
root exudate are able to infect, enter, and establish inside the plant cell. Microbial communities
further specialize their activation spatially and temporally within the plant organs (Akiyama et al.
2005; Reinhold-​Hurek et al. 2015; Ottesen et al. 2013). Dynamicity takes place after infection when
microbes impact plants either beneficially or negatively.

1.4 ROLE OF BACTERIA IN SALINITY TOLERANCE

Salinity refers to increased concentrations of salts within soil which negatively affect the morpho-
logical and physiological growth and development of plants. Due to increased concentrations of
certain salts (Na+​/​Cl–​), hypertonic conditions are generated which impede the uptake of water by
plant cells. A reduction in water contents results in an osmotic stress condition, and if the situation
is prolonged, nutrient imbalance and ion toxicity further exacerbate the situation within the cell.
Ionic toxicity results in an imbalance in the cellular homeostasis and starts damaging the cell wall.
Ultimately, the cell is collapsed physiologically and biochemically gradually, appearing as wilted
phenotypically (Munns 2002). Each plant species has a different salinity stress tolerance capacity
depending upon its built-​in power to compartmentalize salt ions and produce organic solutes suc-
cessfully to contribute to the adjustment of osmotic balance (ionic/​osmotic homeostasis) within the
cell. Plants are therefore categorized as salt-​sensitive vs. salt-​tolerant species (Ruiz-​Lozano et al.
2012; Karakas et al. 2020). As soil salinity changes, the soil physical and chemical properties, and
hence the microbial density/​activity, are also affected, mostly negatively due to the reduction in
organic matter (Canfora et al. 2014; Rath et al. 2019). A reduction in organic matter leads to lowered
air spaces, creating situations that are non-​ideal for microbial establishment, growth, and develop-
ment (Canfora et al. 2014; Enamul et al. 2020; Gopakumar et al. 2020; Zia-​ur-​Rehman 2020; Ayman
et al. 2020; Al-​Wabel et al. 2020a,b; Senol 2020; Amjad et al. 2020; Ibrar et al. 2020; Sajid et al.
2020; Muhammad et al. 2021; Sidra et al. 2021; Zahir et al. 2021; Sahrish et al. 2022). This presents
an undeniable picture that salinity affects plant productivity both quantitatively and qualitatively.
As with plants, a plethora of microorganisms exist with differential responses to multiple levels
of salt stress conditions. These are also classified as salt-​resistant vs. salt-​sensitive microbes, with
their survival dependent on differential stress conditions. Plants resistant in nature usually establish
an interaction with microbes which can thrive under stressful conditions (Liu et al. 2017; Etesami &
Glick 2020; Ventosa et al. 2008). Bacteria which can withstand salinity stress almost up to 25% are
described as halotolerant bacteria, which are plant growth-​promoting bacteria belonging to Bacillus,
Pseudomonas, Halomonas, Oceanobacillus, and Streptomyceas with very similar mechanisms
(Ventosa et al. 2008; Etesami & Glick 2020). The mechanism of action of halobacterium in miti-
gating salt stress includes protecting the cell wall from damage by synthesizing specific membranes,
maintaining ionic homeostasis with the cell by synthesizing compatible organic solutes, pumping
salt ions out of the cell, and adjusting the osmotic balance. These strategies help to adapt the cell
5

Endophytic Microbes 5

to salt stress by enhancing the production of exoploysaccharides that limit salt entry into the cell
(Ruppel et al. 2013). Overall, cell energy is augmented by adjusting osmotic balance, which relieves
the cell from ionic toxicity and nutrient imbalance, detoxification, and modulation of action and
function of phytohormones present in each plant type (Abd-​Allah et al. 2018; Barnawal et al. 2016;
Egamberdieva et al. 2016).
Endophytic bacteria with indol-​3-​acetic acid (IAA), 1-​aminocylopropane-​1-​carboxylate (ACC)
deaminase, phosphate-​ solubilizing and nitrogen-​ fixing properties helped to promote ionic and
osmotic adjustment in plants (Khan et al. 2019; Jorge et al. 2019; Kang et al. 2019; Khan et al.
2020; Tufail et al. 2021). Endophytic bacterial inoculation (Bacillus firmus, Bacillus pumilus) was
found to have a positive impact on reactive oxygen species (ROS)-​scavenging enzymes CAT, POD,
SOD, MDA, glutathione reductase, and dehydroascordate reductase reported in Solanum tuberosum
(Egamberdieva et al. 2017; Gururani et al. 2013). This modified level of enzymes had a positive
modulating effect on the process of photosynthesis by safeguarding the chlorophyll pigment, how-
ever the rate of effect depends on the severity of stress, inoculated bacteria used, growth stage of
the plant, and the type and concentration of enzyme produced (Ilangumaran & Smith 2017; Khan
et al. 2017; Sun et al. 2020). Besides the ROS-​scavenging strategy, osmoregulation by synthesis
of osmolytes under salt stress also proved to be a promising route to avoid the harmful effects of
salinity on photosynthetic apparatuses (Sun et al. 2020). Inoculation of Arabidopsis thaliana with
Enterobacter species up-​regulated the salt-​responsive genes and increased the proline contents within
cells (Hasegawa et al. 2000; Kim et al. 2014), which promoted the ionic homeostasis and stabilization
of cellular structure under stress conditions (Maggio et al. 2002). Modulation of ethylene concentra-
tion in cells (lethal in higher concentrations by inhibiting germination and seedling growth) was also
accomplished with the help of endophytic bacteria species Enterobacter P23. It reduces the ethylene
toxicity by activating ACC deaminase, which in turn converts the ACC to ammonia and alpha-​
ketobutyrate, hence reducing ethylene contents in cells (Glick 2014; Kasotia et al. 2016; Sarkar et al.
2018). The modulation of the ionic interaction Na+​/​K+​ was also found to be effective. Na+​ toxicity
interferes with physiological processes (Basu et al. 2021). B. subtilis downregulates the HKT1 (high
affinity K+​ transporter) in roots, thus reducing the uptake of Na+​. Intriguingly, HKT1 expression was
enhanced in shoot which promotes Na+​ shoot–​root recirculation (Liu et al. 2017; Zhang et al. 2008).

1.5 DROUGHT STRESS
T scarcity of water, drought, has a negative impact if imposed during any developmental stage of
plant life. The morphological effects include reduced seed germination, negative effects on root and
shoot architectural features, reproductive disorders associated with flower functioning, and ultim-
ately a yield lower than the expected rate (Abdelaal et al. 2021; Hu et al. 2020; Ilyas et al. 2021; Khan
et al. 2021). All these morphological losses/​impairments are associated with disrupted physiology,
anatomy, and biochemical functioning of plants. The overall impacts from all areas include cell wall
thickening, increased cuticle deposition, variations in vascular and mesophyll cell diameters, and
stomatal closing. Reduced hydraulic conductivity also limits nutrient uptake, stomatal conductance,
relative water content, and an enhanced transpiration rate, which ultimately impairs the photosyn-
thetic apparatus (Abdelaal et al. 2021; Abdelaal et al. 2018; Hafez et al. 2020; Liu et al. 2005; Reddy
et al. 2004; Shao et al. 2008; Ullah et al. 2017). In trying to cope with drought by altering the above-​
mentioned attributes, plants are exposed to another closely related but lethal stress situation, which
is high temperature stress, which ultimately leads to disruption of cellular membranes (Moretti et al.
2010; Rysiak et al. 2021).
Plants use multiple strategies to cope with stress situations which are categorized into three
types: escape, avoidance, and tolerance. Drought escape is defined as the adjustment of life cycle
such as to avoid confronting a stress situation, which mostly occurs by shortening the life cycle.
Avoidance means to continue the life cycle near to the normal rate by managing or delaying the
6

6 Biofertilizers for Sustainable Soil Management

physiological processes. Tolerance describes the built-​in power of the plant genome to resist dehy-
dration with minimal loss of yield (Luo et al. 2018; Manavalan et al. 2009). Multiple strategies
have been developed through crop breeding programs to address this dynamic stress condition with
variable success depending upon the type of plant, environment, and severity of stress. However,
in recently, a great deal of attention has been paid to using the microbial community to infect
and stimulate natural triggers against stress situations in an eco-​friendly manner. The success of
this biological application depends upon the survival, establishment, and development of target
microbes in a given set of environmental conditions. Several drought-​resistant bacteria (B. subtilis,
B. altitudinis, B. mojavensis, and Brevibacillus laterosporus) have been isolated from desert soil or
drought-​tolerant plants (Astorga-​Eló et al. 2021; Milet et al. 2016). Besides the Bacillus species,
other species of bacteria from the Pseudomonas, Klebsiella, and Azotobacter families have shown a
growth-​stimulating effect under drought stress.
All these microbes are accompanied with various properties which include ACC deaminase,
IAA, phosphate solubilization, exopolysaccharide synthesis (EPS), growth on N-​free culture, and
osmolyte accumulation, e.g. HCN, under drought stress conditions in multiple crop species (Kumar
et al. 2016; Mariotti et al. 2021; Niu et al. 2018; Sandhya et al. 2010; Sarma & Saikia 2014). The
bacterial cells and their metabolites promote plant growth under water stress conditions. Plants
infected with inoculants were found to contain higher amounts of chlorophyll a, b, total chlorophyll,
carotenoids, and stress-​related phytohormones like ABA, JA, and SA (Heidari et al. 2011; Mariotti
et al. 2021). A mixture of three PGPR strains (B. subtilis SM21, B. cereus AR156, and Serratia sp.
XY21) alleviated drought stress in cucumber by maintaining root growth, stabilizing osmotic poten-
tial, reducing plasma lemma peroxidation, enhancing production of SOD and APX in leaves, and
ultimately maintaining the net photosynthesis rate without involving the ACC deaminase to lower
the ethylene level (Wang et al. 2012). Pepper plants inoculated with B. licheniformis K11 enhanced
the production of plant small heat shock proteins (sHSP) by upregulating the transcription of Ca-​
PR10, HSP, Cadhn, and VA genes (Kaushal & Wani 2016; Lim & Kim 2013).
Fungi have been found to be more effective under drought stress than bacteria due to their body
structure and properties (Treseder et al. 2018). The main mechanisms involved thickening of the cell
wall, osmolyte accumulation, and melanin, which made them better candidates than bacteria under
drought stress (Treseder & Lennon 2015). The protective cell wall and filamentous nature of fungi
connected and intermingled together allow water and solutes to flow between them. The hyphae
grow longer and deeper into the soil profile, allowing the extraction of water from deep sites in the
soil. Fungi can tolerate and flourish even more under water stress conditions. Due to this property,
they maintain their density under drought stress, continue their decomposition function, and main-
tain the C and N balance and circulation in the soil (Treseder et al. 2018). Endophytic arbuscular
mycorrhizal fungi (AMF) promote plant growth under adverse environmental conditions, particu-
larly drought and salinity. An extensive network of hyphae collect water from distant locations and
also provide mineral nutrition absorbed from the soil to the host plant, especially phosphorous and
ammonia which it can take up more efficiently than plants (Adamec & Andrejiová 2018; Bizos
et al. 2020; Bona et al. 2015; Marulanda et al. 2009; Matias et al. 2009). Phytohormones JA and
SA are also produced by AMF (Dreischhoff et al. 2020). Various plants species inoculated with
multiple fungal strains in various experiments have produced osmolytes (sugar, protein, proline),
ROS-​scavenging enzymes SOD, POX, and CAT under drought stress conditions and proved to
be promising organisms for a strategy to cope with stress situations (Azad & Kaminskyj 2016;
Dastogeer et al. 2018; Liao et al. 2021; Morsy et al. 2020; Sun et al. 2010; Verma et al. 2021).

1.6 HEAVY METAL STRESS

Another environmental contaminant stress that threatens plant growth and development recently is
heavy metal which is gaining momentous attention due to its deadly consequences. In an effort to
7

Endophytic Microbes 7

attain more yield from the agriculture sector and comfortable lives, the enforced use of pesticides,
herbicides, chemical fertilizers, electrical home appliances, industrialization, mining, vehicles, fuel
combustion etc. all release heavy metals in different forms into environment. The most common
heavy metals released from these sources are cadmium (Cd), copper (Cu), nickel (Ni), cobalt (Co),
arsenic (As), zinc (Zn), lead (Pb), mercury (Hg), and chromium (Cr), which are all persistent
and recalcitrant in nature, damaging eco-​dynamics both above and below the ground (Anbia &
Amirmahmoodi 2016). Long-​term exposure to heavy metals induces physiological and biochemical
impairments in plant architecture. The overall range of toxicity includes inhibition of seed germin-
ation, reduction in chlorophyll content and hence photosynthetic activity, variations in the cellular
metabolic profile which result in oxidative stress and a reduction in enzymatic antioxidant activity,
and variations in anatomical features of plant foliage. These all ultimately lead toward abnormalities
in mitotic activities, which cause reductions in root and shoot growth, wilting, and chlorosis (Amin
& Latif 2017; Garg & Singla 2011; Luziatelli et al. 2020; Mallick et al. 2015).
Bacteria promoting growth in plants under arsenic stress belong to the Gram-​positive (firmicute
and action-​bacteria in which Bacillus spp. is prominent) and Gram-​negative (Proteobacteria where
Pseudomonas spp. holds the prominent position) phyla. The mechanisms used by bacteria to mitigate
arsenic stress include biotransformation via enzymatic oxidation and reduction, bioaccumulation on
the surface, arsenite efflux, methylation, and bio-​adsorption (Thomas et al. 2004). The arsenite oxi-
dation system is well characterized in bacterial isolates from Alcaligenes faecalis, which holds an
island of arsenic genes. These genes alone, or in combination, are responsible for the assemblage of
arsenic oxidase enzyme and its translocation from cytoplasm to periplasm by following differential
mechanisms depending upon the organism (Mallick et al. 2015; Phung et al. 2012). Mercuric ions
(Hg2+​) and methyl mercury (CH3Hg+​) are the two bioavailable toxic forms of mercury. The mech-
anism of detoxification by bacteria includes conversion of Hg2+​ into the homodimeric form which
then ultimately reduces to HgO, which being volatile diffuses into the intracellular environment.
CH3Hg+​ is downregulated by proton attack, which breaks the bond and releases Hg2+​ and organic
acids such as methane (Amin & Latif 2017; Begley & Ealick 2004; Silver & Phung 2005; Zhang
et al. 2020). Reduced uptake, membrane efflux pump, and enzymatic reduction are the most impera-
tive mechanisms used to mitigate chromium stress by bacteria. These mechanisms are among the
characteristics of Shewanella sp., Sphingomonas spp., and Variovorax spp. bacterial species (Bilal
et al. 2018; Viti et al. 2014).
Certain heavy metals like cadmium (Cd2+​), zinc, cobalt, and nickel are difficult to detoxify by
bacterial amendments. The reason behind this complexity is the conversion of divalent cations
(reoxidation) into the highly reactive former form which is not volatile in nature like HgO (Bruins
et al. 2000). Due to the reactive nature of metallic ions and their interaction with cellular organelles
it is difficult to predict the exact mechanism of detoxification. However, adsorption on the cell sur-
face, ion exchange, and chelation prevent the metallic ions from gaining entry into the cell. After
entry, activation of the efflux pump in plants infected with certain bacterial species like Bacillus,
Pseudomonas, Enterobacter, and Rhizobium was found to be active as the resistance mechanism
(Bruins et al. 2000; Silver & Phung 2005). The upregulation of metallo-​proteins in proteomic ana-
lysis of a culture infected with Pseudomonas aeruginosa further strengthens the role of bacteria in
cadmium stress management and maintenance of cellular homeostasis (Izrael-​Živković et al. 2018;
Zhang et al. 2019). Many other reports highlighted the role of multiple bacterial families in tolerance
of lead (Pb) (Chen et al. 2005; Feng et al. 2015; Piazza et al. 2012), copper and its derivatives (Asaf
et al. 2018; Hao et al. 2015; Wei et al. 2009), cobalt (Eitinger et al. 2005), zinc (Wang et al. 2020;
Webb et al. 2017), and nickel (Mazhar et al. 2020; Nanda et al. 2019).
Plant growth-​promoting bacteria are found in diverse ecological habitats, such as the rhizo-
sphere, phyllosphere, root nodules, endophytes, industrial and sewage effluents, etc. and hence
have diverse effects under variable stress conditions. Overall, they are promising biological tools
to enhance plant growth under metal stress conditions as well as metal detoxification. Bacillus,
8

8 Biofertilizers for Sustainable Soil Management

Pseudomonas, Sphingomonas, Variovorax, Enterobacter, and Rhizobium spp. are particularly

involved in metal tolerance by adopting various genomic pathways. The induced genetic activation/​
deactivation of genes results in a variation in the profile of phytohormones which act as a front-​line
defense in plants (Chen et al. 2005; Mazhar et al. 2020; Wang et al. 2020). Phytohormones help to
sustain plant growth by modulating the physiological changes which aid in nutrient uptake under
heavy metal stress conditions. These findings are supported by several investigations describing
indole acetic acid increases (Hafez et al. 2020) under stress conditions which ultimately result in
loosening of the cell wall of root cells. This loosening results in the secretion of root exudate, rich
in organic acid and phytochelatins, attracting the microbial community close to the root zone and
hence restricting the entry of heavy metal in the symplastic pathway (Bacilio-​Jiménez et al. 2003;
Etesami 2018; Schutzendubel & Polle 2002). On the other hand, certain bacteria produce ACC
deaminase under metal stress conditions which inhibit the production of ethylene, a worse growth
repressor in plants under stress condition (Manivasagan et al. 2014). Phosphate-​solubilizing bac-
teria (PSB) modulate the pH of soil and increase the concentration of bioavailable phosphate,
which eventually immobilizes certain heavy metals by adsorption and chelation (Wei et al. 2018;
Yuan et al. 2017).

1.7 CONCLUSION
Abiotic stresses pose serious threats to global food security due to their deleterious effects on plant
physiology, biochemistry, and cellular and molecular biology. Besides the intense debate on the
drastic effects of drought and salinity on forums designed to promote plant production strategies,
heavy metal toxicity also has gained huge attention due to its deadly consequences not only on
plant growth but also on human and animal health. This reinforces the need to adopt an eco-​friendly
strategy to combat these stresses with minimal or no compromise of crop yield. Microbe-​assisted
plant growth, being a biological and eco-​friendly tool, has changed the global scenario addressing the
impacts and strategies to secure plant production under stress situations. Several bacterial (Bacillus,
Pseudomonas, Rhizobium, Acinetobacter) and fungal genera have been found to exhibit a multi-​
tolerance ability under different abiotic stress and ecological conditions. Under all types of abiotic
circumstances, microbial infestation modifies the profile of ACC deaminase, IAA, phosphate solu-
bilization, exopolysaccharide synthesis (EPS), osmolyte production, and accumulation at various
rates depending upon the crop species, microbial strain, ecological condition, growth stage of the
plant, and concentration of enzyme produced. Plants infected with inoculants were found to contain
higher amounts of chlorophyll a, b, total chlorophyll, carotenoids, and stress-​related phytohormones
like ABA, JA, and SA. Microbes can also be used in the strategy to adsorb, physical entrapment on
the cell surface, and reduction in uptake, particularly with metal toxicity to plants and altering cel-
lular architecture. Cellular architecture modulations include reduced uptake of metal ions, chelation
with other organic compounds, and efflux pump activation in the case of huge uptake. Microbe-​
assisted plant growth has arisen as a promising eco-​friendly and biological strategy in sustainable
agriculture projects designed under both normal and stressful situations.

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25

2 Legacy Phosphorus
Tracking Budget, Vulnerability,
and Mobility to Ensure
Sustainable Management in the
Agricultural System
Sammina Mahmood* and Umair Ashraf
Department of Botany, Division of Science and Technology,
University of Education, Lahore, Punjab, Pakistan
* Correspondence: [email protected]

CONTENTS
2.1 Introduction............................................................................................................................. 25
2.2 Phosphorus Cycle.................................................................................................................... 26
2.3 Global Phosphorus Reserves and Geological Variations......................................................... 27
2.4 Phosphorus Budget and Vulnerability..................................................................................... 28
2.5 The Phosphorus Balance at the Farm Level and Fertilization................................................. 28
2.6 Factors Affecting Phosphorus Use Efficiency (PUE).............................................................. 29
2.6.1 Role of P Source and Soil Characteristics in Mobility and Bioavailability
of P to Soil.................................................................................................................. 29
2.6.2 Soil P Transformation and Mobilization.................................................................... 29
2.6.3 Organic Matter (Animal Manure, Crop Residues, and Biochar)................................ 30
2.6.4 Organic Acids (Humic Acid, Lignin, etc.).................................................................. 31
2.6.5 Diversity of Bio-​inoculants and Bio-​fertilizers.......................................................... 32
2.7 Bio-​fertilizers..........................................................................................................................33
2.8 Zeolites.................................................................................................................................... 34
2.9 Conclusion............................................................................................................................... 35
References......................................................................................................................................... 35

2.1 INTRODUCTION
The use of phosphorus (P) is critical for plant active growth and development. This essential nutrient
has boosted the use of P-​based fertilizers in the agriculture sector over the last few decades at a
global scale (Sharpley et al. 2018). However, due to a lack of awareness at the farm level, its use has
remained inappropriate and unmanaged, with use over the recommended dose in the quest for yield
increases from limited land areas over a long period of time. These unplanned activities have pushed
the natural P resources towards two uncertain situations: firstly, over-​and unmanaged use either
leads to runoff of surplus P to water resources or sediment build up leads to “legacy P” at the ground
level. Secondly, over-​use has exerted huge pressure on limited and scarce P resources (Mekonnen
& Hoekstra 2018). The first situation results in environmental degradion of water resources and the
second is disturbing the balance between P supply and demand, and due to this, prices of P0based
fertilizers will probably increase in the future (Nesme & Withers 2016)

DOI: 10.1201/9781003286233-2 25
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26 Biofertilizers for Sustainable Soil Management

the elemental form of P is found in two physical forms, the white or yellow form (P4) and red
phosphorus, both of which are allotropes having miscellaneous physical and chemical properties.
White phosphorus has higher reactivity due to its tetrahedral structure compared to red phosphorous
which is polymeric (Pn) in nature and more stable (Pfitzner et al. 2004). However, red P can be
transformed into white P by heating at 300oC in the absence of oxygen or by exposing it to direct
sunlight. P, due to its reactivity, is not found in elemental form in nature. It is mineralized itself
mostly in the form of phosphates (Ca/​Fe/​Al), predominantly in sedimentary rock and dispersed
worldwide (Zhen-​Yu et al. 2013). Rocks formed from volcanic eruptions also act as a source of P,
however this is rare compared to sedimentary rock.
Sedimentary rocks containing phosphorus minerals are unevenly distributed around the world.
Various physical, chemical, and environmental conditions and geological factors are associated with
sedimentary rock formation along the coastal upwelling zones, which vary greatly among countries
at a global level (Filippelli 2011). The most common group of phosphorus-​containing minerals
is apatite. The basic constituent of apatite mineral is calcium phosphate attached with F–​, Cl–​, or
OH–​ and known as fluorapatite, chloapatite, and hydroxyapatite, respectively. Sedimentary rock
or phosphate after processing (the process called beneficiation) is known as phosphate rock (PR).
According to an estimate, the global total PR amount was 68,776 Mt in 2014. Among this, almost
75% is in Morocco and Western Sahara and 5.4% in China (Jasinski 2011). An investigation into
the future of P reserves predicts that “almost 70% of global P production is from P reserves, and
if the same situation continues the P reserves will be depleted within 100 years.” Morocco, having
the biggest resources (about 77%), produces P fertilizers at an increasing rate which will be almost
700% by 2075. This pressure will be due to an increase in the global population with increasing food
demand at an alarming rate, accentuated by depleting P reserves at a global level. This situation will
create geopolitical crises among countries for P production and distribution (Cooper et al. 2011;
Walan et al. 2014)
This suggests that current wasteful practices in the management of P resources and utilization
should be modified, keeping in mind the sensitivity and lethality of the issue. As unwise use on one
side is putting pressure on resource management, on the other side, it is increasing environmental
concerns, particularly about water pollution. Thus, sustainable management of P-​fertilizers in agri-
cultural systems is crucial and could be achieved by adopting strategies based on P-​cycling and
recycling in natural systems, P-​use efficiency, minimizing the losses through runoff, and reducing
the reliance on synthetic P inputs. This chapter is focused on reviewing the current status of the
above-​mentioned factors and find the ommissions hindering their utility towards sustainable agri-
culture in the future.

2.2 PHOSPHORUS CYCLE
The biogeochemical cycle that describes the phosphorus flow/​movement through the lithosphere,
hydrosphere, and biosphere of the modern agricultural system is called the phosphorus (P) cycle. P
does not exist in a stable gaseous form; hence the P cycle is based on sedimentary flow among mul-
tiple components of the environment. In the natural P cycle, with no human intervention, phosphate
rock (PR) releases P ions in response to weathering of rocks due to environmental influences, into
soil and water media. This released P may be categorized in two forms: soluble and insoluble or par-
ticulate forms. The soluble form of P in soil is taken up by plants and then animals or humans (dir-
ectly or indirectly through the food web). P is then returned to soil through animal/​human excretion,
and decomposition of dead bodies of animal/​humans/​plants, as P is one of the basic constituents of
all life forms on Earth. The insoluble form of P within soil is either leached down into deep ores of
the Earth’s crust or a fraction of it may be converted to soluble form by the activity of soil microbes
to make it absorbable by plants. However, both soluble and insoluble forms of P can be drained into
water reserves in response to poor agricultural practices (Liu et al. 2014; Qiu & Turner 2015).
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Legacy Phosphorus 27

P released in water medium in response to weathering of rocks may also be categorized in two
forms: soluble and insoluble or particulate forms. The soluble form of P is quickly be taken up by
phytoplankton and aquatic plants. Competition for and depletion of resources among phytoplankton
causes their death and decomposition within water bodies. These dead bodies have two fates; they
either cause eutrophication or they pollute the water or it sediments along with other dead aquatic
life in the deep ocean. These sediments over many years appear as rock and are exposed in response
to geological catastrophes or shifts. On the other hand, the particulate or insoluble form of P has
only a single fate, i.e. sedimentation. P is either drained from soil into water or directly released
into water; in both situations it deteriorates the quality of water reserves (Capodaglio et al. 2005;
Capodaglio et al. 2003; Capodaglio et al. 2016; Søndergaard et al. 2003)
Human interference in the natural P cycle has raised several concerns. Humans have used syn-
thetic P fertilizers unwisely through agricultural management practices in order to get better yields
from a reduced land area. This practice has disturbed the natural P cycle and reserves in the soil.
Surplus P is either drained into water where it intensifies water pollution crises or it adds to deep
soil unused reserves (Filippelli 2011). These unused reserves are called “P legacy” and they act
as a secondary P source and could be made available by adopting different agricultural and man-
agement practices (Kusmer et al. 2019; Qiu & Turner 2015). P “legacy” denotes surplus P used
by any means (organic or inorganic) that leaches down into the deep soil profile or sediment into
water reservoirs over time. P legacy varies greatly at inter-​and intra-​country levels based on the
mode of use, soil/​land characteristics, consumption and decomposition level, prevailing cropping
patterns, and expected runoff into water reservoirs (Borbor-​Cordova et al. 2006; Kleinman et al.
2011; Kusmer et al. 2019; Qiu & Turner 2015; Adnan et al. 2018a,b; Adnan et al. 2020; Adnan et al.
2019; Atif et al. 2021).
Buffering capacity refers to the ability of a soil or water reservoir to attenuate the P loading
to the surface soil or water by retaining P inputs over time, which varies greatly across human-​
dominated landscapes (Gentry et al. 2007; Ahmad et al. 2017; Kusmer et al. 2019). The history of
P use and accumulation, along with these factors in current P-​based planning are considered as key
P-​regulating services for restricting the excessive use of P in farming systems (Doody et al. 2016).
A correct estimate between vulnerability and buffering capacity requires a deep knowledge of the
following properties, which varies over space and time.

2.3 GLOBAL PHOSPHORUS RESERVES AND GEOLOGICAL VARIATIONS

In soils vs. aquatic environments, P is widely present in different forms and its availability rate
also varies greatly. In soils, P is present in soluble, mineral, adsorbed, and organic forms. The sol-
uble form of P (orthophosphate ion; PO43–​) is the only form which plants can use for their growth
directly; however its percentage of occurrence in soil is very low compared to total phosphorus
(TP). Insoluble P consists of organic and mineral P, and is the dominant form of P present in soils.
This dominant fraction of P is present in the form of iron/​aluminium phosphate in acidic soil and
calcium phosphate in alkaline soils, which limits its solubility within these soils (Zhen-​Yu et al.
2013). Organic phosphates also include the phosphate forms excreted by microorganisms in soil,
which upon microbial action during a mineralization process are broken down into inorganic form.
However, the activity of microorganisms is highly dependent upon the soil moisture content, soil
temperature, and physiochemical properties of soil. Hence, TP is the sum total of mineral, organic,
and adsorbed forms with a negligible amount of soluble P. Soil and fertilizer management practices
should be based on TP rather than soluble P (Frossard et al. 2011; Khan et al. 2014).
Compared to soil, in aquatic environments the concentration of soluble P is higher due to the
lower percentage of Ca/​Fe/​Al ions. In addition, agricultural sewage, industrial waste, domestic
sewage, and detergents are also sources of P which pollute water resources. As P is an essential
nutrient for plant growth and metabolism, its presence in water promotes the growth of plants and
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28 Biofertilizers for Sustainable Soil Management

phytoplankton within water bodies causing eutrophication (Fahad et al 2016; Fahad et al. 2015a,b;
Fahad et al. 2020; Fahad et al. 2021a,b,c,d,e,f; Fahad et al. 2022a,b). P is also a vital component
of all living organisms, as a basic building block of DNA, RNA (in the form of PO43–​), ATP, and
phospholipids. Also, considerable amounts of P are also present in the teeth and bones of biological
bodies (Boskey 2013).

2.4 PHOSPHORUS BUDGET AND VULNERABILITY

The green revolution has boosted agricultural production with the help of commercial fertilizers.
P, as an essential nutrient for crop growth and development, is used as a commercial fertilizer at
an increasingly alarming rate at the global level. Increasing competition among growers with the
objective of obtaining increased yields from reduced land area with an improved nutritional profile
has diverted the mind set from organic fertilizer to synthetic/​commercial fertilizer use. This in turns
has posed several other undesired issues which initially remained unnoticed. (1) The use of organic
fertilizers was reduced, which were not only playing a role in biological nutrient cycling by the pro-
cess of decomposition but also helping to improve the soil physical and chemical properties. (2) The
chief source of commercial P fertilizer is rock phosphate/​mineral. Increased production demand
rapidly depletes the slowly non-​renewable finite resources (Zhen-​Yu et al. 2013). (3) At the geo-
logical scale, rock phosphate/​mineral resources are scattered differently. This situation could create
a socio-​political imbalance between countries in the future (Khan et al. 2014; Alam et al. 2016).
(4) P exhibits the property of leaching down into the deep soil profile in both organic and inorganic
forms. Soil with extensive application of commercial fertilizer has a soil P legacy. This P legacy has
two fates. (i) It can remain in soil unused by chelating with other minerals. This relationship also
makes other nutrients unavailable for plants and sometimes surplus P has caused toxicity in soil
as well. (ii) Surplus P can drain into water reservoirs and deteriorate the water table quality both
physically and chemically (Kusmer et al. 2019; Qiu &Turner 2015). (5) The reduction in organic
fertilizer also necessitates the use of commercial fertilizers with other essential nutrient s,e.g. N, S,
etc. (6) There is as yet no international organization or regulation that sets the standard for the P
dose rate depending upon soil and environmental history, or that manages P resources and provides
legal constraints. All these factors further complicate the situation when estimated on the impacts on
developing and developed countries.
P available resources at regional or international level, P recycling, and the status of P legacy in
soil determine the overall P budget. The P budget helps to manage the balance between availability
and demand. the efficacy of the P budget could be enhanced by improving P use efficiency (PUE)
which can be defined as the use of P at a decreased rate with increased efficiency. PUE varies greatly
at the global as well as regional level depending upon the management practices adopted in its use.

2.5 THE PHOSPHORUS BALANCE AT THE FARM LEVEL AND FERTILIZATION

P application into soil according to the requirements of available crop cover is called the P balance for
that site. However, this ideal situation is rarely met. It is now obvious that in the usual routine, applied
P into the soil for food production is quite high compared to the actual need and use at the field level
(Liu et al. 2008). This disturbs the P balance which is becoming a common issue not only at the
regional level but also attaining global attention. Some general facts reported behind this imbalance
include the following. (1) At the global scale, variable geological and geographical conditions fur-
ther enhance this issue. One site may be exposed to erosion and depletion of resources as in the case
of deserts compared to other sites where resources become accumulated with the passage of time,
such as forests. Due to this variation, the dose rate recommended for one site cannot be applied with
equal success rate at another site. (2) In certain regions, manure from farm animals is the main source
of fertilizer on crop lands. This practice also has been adopted in some regions as good agricultural
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Legacy Phosphorus 29

practice. This trend is not common at the regional or global level. This factor, along with fertilizer use,
strengthens the P reserves already present in the soil. (3) Each crop type has different P requirements
based on the root characteristics and the length of the growth period. However, the application rate
of phosphorus (either in the form of manure or fertilizer), often exceeds the required levels for crops.
This promotes the P status of the soil to higher level. (4) The soil microbial profile for P fixation
characteristics is also variable at regional as well as global levels. This also plays a role in variations
in P requirements based on site conditions (de Bruijne et al. 2009). (5) Phosphate available in soil
may be immobilized through precipitation and adsorption reactions, making phosphorus unavailable
for plant uptake (Syers et al. 2008). All these facts divert researchers’ attention towards the develop-
ment of strategies to determine soil P requirements (application rate/​dose rate) depending on the soil
fertility level (P status) in order to avoid yield depression at the farm level. This could be achieved
by considering the on-​site soil characteristics, past cropping history with plant characteristics, and
current crop type under consideration for plantation (Cordell et al. 2009; Cornish 2009). However, a
single survey cannot be applied for all future applications. This needs to be determined on an annual
basis in order to avoid yield depression (Roemer 2009; Ilahi et al. 2020). Besides these strategies,
public awareness about the scarce resource, application protocol, and enhanced P use efficiency is
also crucial at the regional level to ensure the wise use of this scarce resource.

2.6 FACTORS AFFECTING PHOSPHORUS USE EFFICIENCY (PUE)

2.6.1 Role of P Source and Soil Characteristics in Mobility and Bioavailability of P
to Soil

The fate of applied P is also called P-​cycling in the soil and is determined by P solubility and
reactivity characteristics and topographic characteristics of the particular soil which ensure bioavail-
ability to the planned cropping pattern (Liu et al. 2014). Both P source and soil characteristics are
interdependent on each other. Plants usually are unable to uptake almost 80% of applied P fertilizers
immediately after application due to sorption, precipitation, and microbial immobilization (either
through root or mycorrhizal hyphae) regulated by soil acidity, basicity, pH of soil, etc. (Roberts &
Johnston 2015), e.g. P associated with iron and its oxides in soil is sensitive to reduced conditions of
soil (Beauchemin et al. 2003), while with aluminium (oxides) and calcium phosphate it is more sen-
sitive towards pH fluctuations of soil (Yan et al. 2014). Exudation of phosphatases and organic acids
from roots or microorganisms further play significant roles in accelerating the P cycle by releasing
P from the P source (either P legacy or applied fertilizer) (Frossard et al. 2011).
Further bioavailability is hugely dependent upon the organic and inorganic nature of the applied
P (Khan et al. 2014), e.g. at pH 4–​5 H2PO4–​ becomes dominant while as the pH increases HPO42–​ and
PO43–​ become dominant in the soil (Yadav & Verma 2012). Diversity in soil characteristics is further
added to by soil texture, soil management history, cropping pattern/​vegetation cover, and economic
and social factors associated with vegetation in a particular area. All of these factors in combination
determine the magnitude of P cycling in the plant–​soil system (Frossard et al. 2011).

2.6.2 Soil P Transformation and Mobilization

Transformation and mobilization of legacy P is a complex process governed by multiple factors.
Transformation and mobilization depend upon the power of the activation of variable factors, the
addition or manipulation of which change the physical and chemical properties of soil (i.e. texture,
soil pH, soil particle charge, etc.). These factors are divided into three main groups: (1) organic
matter (animal manure, crop residues, biochar, etc.) including organic acids (humic acid, lignin,
etc.); (2) bio-​inoculants and bio-​fertilizers which include P-​solubilizing microbes and phosphatase
enzymes; and (3) zeolites.
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30 Biofertilizers for Sustainable Soil Management

2.6.3 Organic Matter (Animal Manure, Crop Residues, and Biochar)

In developing countries specifically, and developed ones also, organic matter application into
cropping fields in the form of organic acids, crop residues, animal manure, etc. is a common prac-
tice. Organic matter is used because of its rich nutrient profile, afordability, readily available at
rural sites especially, and as a biological source with few harmful effects. Animal manure, crop
residues, and biochar (charcoal produced from crop residues) are all biological sources of multiple
nutrients. Land application with these to dispose of the agricultural waste and recycle the nutrients
is a predominant farming practice in developing and resource-​deficit countries (Dai et al. 2016).
The availability of nutrients from these practices depends upon two major factors. (1) The addition
and decomposition of these materials influences the overall physical and chemical profile of soil.
These fluctuations have a huge and variable impact on the solubility, mobility, and availability of
various nutrients, again dependent upon nutrient concentration and type of interaction with soil
particles. (2) The type of animal and crop residues added into the soil and the receiving soil envir-
onment determine the nutrient profile, their magnitude, and direction of availability. However, in all
circumstances, these biological sources act as a primary source of nutrients in soil (Damon et al.
2014; Espinosa et al. 2017).
With reference to P being an organic material, all these are rich sources of highly soluble P
(particularly orthophosphate) which could be mineralized in a very short period of time to release
soluble P. However, including the factors mentioned above, several additional key factors govern
the availability of organic matter-​derived P in soil. These factors include: (1) quality of organic
residue; (2) the activity level of soil microbial entities; and (3) sorption reactions of mineralized
P into soil (Damon et al. 2014). Usually, increased organic matter content in soil provides a more
favourable environment to microbes in the soil, which in turn accelerates the P cycling and mineral-
ization (Lone et al. 2015). Crop-​and animal-​based residues showed differential available P contents.
General findings predict almost 2–​20% P transfer from crop sources, which is relatively low and
highly variable. This could be attributed to any of these factors, e.g. the P concentration in animal
or crop residue, physiological maturity of crop residue, soil P status, and soil type (Espinosa et al.
2017). In contrast, P availability from animal manure ranged between 12–​100%. This variation
in range could be attributed to animal age, diet, and the way manure is stored/​preserved (Bahl &
Toor 2002). The addition of organic matter in all these forms has not only proved to be a primary
source of highly soluble and available P but also affects the native P source and changes its mobility
positively. This occurs through chelate formation with insoluble Ca, Al, and Fe forms of P and
converting it into soluble forms during the decomposition process (Hountin et al. 2000).
The application of biochar mainly affects the ion exchange capacity of soil. In biochar, the P form
changes compared to residues due to its bonding with multivalent metal ions. This ability can affect
the P availability either positively or negatively (Chathurika et al. 2016). In acidic soils, biochar
exhibits anion exchange capacity which competes with metal oxides (Al, Fe) for sorption of P, simi-
larly to humic acid and lignin (DeLuca et al. 2015; Hunt et al. 2007), while this response is reversed
in saline and sodic soils which promote precipitation of P into soil (Xu et al. 2016). P solubility
in acidic soils is mainly regulated by its interaction with Al3+​ and Fe2+​/​Fe3+​, while in saline soils
it is with Ca2+​. As soon as the application of biochar in acidic soils is increased, soil pH increases
and P sorption on metal oxides decreases (Chintala et al. 2014). Biochar application also requires
accurate calculation of the dose rate/​application rate depending on the soil type as being an organic
source it is also enriched with other types of mineral nutrients and elements such as Ca and Mg,
etc., with pronounced effects on plant growth and development (Parvage et al. 2013; Vandecasteele
et al. 2016). The magnitude of the effects of biochar on P cycling is highly dependent on sev-
eral key factors, i.e. production method of biochar, climate, variability in feedstock, and soil type.
Biochar derived from agricultural residues, animal residues, and crop residues overall show positive
responses to plant-​available P, while biochar derived from wastewater sludge has a poor response.
Since the total P produced annually from organic sources is higher than the global production of
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Legacy Phosphorus 31

P fertilizer, it seems to be a helpful and economical tool to thermochemically convert manure into
biochar to minimize the load on overall mineral P fertilizer production (Steinfeld et al. 2006). This
recycling of P from biological residues is a continuous source of P for soils and has environmental
benefits compared to direct land application of mineral fertilizers (Dai et al. 2016; Manolikaki et al.
2016; Wang et al. 2015b). It has the potential to enhance the plant-​available P in biochar-​amended
soils and could be a sustainable strategy to complement conventional P fertilizers. Unfortunately,
sufficient research is not available regarding P contents of biochar, application rate, or its correlation
with plant-​available P.

2.6.4 Organic Acids (Humic Acid, Lignin, etc.)

Organic acids are further classified into low-​molecular-​weight organic acids and high-​molecular-​
weight organic acids. Both types are not only act as direct sources of P but also help in the mobil-
ization of reserved or legacy P. These characteristics are further dependent upon their chemical
structure and its reactivity, their solubility, affinity towards other soil elements, and dissociation
power. All these characteristics change the soil physical properties directly or modify the chemical
profile in a cost-​effective way. Both have the property to increase the soil fertility level and improve
the soil nutritional profile, including P (Çimrin et al. 2010).
The low-​molecular-​weight organic acids are derived from two main sources: (1) plants excrete
under P-​deprived conditions into soil or (2) released in response to decomposition (microbial action
and activation) of animal and plant residues (Yuan et al. 2016). Examples of low-​molecular-​weight
molecules include oxalic acid, malic acid, fumaric acid, tartaric acid, succinic acid, tartaric acid,
etc., which all exhibit the property of having at least one carboxylic group attached. The exact role
of organic acid and the mechanism of activation to P source/​P legacy or applied resource remains
unknown and needs experiments in a targeted direction, however few experiments based on the arti-
ficial application of organic acids alone (Tripathi 2005), in combination (Wang et al. 2015a; Wei
et al. 2010; Fakhre et al. 2021; Muhammad et al. 2022; Shafi et al. 2020; Wahid et al. 2020; Zahida
et al. 2017), or in combination with phosphatase enzyme (Guan et al. 2013) have been conducted.
The results overall have revealed that the possible mechanism of action behind this activation might
be any of the following. (1) Addition to soil with specificity may change the soil physio-​chemical
properties in quite a predictable manner, which in returns affects the solubility of P source/​P legacy,
e.g. variation in soil pH can affect the dissolution of sparingly soluble minerals containing P to a
great extent (Andersson et al. 2015). (2) These might form complexes with other elements present in
soil and having great affinity for P to form multiple types of complexes and making it unavailable,
e.g. Al, Fe, Ca, etc. (Ström et al. 2005) with the enhanced organic anion-​driven solubilization of Ca
complex observed compared to plant-​based hydrolysis and microbial phytases (Patel et al. 2010).
(3) An electrostatic competition observed for sorption into soil between organic acid and P. The
presence of two types into the same soil profile affects both antagonistically. However, in all situ-
ations the exact mechanism still needs to be elucidated.
High-​molecular-​weight organic acid compounds include humic acid and lignin. Humic acid is
derived from the decomposition of plant litter, while lignin is an important precursor in the forma-
tion of humic acid as a result of microbial action. Humic acid exist in a range of structures (ali-
phatic or aromatic) with a variety of functional groups (phenyl, carboxylic, etc.), mainly oxygen
containing (Çimrin et al. 2010). Likewise lignin has major functional groups, such as carboxyl,
carbonyl, hydroxyl, and methoxyl. Specifically, both ingredients of organic matter have produced
success stories with remarkable increases in available P compared to total P of soil in calcareous
soils and soils rich in iron oxides and iron-​aluminium oxides (Chen et al. 2003; Zhen-​Yu et al. 2013).
As far as the mechanism of action is concerned, lignin is a precursor of humic acid and they have
almost identical structural and functional properties, and both have very similar mechanisms of
action (Sun et al. 2011; Tahir et al. 2011). (1) Shift in soil pH: Decomposition of humic acid into soil
32

32 Biofertilizers for Sustainable Soil Management

changes the pH level of soil by producing H+​ ions. This either decreases the Ca-​P mineral precipi-
tation or favours the formation of di-​calcium phosphate dehydrate which is the most stable form of
phosphate with the least solubility properties into soil (Grossl & Inskeep 1991). (2) The formation of
organic ligands/​colloidal interactions with metal and metalloids: functional groups present in high-​
molecular-​weight organic acids, e.g. phenyl, carboxylic, etc. make complexes with metal ions, spe-
cifically Fe and Al, which are mostly associated with phosphate activity and fixation into soil. The
affinity of functional groups for these metal ions enhanced their removal from soil, strengthening the
competitive phosphate ratio in soil (Antelo et al. 2007; Regelink et al. 2015).

2.6.5 Diversity of Bio-​inoculants and Bio-​fertilizers

Microbes, particularly bacteria and fungi, have a natural ability to manipulate the organic and
inorganic complexes present in soil and recycle the nutrients present in them into soil. These
microbes are called bio-​inoculants, exhibiting huge potential to replenish soil nutrient levels in
an eco-​friendly and cost-​effective way. Phosphate-​solubilizing microbes (PSMs) are those which
are capable of hydrolysing organic and inorganic insoluble P compounds into soluble form (plant
accessible) (Khan et al. 2009; Owen et al. 2015; Selvi et al. 2017). PSMs may be comprised
of phosphate-​ solubilizing bacteria (PSBs), phosphate-​ solubilizing fungi (PSFs), phosphate-​
solubilizing actinomycetes (PSAs), and cyanobacteria. The role of these microbes in the availability
and acquisition of P by plants has been described by several researches worldwide. Well-​known
examples of bacteria which play a significant role in P replenishment in soil includes Bacillus
megaterium (Satyaprakash et al. 2017), Bacillus circulans (Kumar & Patel 2018), Bacillus subtilis
(Hajjam & Cherkaoui 2017), and Bacillus cereus (Yu et al. 2011). General examples of fungi
known to be able to solubilize P includes Aspergillus awamori (Mittal et al. 2008; Selvi et al.
2017), Aspergillus versicolor (Ho et al. 2013), Aspergillus niger (Li et al. 2014), Aspergillus
clavatus (Alam et al. 2002), Penicillium citrinum (Mittal et al. 2008), and Arthrobotrys oligospora
(Duponnois et al. 2006). Besides bacteria and fungi, actinomycetes and cyanobacteria also have
exhibited influential roles in P recycling; examples include Acinetobacter rhizosphaerae (Gulati
et al. 2010), Streptomyces albus, Streptoverticillium album (Kumar &Patel 2018), and Calothrix
braunii (Sharma et al. 2013), respectively.
Soil is the basal medium which provides ideal conditions for microbial growth and replenish-
ment under natural conditions. According to one estimate, one gram of fertile soil contains 101–​
1010 microbial load which includes 1–​50% bacteria and 0.1–​0.5% fungi, respectively (Khan et al.
2009). However, significant variation is observed in the ability of microbes overall, bacteria vs.
fungi and among other microbes also, to solubilize insoluble P in soil (Jones & Oburger 2011). The
variation in this potential may be attributed to species diversity of particular microbes and plant
type, prevailing soil and environmental conditions/​factors (biotic and abiotic) of a particular site,
efficacy of microbes to produce organic acids and phytohormones, and the ability of microbes to
survive under fluctuating environmental conditions. Generally, PSFs produce more acids than bac-
teria and consequently, irrespective of the lower percentage of their occurrence, they exhibit greater
P-​solubilizing potential (Sharma et al. 2013).
Besides the ability of actinomycetes to produce phytohormones and survive under harsh environ-
mental conditions (biotic and abiotic), their role in P availability and acquisition by plants also has
attracted attention worldwide. The properties and biological role of actinomycetes in soil is paving
the way to prove them as more influential microbial entities than bacteria and fungi, particularly
under harsh environmental conditions (Hamdali et al. 2008). The overall mechanism of action of
microbes may likely include the production of organic acids after inoculation, changing soil phys-
ical and chemical properties, ion chelation, promoting root growth, penetration and permeability
in establishing a mycorrhizal association among roots and microbes, and strengthening the micro-
bial population with the passage of time. The whole mechanism establishes a microbe–​soil–​plant
33

Legacy Phosphorus 33

triangle and the role after microbial inoculation in P recycling depends directly or indirectly on the
properties of the components of this triangle (Suri et al. 2011).
Organic acid production by microbes lowers the soil pH (Kumar & Patel 2018; Selvi et al. 2017).
Acidic and basic soils react differently. In basic/​alkaline soils, phosphate can precipitate with cal-
cium to form calcium phosphate which is insoluble into soil. an increase in pH is associated with the
production of divalent and trivalent forms of inorganic P (HPO4–​2 and HPO4–​3), making P unavail-
able for plants. However, their solubility can be increased by lowering the soil pH. Organic acids
produced by microbes play a role in lowering the pH and consequently improving the P solubility
in the soil (Satyaprakash et al. 2017).
A positive correlation exists between organic acid concentration and P solubility in soil regulated
by the pH profile. pH is lowered with the evolution of CO2 and soil becomes acidic, which in turn
acts as a positive regulator of P solubility (Alam et al. 2002; Selvi et al. 2017; Walpola & Yoon 2012;
Yousefi et al. 2011). The efficacy of organic acid to lower soil pH is directly dependent on its type
and concentration, which determines the strength of the acid. For example, di-​and tri-​carboxylic
acids are more effective than monocarboxylic and aromatic acids. Aliphatic acids have been found
to be more effective than citric, fumaric, and phenolic acids (Mahidi et al. 2011; Walpola & Yoon
2012). The main organic acids include citric, oxalic, lactic, glyconic, gluconic, malic, succinic,
acetic, tartaric acid, malonic, glutaric, butyric, and adipic acids (Ahmed & Shahab 2011; Kumar &
Patel 2018; Satyaprakash et al. 2017; Selvi et al. 2017; Walpola & Yoon 2012; Yousefi et al. 2011),
with each exhibiting variable potential in P solubilization. These organic and inorganic acids com-
pete with phosphate for the sorption site by chelation with cations (Al and Ca oxides) present in
soil, and due to this characteristic, they are also known as “chelators.” The hydroxyl and carboxyl
group of acid chelates with cation initially bound to phosphate, thereby converting it into a soluble
P form. These acids stabilize Al and Ca oxides in reaction with them and compete for their reaction
sites (Khan et al. 2007; Walpola & Yoon 2012).

2.7 BIO-​FERTILIZERS
Various fertilizers are constituted by adding microbes to them, and are called “bio-​fertilizers” as
the role of microbes is very clearly established in P recycling in an environmentally friendly way
without any harmful effects. Hence, the development of bio-​fertilizers also serves the same role as
microbes in soil. The trend to use bio-​fertilizers as an alternative to chemical fertilizers is acceler-
ating currently due to their cost-​effectiveness and improved efficiency in P recycling. Microbes used
in bio-​fertilizer preparation include bacteria (Bacillus spp., Pseudomonas spp., and Rhizobium spp.),
fungi (Aspergillus spp., Penicillium spp., and Rhozopus spp.) and actinomycetes (Staphylomyces
spp. and Streptomyces) (Mahajan & Gupta 2009). Bio-​fertilizers, compared to chemical or synthetic
phosphate fertilizers, are based on mycorrhizal infection at root level which not only promotes
the conversion and mobilization of P from soil to plant but also serves to improve plant growth
and development by secreting a range of exudates (e.g. metabolites, H+​, organic acids and several
enzymes including phosphatase, etc. depending upon the species and strain used in the synthesis of
bio-​fertilizer). The use of dissolved P mixed microbe fertilizers has significantly reduced the depend-
ency on chemical P fertilizers in modern agricultural practices (Behera et al. 2014; Mukherjee &
Sen 2015).
The mechanism of action is similar to bio-​inoculants by means of the release of organic
acids, phosphatase enzymes, and H+​ and finally converting the insoluble forms of P [(tricalcium
phosphate(Ca3(PO4)2, iron phosphate (FePO4), and aluminium phosphate(AlPO4)] (Selvi et al. 2017)
into soluble forms (Khan et al. 2014). Particularly, the enzymes secreted by PSMs which include a
broad range (e.g. C-​P lyase, phytase, phosphatse, phosphonatase, and phosphohydrolase) catalyse
the hydrolysis of both esters and anhydrides of phosphates (Jones & Oburger 2011). This enzyme
range is particularly important in soils where plant-​or animal-​based manure is applied as the base
34

34 Biofertilizers for Sustainable Soil Management

organic fertilizer. As a consequence, huge amounts of sugars, sugar phosphates, and nucleotides are
available in those soils, which serve as huge raw sources of P. However, the reactivity cycles of each
enzyme and consequently metabolic products released from the hydrolysis of biological compounds
are variable (Khan et al. 2010; Sharma et al. 2013). The effectiveness of bio-​fertilizers is dependent
upon the type of plant, microbial species and strain used, growth stage of plants, and environment
including the soil conditions (biotic and abiotic factors). Hence, plants also act as biocontrol agents
for microbes in soil (Behera et al. 2014).
In addition, mechanical agricultural practices also have a huge impact on P solubilization and
availability through PSMs. It has been observed that deep tillage affects positively microbial growth
and phosphatase activity in clay and loam soils. Deep tillage promotes the growth conditions for
microbes and helps to increase their growth rate in soil as well as promoting phosphatase activity.
This may be because deep tillage loosens the soil particles, aerates the soil, and mixes organic matter
to deep layers of soil, thereby increasing the microbial profile deep in soil. Clay soils are reported to
exhibit more PSM activity compared to loam, which may be because clay soils offer a greater sur-
face area and small pores for incorporation, mixing, and manipulation of organic matter compared
to loam soils. Both situations reveal that deep tillage practice has been found to have a positive cor-
relation with PSM activity (Ji et al. 2014). However, by increasing the soil depth several factors also
fluctuate, including the temperature of the soil. In some reports high temperature has been found
to increase phosphatase activity. However, there is a complicated relationship between tempera-
ture and phosphatase activity, which cannot be sufficiently explained with only a few examples, as
several other factors like soil type, cropping history, environmental conditions, microbe type, and
soil chemical profile can complicate the situation and impede prediction of the exact relationship
between temperature and phosphatase activity (Das et al. 2014; Štursová & Baldrian 2011).
Abiotic stresses, e.g. salinity and heavy metals, are also found to interact negatively with phos-
phatase activity (Fahad et al 2021f). At increased salinity and sodicity levels of soil, alkaline phos-
phatase activity is reduced due to denaturation of enzyme protein and a reduction in microbial growth
(Rietz & Haynes 2003). Likewise, heavy metals interact with protein functional groups and bring
conformational changes to the structure (Karaca et al. 2010). To mitigate these stresses, the addition of
P activators may help in enzyme activity, but insufficient literature is currently available on this topic
to describe the mechanisms and effects in detail. Intensive research work is required in this direction
in future so that a successful strategy can be developed to promote P resource utilization through bio-
logical means before their depletion. All these are organic amendments to enhance P utilization.

2.8 ZEOLITES
Zeolites are microporous, hydrated, and crystaline calcium, sodium, potassium, and barium
aluminosilicates used as an inorganic amendment in soil to improve P fixation and utilization. The
uses of inorganic zeolites however vary. Zeolites are formed in nature by the chemical reaction of
volcanic lava with saline water. It is characterized by a large sorption site, ion exchange capacity and
selectivity, catalytic activities, and most importantly thermal stability for a temperature range of up
to 700–​750oC (Yangyuoru et al. 2006). These properties impart strength to zeolite, making it diffi-
cult to break down. It can be retained in soil for a long period of time as a source of minerals required
by plants for their growth, and hence plays a significant role in soil management. The hydrated and
amorphous silica skeleton helps to retain water and nutrients at the root zone level, which ultimately
reduces the water and fertilizer costs (Polat et al. 2004).
Zeolites are found to have an effect in both acidic and saline soils with a range of soil textures, i.e.
sand to clay, however, under differential soil chemical and textural conditions, zeolite exhibit vari-
able catalytic activity to ameliorate the stress situation and enable a balance between nutrient avail-
ability and uptake (Aainaa et al. 2014; Ahmed et al. 2017; Ahmed et al. 2010; Al-​Busaidi et al. 2008;
Omar & Ahmed 2011). A change in soil nutrient availability chiefly depends on three factors: (1) P
35

Legacy Phosphorus 35

addition from the applied source; (2) shift in pH-​dependent soil chemical equilibrium; and (3) the
effect on microbial growth and activity (Demeyer et al. 2001). In terms of their abundance, low cost,
and ecofriendly nature, zeolites are considered as a good source that reduces the load on commercial
fertilizers needed for plant in a cost-​effective way.
Another aspect of zeolites which has attracted attention is that natural zeolites can be modified
to enhance their ion exchange capacity and ion exchange selectivity. These modifications have been
found to have a significant effect on P availability (Yang et al. 2015). Acid-​modified zeolites in soil
can reduce pH, and alkali-​modified zeolites can enhance cation exchange through alkali metal ions,
with both situations promoting the release of available P into soil (Fadaeerayeni et al. 2015). The
combination of rock phosphate (RP) with zeolite acts as an exchange-​fertilizer (exchanging Ca2+​
onto the zeolite). Also, plant uptake of NH4+​ or K+​ further frees the exchange sites which can be
occupied by Ca2+​, which ultimately lowers the soil Ca2+​ concentration and enhances further dissol-
ution of RP. Both conditions strengthen the available P concentration from source P into soil and
promote its uptake by plants (Pickering et al. 2002):

RP +​NH4+​ –​ Zeolite ⇌ Ca2+​ –​Zeolite +​NH4+​ +​PO43

2.9 CONCLUSION
The importance of phosphorus in agricultural production systems cannot be denied. The P issue
is clearly a double-​edged one. It concerns both the depletion of phosphorus reserves and envir-
onmental pollution, particularly water pollution. Water scarcity is already a global issue, which
is getting worse due to factors which are deteriorating the quality of this already scarce resource.
Human interference is breaking the natural P cycle and releasing more P-​containing wastes into
water. This acceleration in the natural cycle is posing disastrous effects on water quality and aquatic
life. It is changing not only the physiochemical properties of water but also biological processes in
the marine environment, causing eutrophication. As almost 95% of P pollutants enter water from
poorly managed agricultural practices and the rest comes from other sources including industries,
detergents, etc., it is clear that current agricultural practices waste a large amount of these resources,
as only 16% of the total is used effectively. By adopting best management practices (BMPs) in the
agriculture sector the potential pressure on P reserves and threats to water sources can be reduced.
On the other hand, strikingly, no international organization currently exists to devise rules and
regulations about the use of P keeping in view the scarcity of these resources. Legislation at the
international level chiefly concentrates more on the environmental aspects of P use than on its finite-
ness and the recovery of resources. It therefore seems that environmental issues are higher on the
global agenda than the development of strategies to enhance P use efficiency or reuse, keeping in
view the future scarcity. P use is determined by keeping in focus the rapid growth in population,
land use, yield quality parameters, etc. rather than taking into account the divergent plant phos-
phorus availability explicitly on an eco-​regional basis. The increase in the world’s population is
also not being matched by expanding agricultural land. These facts accentuate the need to consider
the P status of soil as an important factor for estimation of its impact on food quality and quantity
under divergent eco-​regional conditions, i.e. comparable ecological conditions at a regional level
are important to determine P status rather than at a global level. The focus henceforth should be on
technologies and approaches allowing the least use of synthetic P fertilizers, increasing PUE, and
reduced P leaching and runoff, to ensure the sustainability of this element.

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38 Biofertilizers for Sustainable Soil Management

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43

3 Bio-​fertilizers for Improving

Micronutrient Availability
Nazish Huma Khan,*1 Muhammad Sufyan Khan,1
Tooba Saeed,2 Fazli Zuljalal,1 and Muhammad Adnan3
1
Department of Environmental Sciences, University of Swabi, Pakistan
2
National Centre of Excellence in Physical Chemistry, University of
Peshawar, Pakistan
3
Department of Agriculture, University of Swabi, Pakistan
* Correspondence: [email protected]

CONTENTS
3.1 Introduction............................................................................................................................. 43
3.2 Bio-​fertilizers..........................................................................................................................44
3.3 Need for Bio-​fertilizers........................................................................................................... 45
3.4 Types....................................................................................................................................... 46
3.4.1 Nitrogen Fixers........................................................................................................... 46
3.4.1.1 Rhizobium................................................................................................... 46
3.4.1.2 Azospirillum............................................................................................... 46
3.4.1.3 Azotobacter................................................................................................ 46
3.4.1.4 Blue Green Algae....................................................................................... 46
3.4.2 Mycorrhiza................................................................................................................. 46
3.5 Impacts of Bio-​fertilizers on Agriculture................................................................................ 47
3.6 Applications of Bio-​fertilizers................................................................................................. 48
3.6.1 Seed Treatment........................................................................................................... 48
3.6.2 Seedling Root Dipping............................................................................................... 48
3.6.3 Soil Application.......................................................................................................... 48
3.7 Environmental Stress and Bio-​fertilizers................................................................................. 48
3.8 Bio-​fertilizers in the Ecosystem.............................................................................................. 49
3.9 Advantages of Bio-​fertilizers.................................................................................................. 50
3.10 Disadvantages of Bio-​fertilizers.............................................................................................. 50
3.11 Bio-​fertilizers: A Way towards Sustainability......................................................................... 50
3.12 Constraints in Bio-​fertilizer Technology................................................................................. 51
3.13 Bio-​fertilizer Strategies........................................................................................................... 52
3.14 Conclusion............................................................................................................................... 52
References......................................................................................................................................... 52

3.1 INTRODUCTION
The global population continues to grow, and by 2050 it is expected to reach 9.7 billion people
(Ehrlich and Harte 2015). Intensive industry, urbanization, and crop yields are closely linked to this
rapid expansion (Saman et al. 2020; Muhammad Tahir et al. 2020; Jakirand Allah 2020; Mahmood
et al. 2021; Farah et al. 2020; Sadam et al. 2020; Unsar et al. 2020; Fazli et al. 2020; Enamul et al.
2020; Gopakumar et al. 2020; Zia-​ur-​Rehman 2020; Ayman et al. 2020; Mohammad I. Al-​Wabel

DOI: 10.1201/9781003286233-3 43
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44 Biofertilizers for Sustainable Soil Management

et al. 2020a,b; Senol 2020; Amjad et al. 2020; Ibrar et al. 2020; Sajid et al. 2020; Muhammad et al.
2021; Sidra et al. 2021; Zahir et al. 2021; Sahrish et al. 2022). Agricultural production is essential
to meet the nutrient needs of humanity, which were expected to reach, by 2020, 321 million tons
of edible cereals (Al-​Zahrani et al. 2022; Rajesh et al. 2022; Anam et al. 2021; Deepranjan et al.
2021; Haider et al. 2021; Amjad et al. 2021; Sajjad et al. 2021a,b; Fakhre et al. 2021; Khatun et al.
2021). Traditional farming methods, on the other hand, rely heavily on the extensive use of synthetic
pesticides and fertilizers to feed crops and prevent infections (Vesile et al. 2015; Afzal et al. 2017;
Ibrar et al. 2021; Bukhari et al. 2021; Haoliang et al. 2022; Sana et al. 2022; Abid et al. 2021; Zaman
et al. 2021; Sajjad et al. 2021a,b; Rehana et al. 2021; Yang et al. 2022; Ahmad et al. 2022; Shah et al.
2022; Muhammad et al. 2022; Wiqar et al. 2022). The proper application of synthetic chemicals has
undeniable benefits not only for plant growth, yield, and quality, but also the farmer’s income is also
important. However, the increasing use of synthetic supplies may end up polluting the air, water,
and soil, posing a significant threat to the natural environment (Rehman and Zhang 2018; Farhat
et al. 2020; Farhat et al. 2022; Niaz et al. 2022; Ihsan et al. 2022; Chao et al. 2022; Qin et al. 2022;
Xue et al. 2022; Ali et al. 2022; Mehmood et al. 2022; El Sabagh et al. 2022; Ibad et al. 2022). The
thoughtless use of agrochemicals and their inability to biodegrade leads to their accumulation in
the soil, leading to negative changes in soil properties such as structure, fertility, and water-​holding
capacity. Excessive use of artificial fertilizers is also linked to algal blooms in water supplies, green-
house gases, and the buildup of toxic substances like As and Cd (Atafar et al. 2010; Deepranjan et al.
2021; Haider et al. 2021; Huang Li et al. 2021; Ikram et al. 2021; Jabborova et al. 2021; Khadim
et al. 2021a,b; Manzer et al. 2021; Muzammal et al. 2021; Abdul et al. 2021a,b; Ashfaq et al. 2021;
Amjad et al. 2021; Atif et al. 2021).
Organic farming is a viable alternative to traditional farming, as it helps us to provide quality
food without adversely affecting the soil’s health or the environment. It incorporates environmen-
tally friendly agronomic approaches and enables contamination-​free food production while ensuring
soil quality and biodiversity (Niggli 2015; Athar et al. 2021; Adnan et al. 2018a,b; Adnan et al. 2019;
Akram et al. 2018a,b; Aziz et al. 2017a,b; Chang et al. 2021; Chen et al. 2021; Emre et al. 2021;
Habib et al. 2017; Hafiz et al. 2016; Hafiz et al. 2019; Ghulam et al. 2021; Guofu et al. 2021).
The-​above mentioned dangers of overloading the soil with chemically synthesized agrochemicals,
along with increased consumer awareness of the importance of protecting the natural environment
and human health, have prompted researchers to seek alternatives that would be just as effective
while posing no risk to the environment (Geiger et al. 2010; Hafeez et al. 2021; Khan et al. 2021;
Kamaran et al. 2017; Muhmmad et al. 2019; Safi et al. 2021; Sajjad et al. 2019; Saud et al. 2013;
Saud et al. 2014; Saud et al. 2017; Saud et al. 2016; Shah et al. 2013; Saud et al. 2020; Saud et al.
2022a,b; Fahad and Bano 2012; Fahad et al. 2017; Fahad et al. 2013; Fahad et al. 2014a,b;Fahad
et al. 2016a,b,c,d; Fahad et al. 2015a,b; Fahad et al. 2018a,b; Fahad et al. 2019a,b; Fahad et al. 2020;
Fahad et al. 2021a,b,c,d,e,f; Fahad et al. 2022a,b; Hesham and Fahad 2020). These microbial strains
have great potential in agriculture as they promote crop growth by increasing the supply of natural
nutrients (Tyota and Watenabe 2013). Compared to artificial chemicals, bio-​fertilizers are an eco-
nomical, ecologically benign, and sustainable supplier of nutrients for crops, and therefore, they are
gaining attractiveness and importance in agricultural production (Swapna et al. 2016; Qamar et al.
2017; Hamza et al. 2021; Irfan et al. 2021;Wajid et al. 2017; Yang et al. 2017; Zahida et al. 2017;
Depeng et al. 2018; Hussain et al. 2020; Hafiz et al. 2020 a,b; Shafi et al. 2020; Wahid et al. 2020;
Subhan et al. 2020; Zafar-​ul-​Hye et al. 2020a,b; Zafar et al. 2021).

3.2 BIO-​FERTILIZERS
The term “bio-​fertilizer” has several meanings. Seaweed extracts, compound municipal waste,
microbiological mixtures with undetermined ingredients, organic fertilizer products, as well as
mineral fertilizer products that have been augmented with organic compounds are all examples of
45

Bio-fertilizers for Improving Micronutrient Availability 45

FIGURE 3.1 Sources of bio-​fertilizers.

bio-​fertilizers. In 2003, Vesey defined bio-​fertilizer as “a product containing microbes that colonizes
the rhizosphere (interior) of the plant and stimulates development when administered to the surface
of roots, seeds or soil, by improving the supply or availability of essential minerals to the
host plant” (Vesey 2003; Ali et al. 2018). Another study (Fuentes-​Ramirez and Caballero-​Mellado
2005) described the fact that bio-​fertilizer contains live microbes that exert a favourable impact on
crop yield and agriculture through several methods (Figure 3.1). Products that include beneficial
microorganisms used to reduce plant pathogens can be classified as bio-​fertilizers, although they
are more commonly referred to as bio-​pesticides (Fuantes-​Ramireez and Cabellero-​Mellado 2005).
In science, bio-​fertilizer is a single microorganism with plant growth-​promoting abilities, but
in agronomy the term refers to a product composed of many beneficial strains, which are useful
for nutrient mobilization, are enclosed in a container, have properties allowing conservation at the
time specified by the producer, and are ready for planting or application to the soil. Bio-​fertilizer
can also allow for the addition of chemicals to help microorganisms work better. The term “bio-​
fertilizer” should not even be confused with terms like plant or animal compost, intercropping, or
fertilizers, which refer to a mixture of mineral and organic chemicals, as well as to bio-​stimulants
from microorganisms (Reddy 2014). The main objective of using bio-​fertilizers is to promote plant
development while minimizing negative environmental consequences and improving crop yields.
They also improve plant–​water relationships, protect plants from dehydration, reduce the incidence
of insect pests, and make plants more resistant to several soilborne diseases, such as those caused
by fungi that also create mycotoxins (Dey et al. 2014). In addition, microbial fertilizers need to be
in a greater quantity to supply sufficient nutrient content to plants, their efficacy is dependent on the
application zone’s soil conditions (Macik et al. 2020).

3.3 NEED FOR BIO-​FERTILIZERS

Synthetic fertilizers used haphazardly contaminate soil, pollute water basins, eliminate beneficial
microorganisms and insects, increase disease susceptibility and decrease soil fertility (Mishra et al.
2013; Adnan et al. 2020; Ilyas et al. 2020; Saleem et al. 2020a,b,c; Rehman 2020; Frahat et al. 2020;
Wu et al. 2020; Mubeen et al. 2020; Farhana 2020; Jan et al. 2019; Wu et al. 2019; Ahmad et al.
2019; Baseer et al. 2019; Hafiz et al. 2018; Tariq et al. 2018). The demand is far greater than the
supply. By 2020, 28.8 million tons of minerals were needed to accomplish the 321 million tons of
grain that needed to be produced, while only 21.6 million tons were available, resulting in a deficit
46

46 Biofertilizers for Sustainable Soil Management

of 7.2 million tons (Arun 2007). Depletion of raw materials/​fossil fuels (energy crisis) and increased
fertilizer costs result. Besides the above, their long-​term use means it costs more to use than bio-​
fertilizers, which are environmentally friendly, efficient, and work well, while synthetic fertilizers
are more expensive and inaccessible to the wider agricultural community (SubbaRoa 2001).

3.4 TYPES
Nitrogen fixers and phosphate absorbers are two types of bio-​fertilizers.

3.4.1 Nitrogen Fixers
3.4.1.1 Rhizobium
Rhizobium is a symbiotic bacterium that exclusively fixes nitrogen with legumes (50–​100 kg/​ha).
Rhizobium’s ability to nodulate leguminous crops is primarily dependent on the availability of suit-
able strains for each legume. It takes up residence in the roots of some legumes, producing root
nodules, which are tumour-​like growths that operate as NH3 manufacturers. In a symbiotic asso-
ciation with legumes, Rhizobium may improve soil fertility and growth of non-​legumes such as
Parasomnia. Some Rhizobium strains have been shown to operate as bio-​control agents and stimu-
late crop development (Kumar et al. 2011).

3.4.1.2 Azospirillum
Azospirillum is a fungus that is both heterotrophic and associative, and it belongs to the Spirilaceae
family. It is a growth-​regulator, in addition to having an ability to fix nitrogen at a rate of 20–​40 kg/​ha,
and it can also produce compounds. There are several varieties in this genus, such as A. amazonense,
A. lipoferum, and A. brasilense which have been shown to have a worldwide range and provide
inoculation benefits. The Azotobacter that colonizes roots does not just stay on the surface; a sig-
nificant fraction enters the cellular tissues of the roots and coexists with the crop (Chavan 2019).

3.4.1.3 Azotobacter
Azotobacter is a type of bacteria that lives in aerobic, free-​living environments; they are hetero-
trophic bacteria from the Azotobacteriaceae family. Azotobacter can be found in both neutral and
alkaline soils, with A. chrococcum being the most common species in arable soils. Other species
include A. vinalandii, A. beijerinckii, A. insignis, and A. macro-​cytogenes. Antifungal antibiotics are
made by this bacterium which minimizes seedling mortality by stifling the growth of a large number
of organisms susceptible to the presence of fungus in the root zone (Subba Rao 2001).

3.4.1.4 Blue Green Algae

This is also known as “paddy organism” being plentiful in paddies. For lowland rice production, con-
siderable amounts of nitrogen are required, which are provided by this type of algae. Sustainability
is important for food production; BNF must gradually replace industrial nitrogen fixation as a source
of fixed nitrogen. The 50–​60% N condition is fulfilled by a hybrid of unrestricted and rice-​growing
plant-​related microbes mineralizing soil organic N and BNF (Roger and Ladha 1992).

3.4.2 Mycorrhiza
Mycorrhiza means “fungus roots.” Host plants have a symbiotic connection and a type of fungus
found in the roots, in which the fungus partner reaps the rewards by obtaining its carbon requirements
from the photosynthetic activity of the host. As a result, the host benefits by getting much-​needed
nutrients such as P, Ca, Cu, Zn, and other elements with the help of the fungus’ fine absorbing
hyphae, as it may penetrate areas that are ordinarily inaccessible. Glomus, Gigaspora, Acaulospora,
47

Bio-fertilizers for Improving Micronutrient Availability 47

FIGURE 3.2 Agricultural impacts of bio-​fertilizers.

Sclerocysts, and Endogone are the most prevalent genera employed in the manufacturing of bio-​
fertilizers (Bagyaraj 1992).

3.5 IMPACTS OF BIO-​FERTILIZERS ON AGRICULTURE

Bio-​fertilizers are crucial in agricultural techniques to boost crop yields. Fertilizers are required
to suit the needs of plants. Nitrogen-​fixing fertilizers (Rhizobium) are responsible for fixing 50–​
100 kg of nitrogen per acre in vegetables. Beat vegetables, red gram, and pea can all benefit from
these fertilizers. Chickpeas, lentils, green gram, black gram, and other legumes are also beneficial
to oil-​seed legumes like soybean and groundnut (Mahdi et al. 2010; Khan et al. 2016). Similarly,
agriculture is interested in Azotobacter because of its potential to fix nitrogen. It can create vitamins
and plant hormones like thiamine and riboflavin. In addition to helping with nitrogen fixation, plant
development hormones are produced by phosphate-​solubilizing bio-​fertilizers which promote plants
growth (Figure 3.2; Madhav and Hina 2020).
A soil solution is used to raise the phosphate level in soils. Plants are able to absorb sufficient
amounts of dissolvable phosphorus, resulting in a 10–​20% increase in yield.
The combination of G. fasciculatum +​ A. chroococcum +​half of the planned P rate produced the
best root length, plant stature, bulb size, new weight, root colonization, and P uptake in a nursery pot
experiment with onion (Carter 1967). K-​solubilizing bacteria, such as Frateuria aurantia, are respon-
sible for converting a potassium (K) mixture into a useful structure for plants, and is applied to crops
in conjunction with various bio-​fertilizers with no adverse effects. Despite the fact that a wide range
of agricultural primers have been shown to be effective, there is a notable disparity in responses
among yields, geographies, and situations (Ghosh 2004). In farming, Arbuscular mycorrhiza (AM)
48

48 Biofertilizers for Sustainable Soil Management

parasites are often abundant. They make up around half of the biomass of soil organisms, and some
products formed by them may account for another 3000 kg (Lovelock et al. 2004).

3.6 APPLICATIONS OF BIO-​FERTILIZERS
The majority of bio-​fertilizers are sold as inoculants based on conventional carriers, which are less
expensive and less difficult to manufacture (Figure 3.3). Culturing microbes, preparing the transport
ingredient, combining it with the culture broth, and packing are all steps in the mass production
of bio-​fertilizers. The ideal carrier materials for bio-​fertilizer preparation must be less expensive,
more readily accessible, as well as being easier to handle; they must have an organic structure and
not be poisonous and they must have the ability to transport additional bacteria and the ability to
stay alive for prolonged periods of time. However, they have the disadvantages of having a shorter
shelf life, being more sensitive to temperature, being more susceptible to contamination, and being
less effective as cell counts drop. As a result, liquid formulations for Rhizobium, Azospirillum,
Azotobacter, and Acetobacter have been created, which, while more expensive, have the benefit of
being more convenient to manufacture as well as providing increased soil efficiency (Ngampimol
and Kunathigan 2008). The practices listed below are crucial.

3.6.1 Seed Treatment
This is the most successful, cost-​efficient, and widely used approach for inoculants of all types
(Sethi et al. 2014). The seeds are mixed together and evenly coated in slurry before even being
shade-​dried for 24 hours before they are used. The coating of liquid bio-​fertilizers is possible to do
in a plastic bag or bucket, depending on the amount of seeds. Two or more microorganisms can be
used to treat seeds without causing an antagonistic reaction, and the greatest amount of bacteria on
each seed is necessary for the best outcomes (Chen 2006).

3.6.2 Seedling Root Dipping

Water is used to soak newly planted crop roots in a mixture of bio-​fertilizer, such as grains, plants,
berries, trees, sisal, silk, grapes, bananas, and tobacco. Various crops require different treatment
times; for example, vegetable crops are sprayed for 20–​30 minutes, and paddy is sprayed for 8–​12
hours before transplantation (Barea and Brown 1974).

3.6.3 Soil Application
In this procedure, bio-​fertilizer is applied directly to the roots, either individually or jointly. Cow
dung is a phosphate-​solubilizing microbial bio-​fertilizer and rock phosphate mixture is maintained in
the shade overnight and applied to the soil with a moisture level of 50%. Rhizobium and Azotobacter
are two examples of bio-​fertilizers that are applied to soil (Hayat et al. 2010).

3.7 ENVIRONMENTAL STRESS AND BIO-​FERTILIZERS

Microbial physiology is influenced significantly by environmental stressors (Iqra et al. 2020; Akbar
et al. 2020; Mahar et al. 2020; Noor et al. 2020; Bayram et al. 2020; Amanullah, Fahad 2018a,b;
Amanullah, Fahad 2017; Amanullah et al. 2020; Amanullah et al. 2021; Rashid et al. 2020; Arif
et al. 2020; Amir et al. 2020). Radiation exposure has sufficient energy to allow electrons to escape
from a cell’s molecules, resulting in the formation of free radicals that can oxidize DNA or RNA,
and non-​ionizing radiation (ultraviolet radiation), by activating electrons in molecules, has a car-
cinogenic impact, resulting in pyrimidine formation dimers, which frequently change the structure
49

Bio-fertilizers for Improving Micronutrient Availability 49

of DNA in cells, which can lead to complications during pregnancy. Ionizing radiation penetrates
cells and endospores easily because of its high frequency. UV light has a negative impact on
cyanobacteria’s expansion, continued existence, photosynthesis, CO2 uptake, and nitrogen metab-
olism, among other things (Sinha 2005). Many researchers believe that UV-​B radiation destroys
cellular constituents that absorb light between 280 and 315 nm, influencing the porosity of cel-
lular membranes and protein breakdown, finally leading to the death of the cephalopod (Vincent
1993). When exposed to UV-​B, reactive oxygen species (ROS) decrease gene expression of critical
photosynthetic proteins. Reduced photosynthetic activity (Fv/​Fm) and elevated reactive oxygen
species were found in Microcoleus vaginatusa formation after being exposed with UV-​Chen B
radiation (Chen et al. 2009). The assimilatory enzyme nitrate reduction is found in almost all bio-​
fertilizers. It has been proposed that nitrate reductase is linked to membrane fractions that contain
chlorophyll, and thus inhibiting chlorophyll directly affects nitrate reductase function. Because
their aromatic amino acids absorb UV-​B, ultraviolet radiation disrupts the complex organization of
phycobilisomes. In cyanobacteria, chlorophyll-​binding proteins and phycobilisomes capture and
over 99% of UV-​B (Sinha 2005). Exposure of Synechocystis cells (phycoerythrin-​deficient) to
moderate UV-​B (1.8 Wm–​2) causes phycocyanin loss, which could be attributed to the two bilins
present in α-​phycocyanin versus single bilins in other biliproteins. Both α-​ and β-​phycocyanin
were photodegraded when Synechococcus sp. PCC 7942 phycobilisomes were subjected to UV-​B
light (Sah et al. 1998). DNA is one of the most significant targets of solar UV radiation in all living
things (Jans 2005).

3.8 BIO-​FERTILIZERS IN THE ECOSYSTEM

Despite the fact that bio-​fertilizers have been routinely employed in agriculture for a number
of years, information on their colonization and ecology is lacking. Furthermore, the pro-
cess underlying their interactions with plants and the local microbial population continues
to pique people’s interest. Microflora that is native to the area in the rhizosphere is one of
the primary elements that influences a bio-​fertilizer’s efficiency in a living ecosystem. This
highly competitive rhizosphere community with a varied range of organisms may have an
impact on the bio-​fertilizer’s survival and qualities that encourage crop growth (Hibbing et al.
2010). Furthermore, seeds and seedlings are bacterized, and also soil additives may cause
local microflora to change structure, which must be taken into account when determining
the risk of microorganisms being released into the environment (Dey et al. 2012). Finally,
microbial bio-​fertilizers’ effect on non-​target species, the biogeochemical processes affected,
changes in soil texture, soil qualities such as permeability, richness, and water-​holding ability,
as well as erosion avoidance should all be carefully studied (Pereg and McMillan 2015). As
a result, prior to the release of bio-​fertilizers into the surroundings, it is critical to evaluate
their unintended consequences for local microbiota communities and, as a result, ecosystems,
as well as a thorough examination of bio-​fertilizers and their effects before changing agri-
cultural methods. Although the impacts of bio-​fertilizers on non-​target members of the soil
rhizosphere and food web have been examined to some extent, the effects of bio-​fertilizers
on non-​target members of the soil rhizosphere and food web have yet to be fully investigated.
Despite the fact that most research found quantifiable changes as a result of introducing
bio-​inoculants into the rhizosphere, the extent of the alterations and their implications for
ecological functions have yet to be published (Martinez-​Viveros et al. 2010). According to
reports, the amount of influence in residential areas, of bio-​fertilizers being implemented
is dependent on a variety of elements such as soil characteristics, bio-​fertilizer application
technique, environmental circumstances, and so on (Dey et al. 2012). However, relatively
few trials have been conducted over a longer length of time, which is critical for drawing
conclusions about the efficacy and risk aspects of bio-​inoculants. As a result, more thorough
50

50 Biofertilizers for Sustainable Soil Management

research on the longer term effects of non-​target creatures and bio-​fertilizers are needed prior
to the use of bio-​inoculants that can be classified as “safe” for commercial use. Additionally,
bio-​fertilizer’s impact on non-​target groups have been investigated using methods that are
both culture-​dependent and culture-​independent, as well as genetic and physiological assays.
Both plating and cytochemical methods for assessing microbial community structures are
appropriate approaches to investigate the effects of bio-​fertilizers on the microbiota of the
local environment. The application of new technology must be done while evaluating the
influence on resident microbiota as well as soil function. Although DNA is a trustworthy as
a useful criterion for evaluating the diversity and potential of a community, recent successes
in extracting messenger RNA (mRNA) from soil are significant enough to include the experi-
ment in bio-​fertilizer risk and efficiency assessments. Furthermore, an mRNA-​based method
would take into account the real functional variety of bio-​fertilizers at any given time in the
system under study. As a result, high-​throughput, high-​resolution methodologies must be used
in conjunction with established multi-​dimensional inspection approaches to bio-​fertilizer per-
formance, diversity, and risk assessment studies before they are released into the environment
(Sharma et al. 2012).

3.9 ADVANTAGES OF BIO-​FERTILIZERS
Bio-​fertilizers aid in the production of high crop yields by adding nutrients to the soil and benefi-
cial microbes required for crop yield. Chemical fertilizers are no longer used as they are harmful to
plants. Chemical fertilizers stunt plant development and release hazardous chemicals into the envir-
onment, polluting the environment. Bio-​fertilizers are used because they include natural ingredients
that do not damage but rather benefit plants, and crop yield can be accelerated. They are environ-
mentally beneficial because they safeguard the environment from contaminants. The soil will pre-
serve its fertility if it is free of chemicals, which will benefit both the plants and the ecosystem, as the
crops will be disease-​free and there will be no pollution in the ecosystem. Bio-​fertilizers eliminate
the toxic components in soil that cause plant illnesses. Plants can be safeguarded in the same way
from famine and other situations by employing bio-​fertilizers. Bio-​fertilizers are affordable, and
they can be used by even low-​income farmers (Chavan 2019).

3.10 DISADVANTAGES OF BIO-​FERTILIZERS
Because bio-​fertilizers are alive, they require careful handling when storing them for lengthy periods
of time. They have to be utilized before they expire. Bio-​fertilizers can be used in conjunction with
traditional fertilizers, but they cannot completely replace them. Bio-​fertilizers must be used at the
correct temperature and pH range to be effective; otherwise, they are ineffective and inhibit the
growth of helpful microorganisms. They are less efficient if the carrier medium is polluted with
other microorganisms or if the grower uses the incorrect strain of bacterium. For organisms to thrive
and work, the soil in which the bio-​fertilizer is placed must possess sufficient nutrients in a suitable
form and amount. Due to the slow release rate of nutrients, nutrient deficiency cannot be remedied
quickly with the use of a bio-​fertilizer. Because bio-​fertilizers supply fewer nutrients than traditional
fertilizers, a greater quantity is required to meet the required quantity of nutrients for better plant
production (Kumar et al. 2017).

3.11 BIO-​FERTILIZERS: A WAY TOWARDS SUSTAINABILITY

Bio-​fertilizers are environmentally benign since they are created from readily available natural
sources such as rice husk, leftover vegetables, and organic debris. Bio-​fertilizers are less expensive to
manufacture than chemical fertilizers, especially when nitrogen and phosphorus are used.
51

Bio-fertilizers for Improving Micronutrient Availability 51

FIGURE 3.3 Advantages of bio-​fertilizer.

Bio-​fertilizers are inexpensive and simple to create; in fact, farmers can prepare them themselves.
Bio-​fertilizers improve soil fertility by mobilizing nutrients from organic materials and encourage
long-​term agricultural production. Bio-​fertilizers also boost soil biological activity, which boosts
nutrient mobilization from natural and synthetic sources as well as harmful material breakdown. Bio-​
fertilizers increase soil organic matter content, and boost water retention, exchangeable cations, and
improve soil and buffering ability against soil acidity, salinity, alkalinity, pesticides, and toxic heavy
chemicals. Bio-​fertilizers provide nourishment for beneficial microorganisms and earthworms, while
also encouraging their growth. Bio-​fertilizers boost crop yields by 10–​20%. The release of growth-​
promoting hormones by bio-​fertilizers promotes plant root multiplication. Fungi, bacteria, and other
parasites in the soil are suppressed by the presence of bio-​fertilizers. Plants are more resistant to stress
and survive longer. Antibiotic compounds are released by some bio-​fertilizers, such as Azotobacter,
in response to biotic stressors. In the soil, cyanobacteria release proteins, amino acids, vitamins, and
other compounds that enhance organic carbon and aid nitrogen fixation.

3.12 CONSTRAINTS IN BIO-​FERTILIZER TECHNOLOGY

Regardless of the fact that bio-​fertilizer innovation is an ecologically friendly option, it is not without
its drawbacks, and due to a variety of issues, its application or installation is restricted. These include
issues with infrastructures, such as a lack of major equipment, energy, and so on. Other restrictions
include financial constraints and difficulties acquiring bank loans. Seasonal requirement for bio-​
fertilizers, simultaneous cropping activities, and a short period of sowing/​planting in a specific loca-
tion are all examples of environmental restrictions. Due to challenges in farmers implementing this
device, there is an unawareness of the technology’s benefits due to diverse techniques of inocula-
tion, when compared to inorganic fertilizers, and there is no discernible difference in crop growth.
For manufacturers, there aren’t enough retail shops or a market network, as well as the lacking of a
correct time and place when the right inoculant is administered, which are all marketing obstacles.
Different restrictions influence manufacturing, marketing, and usage techniques in one way or
another (Kumar et al. 2017).
52

52 Biofertilizers for Sustainable Soil Management

3.13 BIO-​FERTILIZER STRATEGIES
To get updated knowledge about soil and climatic conditions in relation to a specific crop, it is
necessary to strengthen research and technology. The applications of biotechnological technologies
and procedures are important to improve nitrogen fixation, phosphorus solubilization, and other bio-​
fertilizers. Bio-​fertilizer dose standardization in a specific crop and soil and finding a better carrier
medium for strains to extend their shelf life are needed. Monitoring of bio-​fertilizer manufacturing
units on a regular basis to ensure proper viable count, production process, storage, and other factors
is also required. Bio-​fertilizers for agricultural crop production have received a lot of attention. In
this regard, the efforts of scientific training, farmer fairs, exhibitions, and the media are to be highly
praised (Singh et al. 2016).

3.14 CONCLUSION
Bio-​fertilizers play an important role in integrated nutrition management and a renewable supply of
plant nutrients that can be used to augment chemical fertilizers in long-​term agricultural systems.
As ecofriendly and cost-​effective inputs for farmers, biological fertilizers could play a major role
in increasing soil productivity and sustainability, while also protecting the environment. In an apple
orchard, beneficial microbes can be employed as a tool to boost growth, yield, and fruit quality. The
goal of biological pest control is to limit the use of insecticides while also maintaining a clean envir-
onment, food safety, and ultimately human health.

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978-​3-​030-​49732-​3
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4 Role of Plant Bio-​stimulants

and Their Classification
Waseem Ahmed,1 Adnan Noor Shah,2* Asad Abbas,3
Muhammad Nawaz,2 Abdul Qayyum,4 Muhammad Umair
Hassan,5 Muther Mansoor Qaisrani,6 and Jallat Khan7
1
Department of Horticulture, The University of Haripur, Pakistan
2
Department of Agricultural Engineering, Khwaja Fareed University of
Engineering and Information Technology, Punjab, Pakistan
3
School of Horticulture, Anhui Agricultural University, Hefei, China
4
Department of Agronomy, The University of Haripur, Pakistan
5
Research Center on Ecological Sciences, Jiangxi Agricultural University,
Nanchang, China
6
Department of Bioscience & Technology Khwaja Fareed University of
Engineering and Information Technology, Punjab, Pakistan
7
Department of Chemistry, Khwaja Fareed University of Engineering &
Information Technology, Punjab, Pakistan
*Correspondence: [email protected]

CONTENTS
4.1 Introduction............................................................................................................................. 66
4.2 Major Classification of Bio-​stimulants................................................................................... 68
4.2.1 Effect of HS on Soil.................................................................................................... 69
4.2.2 Effect of HS on Plant Physiology............................................................................... 69
4.3 Protein Hydrolysate................................................................................................................. 69
4.3.1 Classification of Protein Hydrolysates (PHs)............................................................. 70
4.3.2 Effect of Protein Hydrolysates (PHs)......................................................................... 70
4.3.2.1 Direct Effect of Protein Hydrolysates (PHs).............................................. 70
4.3.2.2 Indirect Effect of Protein Hydrolysates (PHs)........................................... 70
4.4 Seaweed Extracts..................................................................................................................... 71
4.4.1 Application of Seaweeds............................................................................................ 71
4.4.2 Microalgae.................................................................................................................. 71
4.4.2.1 Activity of Microalgae as Bio-​stimulants.................................................. 72
4.5 Plant-​derived Smoke............................................................................................................... 72
4.6 Chitin and Chitosan Derivatives.............................................................................................. 73
4.6.1 Chitin (CH)-​based Polymers...................................................................................... 74
4.6.1.1 Chitin as a Bio-​stimulant............................................................................ 74
4.6.1.2 Bio-​stimulatory Effect of Chitin in Vegetables (Table 4.1)........................ 75
4.6.2 Chitosan (CHT)-​based Polymer................................................................................. 76
4.6.2.1 Chitosan as a Bio-​stimulant....................................................................... 76
4.6.2.2 Bio-​stimulatory Effect of Chitosan in Vegetables (Table 4.2).................... 77
4.7 Beneficial Microbes as Bio-​stimulants.................................................................................... 77
4.7.1 Beneficial Fungi.......................................................................................................... 77
4.7.2 Beneficial Bacteria...................................................................................................... 78

DOI: 10.1201/9781003286233-4 65
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66 Biofertilizers for Sustainable Soil Management

4.8 Inorganic Compounds............................................................................................................. 79

4.9 Conclusion............................................................................................................................... 80
References......................................................................................................................................... 80

4.1 INTRODUCTION
Agriculture faces numerous obstacles and issues, including scarcity of natural resources and
destruction caused by over usage of heavy metals (Le Mire et al. 2016). Furthermore, it has
been forecast that, in the year 2050, the world’s population will have grown to 9.7 billion people
(Fahad et al. 2016). As the population depends on agriculture to meet their daily food demands,
the best way to fulfill these demands is to make efficient use of available resources to grow healthy
products and maintain a healthy diet. Over the past few years, different types of technological
revolutions have been proposed to boost immunity to the production system. This is due to the
significant reduction of agriculture chemicals. According to the report of environment forums,
one in a million deaths occur per year as a result of fatal diseases caused by pesticide poisoning.
Fertilizer application is associated with pollution, aberrant plants, groundwater contamination,
microbe development, acidity, and soil mineral depletion (Rahman and Zhang 2018; Adnan et al.
2018a,b; Adnan et al. 2020; Adnan et al. 2019; Atif et al. 2021). In this regard, there is a need
for new farming methods that are eco-​friendly, flexible, and efficient in the production of food
(Rouphael et al. 2018; Xu and Geelen 2018; Fahad et al. 2015a,b; Fahad et al. 2020; Fahad et al.
2021a,b,c,d,e,f; Fahad et al. 2022a,b). For this purpose, the use of chemicals or microbe develop-
ment is a promising method to accelerate plant growth, increase to the prevention of damage to
the environment or soil conditions, and increase the efficiency of resources to be used in a more
effective way for betterment of plant growth and development (Fakhre et al. 2021; Muhammad
et al. 2022; Shafi et al. 2020; Wahid et al. 2020; Zahida et al. 2017). The term “bio-​stimulants”
was proposed for these chemicals and microorganisms (Zhang and Schmidt 1997). In agriculture,
bio-​stimulants are natural substances that improve crop growth and quality, and helps the crop
to pass through conditions of biotic and abiotic stress (Parađiković et al. 2019). Bio-​stimulants
will encourage growth of a plant from seed germination to its maturity by increasing its nutrient
absorption, intensive growth, improvement in the quality of the product and its features (such as
sugar content, colour, frui,t and seed), a complex of soil–​microbial interactions, improvements
in physical and chemical characteristics of the soil, and increased tolerance to extensive abiotic
stress and stress-​related disorders.
Humic substances, plant extracts, smoke-​ derived plants, hydrolysed protein and amino
acids, chitosan, and polysaccharides (agar, alginates, and carrageenans), as well as inorganic
compounds (Al, Co, Na, Se, and Si) or microorganisms (bacteria or fungi) can all be used as
plant bio-​stimulants for seed, plants, and soil (Pilon-​Smits et al. 2009; Rouphael and Colla 2018).
Bio-​stimulants, which are created in the form of natural or synthetic ingredients and comprise
hormones or plant hormone precursors, are the most common. They have a direct impact on
physiological processes in the delivery of possible advantages to growth, development, and
responses to stress, water, salt, and toxic substances, such as toxic aluminium, when employed in
different cultures (du Jardin 2012).
In the scientific literature, Kauffman et al. (2007) was the first to define the term “bio-​stimulants”
as bio-​stimulants being materials that are different from the fertilizers that help crops to develop
when used in tiny amounts. Russo and Berlyn (1991) described bio-​stimulants as “the substances or
products that are not fertilizers but have a favorable impact on plant growth”. Most bio-​stimulants are
made from organic commodities which contain no additional chemicals or laboratory-​manufactured
growth regulators. According to Basak (2008), bio-​stimulants can be divided into various categories
depends on the mechanism of action and the origin of the actively used ingredients. Bulgari et al.
(2015) stated that bio-​stimulants can also be ranked on the basis of their action or impact on the
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Role of Plant Bio-stimulants and Their Classification 67

physiological parameters of plants other than their state of composition. Yakhin et al. (2017) defined
bio-​stimulants as genetic agents that boost agricultural production by new or derivative capabilities
of complicated substances, rather than solely through the presence of known important mineral
elements, bio-​fertilizers, or plant chemicals.
The Association of American Plant Food Control Officials (2019) defined bio-​stimulants as
compounds other than primary, secondary, and micronutrients for plants which may be proposed
by scientific researchers for the benefit of many species of plant when applied to the plant or
soil. The European Biostimulants Industry Council (2012) proposed that they are plant-​based
products containing chemicals or microbes whose goal is to promote biological responses in
plants and soil to improve nutrient absorption, nutritional efficiency, abiotic stress tolerance, and
crop productivity.
Plant bio-​stimulants are commonly used to improve the productivity and quality of crops
having great economic value such as greenhouse crops, fruit trees, plants, vegetables, flowers,
and ornamental plants. Also, their beneficial effects have been realized on cereals, grains,
oilseeds, pulses, and other crops. However, there is a lack of information on the method of appli-
cation and their expiry. Bio-​stimulants do not have a direct effect on pests and are therefore not
included in the regulation of pesticides (European Biostimulants Industry Council and Ertani
2012). The mechanism triggered by bio-​stimulants is difficult to measure and still being analysed
(Paul et al. 2019).
In past years, the expression “bio-​stimulator” has gradually begun to be utilized in scientific
publications along with the expansion of the range of organic materials and microorganisms. The
bio-​stimulants market is increasing daily. Figure 4.1 shows the market size of bio-​stimulants.
The current issue addresses the latest development of bio-​stimulants in horticulture from various
experiments showing that the utilization of materials and useful microbes can be both a beneficial
and viable tool for horticultural cultivation. A unique release of bio-​stimulants in horticulture
includes 10 revisions on the plant bio-​stimulants’ concept, strategy, basic classes, and management
(Du Jardin 2015), humorous and fulvic acids (Canellas et al. 2015), protein hydrolysates (Canellas
and Olivares 2014), marine plants release (Battacharyya et al. 2015), chitosan (Pichyangkura and
Chadchawan 2015), silicon (Adnan et al. 2015; Savvas and Ntatsi 2015), phosphites (Gómez-​Merino
and Trejo-​Téllez 2015), arbuscular mycorrhizal mould (Rouphael et al. 2015), Trichoderma (López-​
Bucio et al. 2015), and rhizobacteria that promote plant development (Ruzzi and Aroca 2015).

FIGURE 4.1 Market size of bio-​stimulants globally.

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68 Biofertilizers for Sustainable Soil Management

4.2 MAJOR CLASSIFICATION OF BIO-​STIMULANTS

Apart from recent initiatives to define the regulating nature of bio-​stimulants, no official or legisla-
tive concept of plant bio-​stimulants exists elsewhere in the world, including in the European Union
and the United States. These circumstances prevent comprehensive description or ranking of certain
materials as well as microorganisms. Despite this, bio-​stimulants are divided into various classes
according to their role or action on plant growth and development (Calvo et al. 2014; Rouphael et al.
2017; Ahmad et al. 2021) giving certain groups of bio-​stimulants that include:

• Organic compounds (humic and fulvic acid, animal, vegetal protein hydrolysates, seaweed
extracts, and smoke-​derived compounds)
• Chiton and chitanose derivatives
• Beneficial microorganisms (N2-​fixing bacteria, mycorrhizal fungi, and plant growth-​promoting
rhizobacteria)
• Inorganic compounds (copper, silicon, sodium, selenium, and aluminium).
• They are discussed below.

Humic acid and fulvic acid:

Humic substances (HS) are naturally occurring nutrients in the soil that are produced by the
decomposition of plant, animal, and microbial wastes, as well as the metabolic process of soil-​
dwelling insects that consume certain subterranean parts. HS is a group belonging to contrasting
substances, divided by the number of molecules and their stability into humins, humic acid, and
also fulvic acid. Humic substances are found to affect microbial metabolism as well as chemical
and biological modifications of living organisms (including mainly plant and animal waste) that
exist also in the atmosphere and water (Canellas et al. 2015; Graber and Rudich 2006; Salma
et al. 2010). The most comprehensive content of HS comes from prehistoric deposits when flora
and fauna were submerged in wetlands. Within the Earth’s atmosphere, humic organisms con-
tain large (about 60%) compounds of organic matter and seem to be largely responsible for soil
quality by affecting physio-​chemical soil boundaries such as form and permeability (Bronick
and Lal 2005; Du Jardin 2015; Trevisan et al. 2010; Ali et al. 2016). Macromolecular formations
of diverse materials (such as sugars, essential fats, peptides, hydrocarbon chains, and aromatic
rings) are held together by hydrocarbons (such as van der Waals and ion–​dipole forces) and
ionic bonds (Piccolo 2001; Trevisan et al. 2010). Humic material and its structures in soil are
therefore affected by interactions between microorganisms and plant roots. Any experiment to
use numerous materials to promote plant growth and crop harvesting requires the expansion of
these interactions to obtain the desired results. The major constituent of humic material is humus
(containing carbon compounds), which is created by a series of chemical as well as biological
fertilizers for soil and organic matter under controlled temperature, time period, water, aeration,
and moisture (Canellas and Olivares 2014). Humus is the main part of organic matter found in
soil (65–​75%) and can remain in the soil for hundreds of years. A recent randomized conceptual
analysis of humic substances sprayed on plants showed that dry growth of 24% of shoots and 26%
of roots was achieved (Rose et al. 2014).
Humic compounds are classified as alkaline (high pH) or acid (low pH) based on soluble
differences in alkaline and alkaline pH (low pH) humin-​insoluble (carbon-​based macromolecular
substances); alkali-​soluble-​humic acid and alkali-​and acid-​soluble-​fulvic acid (humic and fulvic
acid are the final elements in the decomposition of animals along with plants fossils) (Trevisan
et al. 2010). Humic acid contains molecules of dark brown cells, has a high molecular weight,
and dissolves in alkaline solutions. It is produced by microorganisms and can break down dead
organisms. Fulvic acid is a category of chemicals that are formed by the decomposition of plants
and animals. It is found in humus. Fulvic acids help to absorb essential nutritional elements because
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Role of Plant Bio-stimulants and Their Classification 69

they can quickly enter via the cellular membrane. While humic acids can enhance nutrient absorp-
tion from magnificent properties of removing heavy metals and water loss. Humic acids have also
been noticed to raise soil structure and microbial activity.

4.2.1 Effect of HS on Soil
Soil organisms, including humic substances, are accepted as a major component in soil fertility,
including having physical and chemical functions (Bronick and Lal 2005; Khan et al. 2017). Therefore,
HS causes the formation of stable soil compounds, soil aeration, and hydration for the absorption and
discovery of nutrients. Canellas et al. (2015) stated that humic substances work on the carbon exchange
capacity in chemical reactions and neutralize the pH of the soil. It has been shown that plants that grow
in soil containing enough humin, humic acid, and fulvic acid are healthy with high production along
with providing good-​quality nutritious food. HS can also work to obtain phosphate in plants by storing
phosphorus solution in the plant but this useful effect depends on the pH of the soil and the concen-
tration of calcium (Delgado et al. 2002). The activation of cytoplasmic membrane H±ATPs, which
convert available energy supplied by hydrolysis of ATPs to electrical and chemical energy utilized to
acquire nitrates and other nutrients, is another contribution of HS to root feeding.

4.2.2 Effect of HS on Plant Physiology

Humic substances directly affect plant physiology which means that the impacts are not related to
soil characteristics and the availability of nutrients but include regulation of cellular activities such as
mutations, genetic modification, and hormonal actions (Canellas et al. 2008). The processes by which
some humic chemicals behave as external plant hormones that alter root expansion are unknown. HS
are reported to have a significant impact on the formation of the root system (Canellas et al. 2002;
Khristeva 1957; Ramos et al. 2015; Zandonadi et al. 2007). Zandonadi et al. (2007) revealed that
humic substances are not involved in root elongation at lateral root emergence sites (Kulkarni et al.
2011). Humic substances are also reported to form genital roots with identified plasma lemma and
the function of H+​tonoplast pumps (Zandonadi et al. 2007). Jannin et al. (2012) explained that humic
acid utilization in Brassica napus brought about a resurgence of root development and increase in
N and S intake due to the development of genomes found in roots including a sulphate carrier and
a nitrate carrier. As claimed by various researchers, humic substances can also have a significant
impact on important metabolic processes and in reducing stress levels (De Pascale et al. 2017; Khan
et al. 2016). It has been reported that humic substances contain hormone-​like substances such as
auxins, gibberellins, cytokinins, and polyamines (Nardi et al. 2009). They also have been noticed to
have a beneficial impact on some other plant body procedures such as the chlorophyll level. It is also
reported that humic substances trigger several cellular actions in plant cells and increase plant resist-
ance to many types of abiotic stresses, e.g. in tomatoes and okra, and HS has been already proven to
improve plant salt stress (Paksoy et al. 2010; Türkmen et al. 2004; Van Oosten et al. 2017).

4.3 PROTEIN HYDROLYSATE
Protein hydrolysates (PHs) are a fusion of polypeptides, oligopeptides, and amino acids generated by
the incomplete breakdown of plant proteins and common bio-​stimulants (Schaafsma 2009). Amino
acid and peptide composition are acquired by protein breakdown through biochemical means via
agricultural and industrial materials derived from plants (plant wastes) and animal faeces (e.g. col-
lagen, epithelial tissue) (Calvo et al. 2014; du Jardin 2012; Halpern et al. 2015; Zeeshan et al. 2016).
PHs have obtained importance as bio-​stimulants due to their potential to boost the development,
yield, and durability of a variety of horticulture crops. The use of PHs can also reduce the negative
outcomse of pressure on abiotic plants due to salinity, drought, and heavy metals.
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70 Biofertilizers for Sustainable Soil Management

4.3.1 Classification of Protein Hydrolysates (PHs)

The chemical properties of PHs are determined by the protein supply and the technique of pro-
tein breakdown in the presence of water (chemical, thermal or enzymatic hydrolysis) (Colla et al.
2015a; Du Jardin 2015; Halpern et al. 2015). Chemical degradation using H2SO4 or HCl at extreme
temperatures (> 121°C) and compression (> 220.6 kPa) is used to oxidize animal-​derived proteins
in acidic circumstances (protein processing continued by specified heating has incorporation of
alkaline agents such as Ca, Na, or KOH) (Colla et al. 2015b; Pasupuleti and Braun 2008). Some
researchers have described a decreased growth of fruit plants as a result of PHs found in animals that
may be due to unequal amino acid formation or high density of amino acids and high salts (Lisiecka
et al. 2011). Companies should carefully inspect the stability of mammal PHs before releasing them
onto the market (company safety testing). European Regulation (No. 354/​2014) restricts the usage
of PHs found in animals in edible parts of living plants due to growing concerns about food safety,
however they can be used in the inedible part of plants.

• Enzymatic hydrolysis is appropriate for the generation of protease, proteinase, or peptidase

via animals (pancreatin, pepsin), plants (papain, ficin, bromelain), or microbes (alcalase,
flavourzyme). Protein hydrolysates are present in powder form and with water added can be
used in leaves such as foliar applications or extracts that are taken in proximity to plant roots
(Colla et al. 2017).

The emergence of degradation of certain molecules and enzymes can conserve vitality and maintain
the formation of amino molecules (Niculescu et al. 2009).

4.3.2 Effect of Protein Hydrolysates (PHs)

4.3.2.1 Direct Effect of Protein Hydrolysates (PHs)
The latest studies show that protein hydrolysates affect plants directly by stimulating the mech-
anism of nitrogen and carbon by regulating the enzymes involved in the formation of N, its genes,
and by acting in a signature form of N in roots. The elimination of oxidants by other organic matter
substances, involving glycine betaine or proline, can provide antioxidant action that helps to reduce
environmental stress. Some organic molecules like proline have solubilizing properties that can pro-
tect flora against toxic chemicals while providing the movement and availability of micronutrients.

4.3.2.2 Indirect Effect of Protein Hydrolysates (PHs)

In the farming industry, secondary impacts on crop nutritive value are also significant where protein
hydrolysate is used in plants and soils. PHs can also play a role in increasing nutrient availability
in low-​growing plant areas, increasing nutrient intake and nutrient utilization of plants. They boost
microbiological biomass production cellular processes and total crop production. Certain amino
molecules and peptides are believed to help with nutrient uptake along with root access due to their
chelating and complex activities.
Hydrolysate proteins are involved in the formation of macro-​chelate (such as K, Ca, and Mg)
as well as micronutrients (Cu, Fe, Mn, and Zn) utilized in industry for increasing potent nutrient
intake (De Pascale et al. 2017). They contributeg to plant enlargement by amendments in the
phytohormone balance (Colla and Rouphael 2020; Ertani et al. 2009). They are also reported to
promote phytonutrients (such as polyphenols, carotenoids, flavonoids) with the antioxidant action
of portable herbs (Ertani et al. 2009; Tsouvaltzis et al. 2014). Regulation Commission (EU) No.
354/​2014 lays down health rules as regards animal by-​products and derived products in consumable
plant components not intended for human consumption. Therefore, bio-​stimulants based on protein
hydrolysate have positive effects and enable productive methods for organic farming. Therefore,
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Role of Plant Bio-stimulants and Their Classification 71

the formation of PHs must be inhibited by the utilization of basic resources, substrate catalysts, and
degradation situations.

4.4 SEAWEED EXTRACTS
Seaweeds are a large group of species divided into distinct phyla compromising of brown, red, and
green macroalgae and are utilized as manure in agriculture. Ascophyllum nodosum, Ecklonia maxima,
Durvillea potatorum, and Macrocystis pyrifera are the most common sources of seaweed extract.
They are mostly obtained from carbohydrates (such as laminar, fucoid, and alginates) (Battacharyya
et al. 2015), phenolic compounds (Audibert et al. 2010), phlorotannins (Rengasamy et al. 2015),
ACC (1-​aminocyclopropane-​1-​carboxylic acid) (Nelson and Van Staden 1985), osmolytes (Reed
et al. 1985), and phytohormones (such as abscisic acid, auxin, cytokinin, and gibberellins) for the
enhancement of plants (Stirk and Van Staden 2014). They can be given to the plant as a foliar
treatment. Many of these compounds are distinct from their algal source, so they are defined by the
interests of the scientific community and industries into these groups of species.

4.4.1 Application of Seaweeds
• The use of seaweed bio-​stimulant is known to enhance seed sprouting (Masondo et al. 2018),
photosynthetic components (Di Stasio et al. 2017; Patel et al. 2018), nutrient absorption (Di
Stasio et al. 2017), blooming of flowers and fruits (Pohl et al. 2019), tolerance to abiotic
and biotic suppression (Bajpai et al. 2019; D’Addabbo et al. 2019; Jayaraj et al. 2008), and
increased production and life span of fruit juices (Kamel 2014; Renaut et al. 2019).
• The positive effect on soil microflora is also explained by pathogens being encouraged to
increase plant development and pathogen resistance in repressive soils (du Jardin 2012).
• The use of liquid seaweed bio-​stimulant has been shown to encourage flower initiation (flower
onset) and growth in many plant species. The phytohormone content in bio-​stimulants may
affect the setting of flowers and is also associated with early fruits.
• Useful effects and defensive substances found in seaweed include antioxidants as well as a
genetic regression response that may be involved (Calvo et al. 2014).
• The exceptional effects of gelation, fluid retention, and moisture content in soil are due to sea-
weed polysaccharides present in soil (du Jardin 2012).
• The polyionic compounds of seaweeds take part in the preparation and exchange of cations
which are also of interest in heavy metal complexes and soil preparation (du Jardin 2012).

Commercial seaweed extraction from agrochemical companies has gained widespread acceptance
in agriculture as natural or plant bio-​stimulants over the past few years. Commercial liquid extrac-
tion such as Stimplex (a marine commodity product) has shown a significant increase in the range of
ornamental Capsicum annuum (Ozbay and Demirkiran 2019). Stimplex enabled responses such as
cytokinin in A. thaliana (Khan et al. 2011). The extraction of seaweed (Acadian, Acadian Seaplants
Ltd Canada) with Ascophyllum nodosum increased root growth and resistance to water in pumpkin,
tomatoes, peppers, apricot, petunia, pansy, lettuce, and cosmos (Neily et al. 2010). Seaweeds are
reported to have many useful effects on a variety of plants but there is a need to better understand
how they work and to better utilize them in agriculture.

4.4.2 Microalgae
Recently microalgae, which includes single-​celled eukaryotic and prokaryotic (cyanobacteria) blue
algae are has become well-​known as a bio-​stimulant due to its ability to boost flourishing, plant
expansion, production, mineral utilization, and tolerance to various abiotic stressors. The effective
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72 Biofertilizers for Sustainable Soil Management

use of bio-​stimulants of plant-​based microalgae is limited even though the polyunsaturated fatty
acids, carotenoids, betaines, polyamines, phycobiliproteins, sterols, vitamins, and polysaccharides
are some of the bioactive organisms.

4.4.2.1 Activity of Microalgae as Bio-​stimulants

In some review papers the authors have described the dominant types of microalgae that have
stimulatory action as: Calothrix elenkinii, Acutodesmus dimorphus, Chlorella ellipsoida,
Scenedesmus platensis, Scenedesmus quadricauda, Chlorella vulgaris, Dunaliella salina,
Chlorella infusionum, and Scenedesmus platensis (Colla and Rouphael 2020). The number of
key substances (protein, lipids, carbohydrates), vitamins, amino molecules (tryptophan, arginine),
polysaccharides (glucan), and osmolytes (proline and glycine betaine) is linked to the bio-​stimulant
scheme of microalgal extracts (Chiaiese et al. 2018). A polysaccharide found in microalgae has
been revealed to contain a variety of biological agents which are used in farming to boost plant
productivity, nutrient availability, and biological and eco-​physiological stress resistance. The
microalgal exopolysaccharides are well-​known to reduce salt pressure and can be utilized under
severe environmental circumstances. Green alga in Solanum lycopersicum have lowered salt
pressure as well as reduced the size and loss mass of plant seeds and also roots (El Arroussi et al.
2018). Screening and selecting microalgal isolates containing organic plant hormones, especially
cytokinins and auxin, regarded as major sources for developing the possibility of algal econo-
mization in increasing plant maturation, productivity, and immunological responses especially in
abiotic stress (Ronga et al. 2019).
It is vital to remember that algae species, growth circumstances, and extraction processes all
influence the active chemicals identified in microalgal fragments. Physical (mashing, autoclaving,
liquid nitrogen), chemical (HCL, NaOH, and osmotic shock), and enzymatic (cellulase and protease)
approaches are used to remove different kinds of toxins from microalgae (Chiaiese et al. 2018). In
addition, bio-​stimulants based on microalgae are commonly distributed to plants and the rhizosphere
by various means such as soil wetness, composting, fertilizer spray, and seed treatment (Colla and
Rouphael 2020). The microalgae’s bio-​stimulant action can be attributed to their ability to alter the
number of microbes in the ground, rhizosphere, and biosphere (Renuka et al. 2018; Xiao and Zheng
2016). The effect of the incorporation of cyanobacteria Calothrix into the genus on the growth of rice
plants and with the pathogen culture is supported by research (Priya et al. 2015). They found that
Calothrix in the region upgraded the growth and formation of rice and revived the growing number
of bacteria. Chiaiese et al. (2018) and Ronga et al. (2019) described the methods of bio-​stimulant
use based on microalgae that were provided with various explicit and implicit placement methods,
such as:

• Biochemical (stimulating carotenoids and chlorophyll synthesis) and physical modification

(prevented senescence)
• Increase in the number of key genes involved in plant metabolism
• Increased activity around bacterial communities (rhizobacteria and mycorrhizae) by releasing
low-​and high-​molecular-​weight organic solute is associated with the rhizosphere
• Variations in root structure and as a result an increase in macro-​and micronutrient efficiency
and effectiveness.

4.5 PLANT-​DERIVED SMOKE
Plant smoke is a complex collection of compounds of chemical substances based on heating a
natural blend of flowering plants to encourage germinating seeds and vegetation in a variety of
plant species (Simorangkir 2007). Smoke contains life-​forming compounds that dissolve easily
into freshwater so its extract or smoke water (SW) can be produced to enhance plant health (De
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Role of Plant Bio-stimulants and Their Classification 73

Lange and Boucher 1990; Gupta et al. 2020). This can reduce the issue of air pollution caused
by smoke. Plant smoke has been proven to be a stimulant for several plant-​related conditions
including seed breakdown, accelerated germination, the percentage of seedlings, and health of
seedlings (Aslam et al. 2017). It has been found that SW has a dual control effect, where a low
SW concentration is very effective and there is a negative effect with high concentrations (Daws
et al. 2007; Gupta et al. 2019; Light et al. 2002). This is because smoke water contains both stimu-
lating and suppressive compounds (Flematti et al. 2004; Van Staden et al. 2004). A high volume
of inhibitors (trimethylbutane gold) in unmodified SW has been noticed because SW exhibits
an inhibitory effect on high aggregation (Gupta et al. 2020; Light et al. 2002). It acts to reduce
the inhibition of heavy metals, dryness, salinity, and extremes in temperature on growing plants
(Akhtar et al. 2017).
Like the extraction of seaweed, the major effects of bio-​stimulants from plant-​based fumes
such as smoke water (SW) and karrikin gold (KAR1) (Gupta et al. 2020; Kulkarni et al. 2011;
Light et al. 2002; Masondo et al. 2018) have been recorded in many agricultural plants. Smoke
water is used in unusually small areas until it attains growth-​promoting tasks and post-​emergence
growth (Gupta et al. 2020). An aspect of plant-​derived smoke it that while creating various stress
resistances it qualifies for inclusion in the category of bio-​stimulants. Among the many functions
of plant smoke in plant life are developing stress resistance which helps to eliminate salinity stress
in Zea mays and Oryza sativa, reducing heat stress in Solanum lycopersicum, reducing heavy rain
stress in Glycine max, and reversing abscisic acid stress in Lactuca sativa, which have strongly
supported its use as a bio-​stimulant. Smoke contains antibacterial agents that can help to protect
young plants from dangerous microorganisms (Holley and Patel 2005). Many plant species have
shown hormone-​like responses to chemicals found in SW and interrelated plant hormones in
photoblastic as well as thermos-​dormant seeds have been reported (Gupta et al. 2019; Van Staden
et al. 2004).
These findings suggest that this smoking technology could be useful in commercially cultivated
plants (Van Staden et al. 2004). As a result, plants inoculated with plant-​derived smoke are safe
to consume. It has shown that SW and smoke compounds (KAR1) do not show any mutagenic
genotoxic effects. Smoke influences the expansion and advancement of various plant species and
the use of smoked substances (KAR1) and water from the smoke can provide a new way to improve
crop production and harvest.

4.6 CHITIN AND CHITOSAN DERIVATIVES

Chitosan is a commercially available form of the natural and industrial biopolymer chitin without
an acetyl group. The physical responses of chitosan oligomers in plants are the outcome of this
polycationic substance’s ability to hold together many cellular elements, including genetic
material on the cell surface along with cellular fragments, as well as to attach specific receptors
associated with immune system activation, in a similar way providing plant protection (Hadwiger
2013; Katiyar et al. 2015). Crustaceans (king crab, shrimp, lobster), fungi (filamentous fungus,
black mould), insects (ladybird, insect wax, butterfly), mole oyster, and squid pen are all naturally
occurring sources of chitin and chitosan (Yadav et al. 2019). Microbial proteases have been the most
extensively used method for chitin and chitosan synthesis (Philibert et al. 2017). Crustacean shells
are a significant source of chitin, and chitin improvement involves three stages including demin-
eralization, deproteination, and removal of pigments and lipids (Holley and Patel 2005; Philibert
et al. 2017).
In agriculture, chitosan is utilized as a fertilizer in seed, leaf, fruit, and vegetable crops to increase
crop production and provide protection against various microorganisms.
Because of the polymer’s capacity to break down chitin slowly it is commonly using it as a bio-​
fertilizer, extracting essential nutritional elements from plants without the risk of over-​fertilization.
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74 Biofertilizers for Sustainable Soil Management

4.6.1 Chitin (CH)-​based Polymers

The chemical structure of chitin is given below:

Chitin is the second most frequent carbohydrate found in the plant body, and is a key component of
the formation of fungal cells and bones of invertebrates (Pusztahelyi 2018). According to the latest
market research, the global chitin trade is predicted to reach USD 2900 million by 2027, with signifi-
cant segments including health maintenance, waste, water treatment, and biochemical (Market and
Chitin 2017). It is a highly concentrated compound of b-​1,4-​N-​acetylglucosamine that is stretchable,
therefore, its structural alteration, including chemical variants, is critical to the optimal use and val-
idity of this biopolymer.

4.6.1.1 Chitin as a Bio-​stimulant
Chitin can be employed instantly as a bio-​fertilizer to promote plant expansion because it contains
a high nitrogen concentration and a weak C/​N ratio. Furthermore, adding chitin to soil enhances the
population and composition of microbes. It is identified as a pathogen-​associated molecular receptor
pattern (PAMP) in plants by specific receptors found in the plasma cell membrane (Chandanie et al.
2009). It follows that chitin could be used as a PAMP-​induced immunity which can create immune
responses to fungi, bacteria, and viruses (Pusztahelyi 2018). The commonly produced chitin is
insoluble in most typical liquid chemicals due to its high density.
The main methods of applying chitin to plants include

• Foliar spraying
• Direct application to soil.

Foliar application as a useful impact of CH on plants is linked with its direct action as an obs-
tacle for several microbial diseases and also by an incidental action which triggers antibodies
simultaneously with molecular signaling mechanisms (Eckardt 2008; Ramírez et al. 2010; Wan
et al. 2008).
The effects of applying chitin directly to soil are more complicated than those of foliar applica-
tion and involve the following:

a. Enhanced nitrogen availability because of a higher chitin concentration

b. Enhanced action of the chitinolytic compound which will adversely affect microbes in the soil
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Role of Plant Bio-stimulants and Their Classification 75

c. The positive impact of soil microbes such as mycorrhiza that work in conjunction with planting
and improving plant performance (Li et al. 2007).

In addition, chitin use in consumable coats in agricultural produce can impart a definitive hurdle to
gases interchange and can delay maturation while also reducing water loss and respiratory levels
(Dhall 2013).
The elements of chitin have activity on the exterior surfaces of living cells such as polycationic
and lipid molecules. This leads to the application of water purification but the emergence of chitosan
films on the waxy surface of the plant leaves give rise to its use as an anti-​transpirant take out CH
from yeast and fungi have various benefits (Sun et al. 2018).

• Cannot make humans or animals more susceptible to illnesses, such as CH from vertebrates
• Have physiochemical qualities like eco-​friendly, bio-​stability together with non-​toxicity,
which facilitate its agricultural use.

Some procedures recommend using CH with a low concentration to facilitate melting. An example
of chitin oligosaccharide produced in Arabidopsis thaliana is a genetic expression of vegetable
growth, nitrogen, and carbon survival. Plants medicated with CH oligosaccharide demonstrated
improved weight gain (10%), maximum root length, and overall carbon and nitrogen content when
compared to organic plants (Winkler et al. 2017).

4.6.1.2 Bio-​stimulatory Effect of Chitin in Vegetables (Table 4.1)

TABLE 4.1
The Consequences of Applying Chitin to Vegetables

Vegetables Scientific Name Effect of Chitin References

Cabbage Brassica oleracea Complex infections such as root knot nematode Loganathan et al. 2010
and cottony soft rot have been minimized due
to the composition of chitin and Trichoderma
Chili pepper Capsicum annum Chitin and salicylic acid treatment, as well as Rajkumar et al. 2008
opponents (fluorescent pseudomonads SE21
and RD41), are well regulated for damping off
(Thanatephorus cucumeris) seeds
Eggplant Solanum melongena Chitin-​containing soil supplements found in Inderbitzin et al. 2018
crabs reduce
verticillium pressure in plants
Lettuce Lactuca sativa Soil use of chitin is associated with the use of Lin et al. 2020
betaine paper for improved plant performance
under conditions of water stress
Tobacco Nicotiana tabacum Nanochitin enhanced seed sprouting and Zhou et al. 2017
development, as well as Fusarium species
tolerance
Wheat Triticun aestivum Nanochitin whiskers develop resistance to Liang et al. 2018
Fusarium crown rot of wheat and also
Gibberella zeae
Wheat Triticun aestivum Nanochitin works on the enhancement of crop Xu and Geelen 2018
production, and protein, ferrous, and zinc
contents
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76 Biofertilizers for Sustainable Soil Management

4.6.2 Chitosan (CHT)-​based Polymer

The chemical structure of chitosan is:

Excessive usage of chemical compounds to boost production and germ tolerance have the ability
to trigger permanent environmental damage because of its assembling in the atmosphere around
the plant and affecting biodiversity. To look for new ways to resolve this problem, the use of nano-
technology is very favourable. Chitosan (CHT) of biomaterials is very helpful in nanotechnology
because of their biological degradation, compatibility, and non-​harmful nature. Moreover, CHT can
be easily altered without compromising its reproductive capacity compared to other biopolymers
(Chakraborty et al. 2020). The advantages of the use of chitosan are very dominant because it is
described GRAS (generally recognized as safe) and is easily soaked, cheap, accessible, and easy to
use (Bellich et al. 2016).

4.6.2.1 Chitosan as a Bio-​stimulant

• The key functions of CHT have been strongly linked with improved photosynthetic action and
resistance to abiotic pressures such as drying and salt as well as low or high temperatures, and
increased activity of the antioxidant enzyme and its expression protective genes (Pichyangkura
and Chadchawan 2015).
• Furthermore, the use of chitosan can lead to increased plant growth, especially with an
increase in nitrogen and nutrients, and also can be used as an additional source of carbon in
biosynthetic plant processes (Mondal et al. 2013; Pirbalouti et al. 2017).
• Chitosan’s major agricultural goals rely on its promising impacts on macromolecule produc-
tion in insects and diseases (Chakraborty et al. 2020; Patel et al. 2020).
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Role of Plant Bio-stimulants and Their Classification 77

TABLE 4.2
The Consequences of Applying Chitosan to Vegetables

Vegetables Scientific Name Effect of Chitosan References

Ginger Zingiber officinale Chitosan and oligochitosan improve immune enzyme action on Liu et al. 2016
ginger
Lettuce Lactuca sativa Applying chitosan at 2% to Ni-​contaminated soil can importantly Turan 2019
control Ni bioavailability
Eggplant Solanum Combined nanocomposites enhance nematocidal activity with a Attia et al. 2021
melongena standardized immune response
Okra Hibiscus esculentus Nanochitosan has a positive effect on plant morphogenesis, Mondal et al.
growth, and physiology 2012
Potato Solanum Chitosan spray mixed with humic acid led to extensive tuber Harfoush et al.
tuberosum production and yield 2017
Tomato Solanum Foliar use of salicylic acid and chitosan at 75 mg L–​1 can be used Mondal
lycopersicum at the beginning of high growth, acquiring maximum fruit yield et al. 2016
in summer tomatoes. El Amerany et al.
Application of chitosan +​compost +​arbuscular mycorrhizal 2020
fungus has improved tomato growth

• In plants, several antimicrobial and control functions have been discovered through chitosan
molecules usage. For example, the reproductive elicitor of Oryza sativa L. is chitosan
nanoparticles (CHTNP), which contain CHT structures and nanoparticle properties (such as
interface), surface impact, tiny size effect, and quantum size (Divya et al. 2019), which influ-
ence the germination of seeds and maturation of wheat seed positively (Li et al. 2019).
• It can also expand the roots and lower the respiration rate, leading to improved water absorp-
tion and better water use in plants (Bittelli et al. 2001; Mondal et al. 2016).
• It can be used in a variety of ways including seed coverage, foliar application, along with
being cover-​up factor for vegetable or fruit protection after harvesting (Gómez-​Merino and
Trejo-​Téllez 2015; Pandey et al. 2018; Toscano et al. 2018).

4.6.2.2 Bio-​stimulatory Effect of Chitosan in Vegetables (Table 4.2)

Chitin and chitosan are natural biopolymers that have many functions in plants. The use of these
compounds has shown good results in protecting the biodiversity of horticultural crops as well as in
crop production and growth, especially under natural barriers that emphasize their positive role in
crop cultivation under arid and non-​arid conditions.

4.7 BENEFICIAL MICROBES AS BIO-​STIMULANTS

Microbial bio-​stimulants include microbes or an association of microbes that can be employed to
boost yield, absorption of nutrients, abiotic stress tolerance, crop productivity, and yield in seeds,
young plants, and land. This indludes plant-​growth ptomoting Rhizobacteria (PGPR, soil-​dwelling),
plant growth-​promoting fungi (PGPF, specifically mycorrhizal fungi), and endophytes (both fungi
and bacteria) (Fiorentino et al. 2018; Rouphael et al. 2017).

4.7.1 Beneficial Fungi
Species of plants, as well as fungus, are developed independently from soil, plants, and the mutual-
istic theory. This continuation contributes to the understanding of the diverse range of relationships
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78 Biofertilizers for Sustainable Soil Management

that emerge during evolutionary periods (Bonfante and Genre 2010; Johnson and Graham 2013).
Fungi interconnect with the root systems of plants in a variety of different mutualistic symbioses
(meaning that when two species live near each other they communicate and develop beneficial
partnerships) to parasitic in the living cytoplasm (Behie and Bidochka 2014). Mycorrhizal fungi
are the most numerous genus in symbioses, with more than 90% of all plant species. Plants that
promote fungal growth, such as arbuscular mycorrhiza fungi (AMF; Rhizophagus intraradices and
endomycorrhizal fungus), and Trichoderma species (Teleomorph Hypocrea) can encourage plant
growth by increasing the plant mineral content (Fe, Cu, Zn) as well as minerals in the soil (Fe3+​, Mg2+​
, K+​, etc.) through improved formation of stimulant hormones (such as indole acetic acid) (López-​
Bucio et al. 2015; Rouphael et al. 2015; Smith and Read 2008). AMF is formed between the cortical
cells of the root system and a category of fungus that sets up distinctive structures called arbuscules
and vesicles. AMF requires host plants to form an external hyphae network that can expand over
40 times in space (Krüger et al. 2012). There is growing interest in utilizing mycorrhiza to upgrade
the agribusiness, given the common advantages of efficiently high nutrient markers, water balan-
cing, and protection from biotic and abiotic plant pressures. Metagenomics is an important tool
for tracking and studying the bacterial interrelation in the space around plants. A fusion of plant
particles and soil capacity proceeds these processes (Colla et al. 2015a; Savvas and Ntatsi 2015).
Trichoderma species, Sebacinales and Piriformospora indica, as model organisms are different
from mycorrhiza fungi because they can spend a portion of their life span elsewhere than with their
host plant, attaching roots, transferring nutrients to the moderator, and using various methods (Behie
and Bidochka 2014). Trichoderma-​based bio-​stimulants have reportedly enhanced plant growth,
yield, healthy food quality, and biotic and abiotic stress tolerance (López-​Bucio et al. 2015; Lorito
and Woo 2015). Trichoderma has efficacy against 30 Tricoderma asperellum samples as well as four
different Fusarium oxysporum species (El Komy et al. 2015). Some classes of Trichoderma have a
prominent bio-​stimulant effect, which distinguishes them from their widespread use in agriculture.
They are non-​toxic to himans, animals, and crops, and also to native habitats, where they invade
plant roots without causing harm. The advantageous outcomes of Rhizophagus intraradices and
Trichoderma atroviride application are suitable for the production of auxin-​containing substances
with the release of flexible organic chemicals that improve the branching strength, and the solubility
of essential nutrients (Zn, Fe, P, Mn, etc). (Rouphael et al. 2017). The combination of Hypocrea lixii,
Hypocrea rufa, and Hypocrea virens increases the growth parameters, biomass production, as well
as nitrogen and phosphorus roots assimilation in Cicer arietinum (chickpea) in glasshouse and field
experiments (Rudresh et al. 2005).
The accomplishment of AMF and Trichoderma sp. improved the plant performance under water
stress conditions (Augé et al. 2015; Chitarra et al. 2016). It has been noted that the inclusion of
both AMF and Trichoderma species can have a beneficial impact on plant relationships and growth
suppression (Chandanie et al. 2009; Colla et al. 2015a). These fungal endophytes can be considered
bio-​stimulants, although crops have been generally assisted by the use of bio-​pesticides.

4.7.2 Beneficial Bacteria
Studies show that the use of advantageous bacteria can provide farmers with a solution to reduce
fertilizer application without compromising crop quality and making existing crop management
systems economically and environmentally viable. Bacteria such as Pseudomonas in plant roots
form beneficial relationships with plants that encourage nutrient absorption as well as plant develop-
ment. Successful colonization should reduce the need to re-​install microbial inoculants. Nitrification,
siderophore manufacturing, dissolving phosphate, formation of plant hormones (auxins, cytokinin,
and gibberellin), the activity of ACC, and presence of organic molecules and organic polymer agents
are all reported to promote maturation and production in various plants (Bharti and Barnawal 2019;
Vessey 2003). Bacteria affiliate to the flora in many ways (Ahmad et al. 2008):
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Role of Plant Bio-stimulants and Their Classification 79

I. Mould, where there is a continuity between parasitism and mutualism.

II. Bacterial strips travel from soil to inside tissues, with rhizosphere and rhizoplane regions.
III. Some bacteria are transported upwards by seeds, and this relationship may last for a short
or long time.
IV. Activities affecting plant life span in the biogeochemical cycle, provide nutrients, increase
nutrient efficiency, increase resistance to disease, improve abiotic stress tolerance, and cause
changes in plant morphology through plant growth hormones.

PGPR can also stimulate plant growth in a variety of forms such as increasing nutrient uptake,
and maximizing the root zone and yield of plants (Paungfoo-​Lonhienne et al. 2019). Indirect
enhancements include a decrease in the unfavourable effects of phytopathogenic organisms like
bacteria and fungi through a process called induced systematic resistance (ISR). ISR regulates
phytopathogenic fungi by forming antifungal substances like hydrogen cyanide, phenazines,
pyrrolnitrin, tensin, etc. (Bhattacharyya and Jha 2012). Cakmakci et al. (2007) described PGPR as
enhancing the effects of glutathione reductase (reduces oxidized glutathione for reuse as an antioxi-
dant compound) and activity, and improving plant growth in wheat. Bacillus lentimorbus has also
been reported to increase antioxidant activity in edible components of lettuce, spinach, and carrot
plants (Niculescu et al. 2009). Through the use of bio-​stimulants in cultivation within botanical
variants, two key classes of functional and natural categories should be considered:

(i) Intersection endosymbionts of the Rhizobium type

(ii) Interactions of Rhizobium plant growth-​promoting rhizobacteria (PGPR).

Rhizobium and related genera are sold as organic fertilizers, which are bacterial inocula that help
plants absorb nutrients.

Phosphorus is the second most important macronutrient for the growth of plants and a lack of it
reduces crop production. It is naturally available in the soil but it is unavailable to plants. Through
a process known as mineral phosphate solubilization, phosphate solubilizing rhizobacteria (PSRB)
release chemical compounds and phosphatases to transform soluble phosphates into soluble mono-
basic (H2PO4–​) and dibasic (HPO42–​) ions (Gyaneshwar et al. 2002).
Fe is an important micronutrient in plants, however, it is present mainly in the form of Fe3+​ ions
and forms hydroxides and oxyhydroxides, but is needed in the form of Fe2+​ by plants. By releasing
moderate chelating agents known as siderophores, PGPR sequesters iron as Fe2+​ ions (Fe3+​ is reduced
to Fe2+​ in the siderophore membrane and transmitted to the cell wall). Crops directly incorporate
Fe2+​ from siderophores by exploiting Fe-​siderophore characteristics or by alternating reactions with
a suitable ligand (Novo et al. 2018). In addition, the global economy for bacterial bio-​stimulants is
expanding, and PGPR inocula are increasingly regarded as a type of “probiotic” plants, that is, they
contribute actively to nourishment and immune defence (Berendsen et al. 2012).

4.8 INORGANIC COMPOUNDS
Inorganic compounds are minerals like silica, selenium, cobalt, and others that enhance the develop-
ment of plants, purity of plant foods, and abiotic stress resistance. Out of five beneficial properties,
Si is most widely used as a bio-​stimulant; it reduces salt, drought, and nutrient stress, as well as
climate-​related stress, and also reducing metal toxicity (Yan et al. 2020). Useful chemicals are syn-
thetic components that aid soil fertility which can be significant for certain taxons but may not be
essential for all species (Pilon-​Smits et al. 2009). Aluminium, copper, sodium, selenium, and silicon
are the five most important nutrients, and they can be found in a variety of forms in soil as well as
in crops, including undissolved structures such as amorphous silica (SiO2.nH2O) (Du Jardin 2015).
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80 Biofertilizers for Sustainable Soil Management

Such favourable roles may synthesize, such as nourishing the cellular structure by silica crystals,
or be exposed to specific ecological circumstances, such as selenium microbe attacks and osmotic
stress for sodium. These involve cell wall stiffening, heat reduction by crystallization, radioactivity-​
regulated heat, composition-​influenced functions of enzymes, connections between flora and foods,
as well as other aspects throughout absorption, movability, enzyme preservation, and mutually bene-
ficial contact between two organisms, microbe and herbivore actions, protection against metal tox-
icity, hormone production, and signaling (Pilon-​Smits et al. 2009).
Chlorides, phosphates, phosphites, silicates, and carbonates are synthetic compounds and
important chemicals employed as antifungal agents (Deliopoulos et al. 2010). Many synthetic
chemicals affect pH, osmosis, and oxidation–​reduction stability as well as the biological stress
signatures of hormones and enzymes (e.g. peroxidase). Their roles as bio-​stimulants for plant devel-
opment show they are effective in nutritional efficiency and resistance to abiotic pressure, which
differ from their fungicidal activity and their role as fertilizers, which requires much attention (Du
Jardin 2015).

4.9 CONCLUSION
Bio-​stimulants are not confined to a single substance or microbe. The component can be a synthetic
form of chemical or perhaps a class of chemical with approved biological action, e.g. extruded
plants but not complete arrangements. In this case they correspond to the current definition of a
substance. This substance involves the European REACH Regulation (EC No 1907/​2006) relating
to the certification, testing, authorization, and limitations of chemicals, that acknowledge the group
of flexible materials: “UVCB materials can be recorded as a single material within this Act, regard-
less of their distinct structure”. There is a European Commission guideline document on effective
compounds that protects plant produce. Agricultural plant materials include any compounds iden-
tified in plants and obtained by compressing, digesting, granulating, and/​or eliminating plants or
sections of plants of the same type. To be seen as a bio-​stimulant, a substance has to appear to
alter the plant structure, making it more systematic at using limited water and nutrients resources,
or protecting it from harmful factors, such as reactive oxygen produced by environmental stress,
pests, or microbes.

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5 Approaches for Using

Bio-​fertilizers as a Substitute
for Chemical Fertilizers
to Improve Soil Health and
Crop Yields in Pakistan
Anas Iqbal,1 Muhammad Izhar Shafi,2 Mazhar Rafique,3
Maid Zaman,4 Izhar Ali,1 Waqar-​un-​Nisa,3 Ayesha Jabeen,3
Sofia Asif,5 Bushra Gul,6 Muhammad Adnan,7 Rafi Ullah,7
Nazeer Ahmed,7 Muhammad Haroon,7 Muhammad
Romman,8 and Ligeng Jiang1*
1
College of Agriculture, Guangxi University, Nanning, China
2
Department of Soil and Environmental Sciences, Faculty of Crop
Production Sciences, The University of Agriculture, Peshawar, Pakistan
3
Department of Soil & Climate Sciences, The University of Haripur,
Khyber Pakhtunkhwa, Pakistan
4
Department of Entomology, The University of Haripur, Khyber
Pakhtunkhwa, Pakistan
5
Department of Food Science and Technology, The University of Haripur,
Khyber Pakhtunkhwa, Pakistan
6
Department of Biosciences, University of Wah, Punjab, Pakistan
7
Department of Agriculture, University of Swabi, Khyber Pakhtunkhwa,
Pakistan
8
Department of Botnay, University of Chitral, Khyber Pakhtunkhwa,
Pakistan
* Correspondence: Ligeng Jiang: [email protected]

CONTENTS
5.1 Introduction............................................................................................................................. 90
5.1.1 Current Fertility Status of Pakistani Soils.................................................................. 92
5.2 Bio-​fertilizers..........................................................................................................................92
5.2.1 Types of Bio-​fertilizers............................................................................................... 93
5.2.2 Phosphate-​solubilizing Microbe Bio-​fertilizers (PSBs)............................................. 93
5.2.3 Rhizobium Bio-​fertilizers............................................................................................ 94
5.2.4 Arbuscular Mycorrhizal Bio-​fertilizers...................................................................... 94
5.2.5 Azotobacter Bio-​fertilizers......................................................................................... 95
5.2.6 Azospirillum Bio-​fertilizers........................................................................................ 95
5.2.7 Azolla and Blue-​green Algae Bio-​fertilizers............................................................... 95

DOI: 10.1201/9781003286233-5 89
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90 Biofertilizers for Sustainable Soil Management

5.2.8 Silicon-​solubilizing Microbe Bio-​fertilizers............................................................... 96

5.3 Bio-​fertilizer Effect on Qualitative and Quantitative Attributes: A Case
Study of Cucumber.................................................................................................................. 96
5.4 Market Characteristics Needed for the Release of Bio-​fertilizer............................................ 96
5.4.1 Availability................................................................................................................. 96
5.4.2 Solubility and Mode of Action................................................................................... 96
5.4.3 Stability of Storage..................................................................................................... 97
5.4.4 Effectiveness............................................................................................................... 97
5.5 Pakistan and Bio-​fertilizers..................................................................................................... 97
5.5.1 History of Bio-​fertilizers in Pakistan.......................................................................... 98
5.5.2 Bio-​fertilizer Research and Development in Pakistan................................................ 98
5.5.3 AARI (Ayyub Agricultural Research Institute), Faisalabad....................................... 98
5.5.4 NIAB (Nuclear Institute of Agriculture and Biology) and NIBGE
(National Institute for Biotechnology and Genetic Engineering), Faisalabad............ 99
5.5.5 NARC (National Agricultural Research Centre), Islamabad...................................... 99
5.5.6 ISES (Institute of Soil and Environmental Sciences), University of Agriculture,
Faisalabad................................................................................................................. 100
5.5.7 NFRDF (Nature Farming Research and Development Foundation)........................ 100
5.5.8 Bio-​fertilizer Study in Higher Education Institutes.................................................. 101
5.6 Problems in Mass Production and Commercialization of Bio-​fertilizers in Pakistan........... 101
5.7 Future Scenarios and Recommendations.............................................................................. 102
5.8 Conclusions........................................................................................................................... 102
References....................................................................................................................................... 103

5.1 INTRODUCTION
Soils are one of the world’s most significant natural resources, and protecting, maintaining, and
improving them is crucial for the survival of life on Earth. Soil’s fertility allows for the supply of
critical chemical elements in the quantities and ratios required for the growth of plants (Itelima
et al. 2018; Al-​Zahrani et al. 2022; Rajesh et al. 2022; Anam et al. 2021; Deepranjan et al. 2021;
Haider et al. 2021; Amjad et al. 2021; Sajjad et al. 2021a,b). It is critical for crop production, yet
poor soils and runoff remain a management concern in many regions of the world. The main reason
for this is that researchers and farmers commonly assess soil fertility using different theories and
ambiguous literature findings (Yageta et al. 2019; Fakhre et al. 2021; Khatun et al. 2021; Ibrar et al.
2021; Bukhari et al. 2021; Haoliang et al. 2021; Sana et al. 2022; Abid et al. 2021; Zaman et al.
2021). As a result, understanding soil fertility is critical for enhanced soil production and appro-
priate land management strategies. Soil researchers have created numerous chemical, physical,
and biological methods for measuring soil fertility, but the assessment is not confined to scientific
measurements and is based on farmers’ qualitative judgements (Ali et al. 2020; Iqbal et al. 2021a;
Iqbal et al. 2020; Karlen et al. 2003; Sajjad et al. 2021a,b; Rehana et al. 2022; Yang et al. 2022;
Ahmad et al. 2022; Shah et al. 2022; Muhammad et al. 2022; Wiqar et al. 2022; Farhat et al. 2022;
Niaz et al. 2022).
Disparagement of the ineffectiveness of major technology implementations and scientific allo-
cation of material by extension facilities has increased interest in the importance and incorporation
of farmers’ understanding (Berazneva et al. 2018; Guzman et al. 2018; Ihsan et al. 2022; Chao
et al. 2022, Qin et al. 2022; Xue et al. 2022; Ali et al. 2022; Mehmood et al. 2022; El Sabagh et al.
2022; Ibad et al. 2022). Farmers apply their local soil skills to make day-​to-​day land managerial
decisions by observing and evaluating them (Bado & Bationo 2018; Deepranjan et al. 2021; Haider
et al. 2021; Huang Li et al. 2021; Ikram et al. 2021; Jabborova et al. 2021; Khadim et al. 2021a,b;
Manzer et al. 2021; Muzammal et al. 2021; Abdul et al. 2021a,b; Ashfaq et al. 2021; Amjad et al.
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Bio-fertilizers as a Substitute for Chemical Fertilizers 91

2021; Atif et al. 2021). Incorporating indigenous data assists extension staff in matching their
energies to native requirements and may result in increased uptake of co-​produced technologies
(Khan et al. 2016; Ingram et al. 2018). Farmers’ assessments of soil health are widely reported as
“regional” or “farmer’s soil awareness” in much ethno-​pedological research (Barrera-​Bassols &
Zinck 2003; Athar et al. 2021; Adnan et al. 2018a,b; Adnan et al. 2019; Akram et al. 2018a,b; Aziz
et al. 2017a,b; Chang et al. 2021; Chen et al. 2021; Emre et al. 2021; Habib et al. 2017; Hafiz et al.
2016; Hafiz et al. 2019; Ghulam et al. 2021; Guofu et al. 2021), demonstrating that farmers may
be aware of the mechanism and scientific attributes of soil type but use different connotations or
conceptions to interact and plan their soil productivity. As a result of how local information systems
differ from scientific knowledge systems, shared understanding among farmers and researchers is
difficult to achieve (Agrawal 1995). According to Barrios et al. (2006), while both systems share
indispensable concepts, such as the importance of water in plant growth, each information system
comprises gaps that are filled by others. They also claimed that attempting to strike a balanced
scientific precision and local relevance broadens common information, resulting in a new, hybrid
knowledge base. Farmers and agronomists both begin their appraisal of soil fertility with the same
aim: crop growth efficiency (Murage et al. 2000; Hafeez et al. 2021; Khan et al. 2021; Kamaran
et al. 2017; Muhmmad et al. 2019; Safi et al. 2021; Sajjad et al. 2019; Sajjad et al. 2021a,b; Saud
et al. 2013; Saud et al. 2014; Saud et al. 2017; Saud et al. 2016; Shah et al. 2013; Saud et al. 2020;
Saud et al. 2022a,b; Qamar et al. 2017; Hamza et al. 2021; Irfan et al. 2021;Wajid et al. 2017; Yang
et al. 2017; Zahida et al. 2017; Depeng et al. 2018). In addition, growers also define the qualities
of healthy or unfertile topsoil, primarily through physical and morphological traits like colour and
texture, which are regarded as universal soil fertility criteria (Mairura et al. 2007; Iqbal et al. 2016;
Hussain et al. 2020; Hafiz et al. 2020 a,b; Shafi et al. 2020; Wahid et al. 2020; Subhan et al. 2020;
Zafar-​ul-​Hye et al. 2020a,b; Zafar et al. 2021; Adnan et al. 2020; Ilyas et al. 2020; Saleem et al.
2020a,b,c; Rehman 2020; Farhat et al. 2020; Wu et al. 2020; Mubeen et al. 2020; Farhana 2020;
Jan et al. 2020; Wu et al. 2019). Soil scientists use quantitative analysis to assess soil as a natural
resource, whereas growers assess soils as part of their day-​to-​day work in the field. Producers
have more knowledge or “technical experience” of soil, whereas scientists have more scientific
expertise or understanding of soil (Ingram et al. 2010; Ahmad et al. 2019; Baseer et al. 2019; Hafiz
et al. 2018;Tariq et al. 2018; Fahad and Bano 2012). Such distinctions can be classified into three
parts: awareness of additional environmental knowledge, spatial scale, and timing. Examining the
various approaches taken by growers and researchers reveals the potential worth of increased con-
sciousness regarding indigenous descriptions of soil quality, which indicate full forms of informa-
tion and livelihood knowledge and have implications for developing an integrated soil approach to
management (Yageta et al. 2019).
Sustainable development in the agricultural system could be accomplished without affecting
future generations’ environmental resources or capacity to meet their own needs (Umesha et al.
2018; Fahad et al. 2017; Fahad et al. 2013; Fahad et al. 2014a,b; Fahad et al. 2016a,b,c,d; Fahad et al.
2015a,b; Fahad et al. 2018a,b; Fahad et al. 2019a,b; Fahad et al. 2020; Fahad et al. 2021a,b,c,d,e,f;
Fahad et al. 2022a,b; Hesham and Fahad 2020). Excessive use of synthetic fertilizers depletes
favourable living circumstances since residues that act as secondary contaminants might infiltrate
food chains and eventually people (Kumar et al. 2019; Iqra et al. 2020; Akbar et al. 2020; Mahar
et al. 2020; Noor et al. 2020; Bayram et al. 2020; Amanullah, Fahad 2018a,b; Amanullah, Fahad
2017; Amanullah et al. 2020; Amanullah et al. 2021). Secondary pollutants can linger in the eco-
system for an extended time, posing a health risk (Uosif et al. 2014). The use of bio-​fertilizers rather
than agrochemicals may usher in a new era of industry. Bio-​fertilizers could help plants obtain the
nutrients they need while not harming the environment (Mishra & Dash 2014). This section could
assist as a helpful guide for developing bio-​fertilizers and using them to accomplish agricultural
sustainability.
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92 Biofertilizers for Sustainable Soil Management

5.1.1 Current Fertility Status of Pakistani Soils

Optimum crop yield is dependent on good soil fertility. Soil analysis over time is essential and
provides details on the basic and current soil quality. For several reasons, soils in arid and semi-​arid
parts of the globe are often infertile (Vanlauwe et al. 2011). Nutrient loss reduces soil fertility, while
restoration with organic or inorganic inputs impacts crop development and production (Chukwuka
2009; Luo et al. 2020; Ullah et al. 2020). Deteriorations in soil quality are thought to be a major
contributor to the low productivity of crops such as rice, wheat, sugarcane, maize, and tobacco
(Rahman et al. 2016; Belachew & Abera 2010, Yuan et al. 2022; Rashid et al. 2020; Arif et al.
2020; Amir et al. 2020; Saman et al. 2020; Muhammad Tahir et al. 2020; Md Jakir and Allah 2020;
Mahmood et al. 2021; Farah et al. 2020; Sadam et al. 2020; Unsar et al. 2020; Fazli et al. 2020; Md.
Enamul et al. 2020; Gopakumar et al. 2020; Zia-​ur-​Rehman 2020; Ayman et al. 2020; Mohammad
I. Al-​Wabel et al. 2020a,b).
Pakistan is primarily a dryland region, with 80% of its land area classified as desert or semi-​arid,
12% classified as sub-​humid, and 8% classified as humid (Khan et al. 2013). As a result, soils in arid
and semi-​arid locations are subjected to various degradation processes. The most significant reasons
for soil deterioration and desertification, as well as reduced agronomic productivity, are salinization,
drought stress, soil erosion, and reduction of soil fertility and soil organic matter (SOM) contents
(Smith et al. 2020; Senol 2020; Amjad et al. 2020; Ibrar et al. 2020; Sajid et al. 2020; Muhammad
et al. 2021; Sidra et al. 2021; Zahir et al. 2021; Sahrish et al. 2022). Therefore, understanding the
climate–​soil–​productivity nexus is critical for satisfying the expanding population’s food and nutri-
tion needs. Pakistan’s population grew from approximately 30 million to 201 million from 1947 to
2018 and is expected to reach 244 million in 2030 and 352 million in 2100 (Lal 2018). However,
the current annual growth rate of approximately 2.0% is falling and is anticipated to reach 0.3%
by 2100. As evidenced by the rapid growth of the populations of particular cities, the rise in global
population is indicative of Pakistan’s strong urbanization tendency (Alam et al. 2007). From 1960 to
2018, Pakistan’s population grew by 4.5 million, while overall cereal production (wheat, sorghum,
maize, rice, millet, etc.) increased by 6.5 million metric tons (from 6.6 to 43.0 million metric tons).
Therefore, per capita cereal crop yields increased significantly between 1961 and 1980, but remained
stable between 1980 and 2016 at 220 kg per person. Despite these tremendous improvements, there
is no reason to be complacent because much greater difficulties are already apparent for the near
future. It is not only the populations that doubled between now and 2100, but nutritional tastes may
move towards animal-​based goods because of increasing wealth and overall economic success. The
issue of promoting food security and nutrition is exacerbated further by the ever-​increasing hazards
of soil pollution, expanding suburbanization, global warming, and decreasing aquifers (Lal 2018).
The Indus plains in Pakistan have the lowest soil organic carbon (SOC) contents, ranging from
0.5 to 0.1% in the root zone. A low SOC content has an impact on agronomic production and
input performance (Lal 2018), particularly in Pakistan’s rice–​wheat as well as other crop culti-
vation. However, by implementing effective management techniques, SOC concentration can be
regained. The goal is to improve the soil/​ecosystem by expanding the use of bio-​fertilizers. As a
result, site-​specific best management practices, such as cover crops, irrigation tillage, conserva-
tion tillage, mulches, integrated nutrient management (INM) incorporating manure/​compost input,
usage of biochar, bio-​fertilizers, contour farming, and crop interaction with livestock and plants are
recommended (Sarfaraz et al. 2020).

5.2 BIO-​FERTILIZERS
Bio-​fertilizers are organic in nature and include metabolites derived from microbes or bacteria
(Mishra & Dash 2014). Microorganisms extracted from soil (rhizosphere), air, and water are used to
make bio-​fertilizers, then purified for use in the field. Microorganisms begin creating agriculturally
important metabolites in response to particular environmental conditions, and plants may use these
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Bio-fertilizers as a Substitute for Chemical Fertilizers 93

FIGURE 5.1 A schematic diagram of bio-​fertilizers with various functions enlightened.

metabolites to support numerous biochemical processes (Salar et al. 2017). Microbes and microbial
metabolites facilitate the breakdown of complicated soil minerals/​particles into simpler forms and
the resulting forms work as a growth stimulator for specific crops. Bio-​fertilizers can be applied for
various purposes (Kaur & Purewal 2019; Xie et al. 2021) (Figure 5.1).

5.2.1 Types of Bio-​fertilizers
Bio-​fertilizers are amongst the most effective current agricultural fertility contributors. Organic
fertilizers are used in agriculture as an alternative to traditional fertilizers, including compost,
domestic garbage, and green manure (Khan et al. 2017; Ali et al. 2021; Iqbal et al. 2021c; Mishra
et al. 2013). However, synthetic fertilizers are more successful in this regard. As a result, farmers
frequently use chemical fertilizers for crop production, but on the other hand, their excessive use
is harmful to the ecosystem by polluting water, air, and soil (Iqbal et al. 2019; Iqbal et al. 2021b).
Furthermore, they have the potential to deplete soil health in the long term (Itelima et al. 2018; Wu
et al. 2021). Bio-​fertilizers comprise microorganisms that encourage appropriate nutrient supply to
the host plants and maintain optimal growth and physiological regulation. Organic fertilizers are
made using several living microorganisms (Xie et al. 2021). Only microorganisms with specialized
functions to improve plant growth and reproduction are employed (Gupta et al. 2015). Bio-​fertilizers,
as fundamental constituents of organic agriculture, develop the quality and stability of soil classified
into several types based on their kind, action, and availability (Kaur & Purewal 2019).

5.2.2 Phosphate-​solubilizing Microbe Bio-​fertilizers (PSBs)

Phosphorus is an important macronutrient because it influences root growth, protein synthesis,
signal transduction, respiration, and N fixation in plants (Ahmad et al. 2019; Izhar Shafi et al. 2020).
However, plants cannot utilize it directly as it is present in unavailable forms in the soil. Therefore,
it must be converted to plant-​available forms from unavailable forms (Shafi & Sharif 2019). There
are many strains of useful bacteria that can reduce phosphorus into its most basic form, allowing it
to be easily absorbed by root systems. Phosphate-​solubilizing microbes (PSMs), although naturally
common, vary in number depending on the soil type and location (Awais et al. 2017). In developing
nations, PSBs, in combination with rock phosphate (RP) of poor quality, could be a substitute for
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94 Biofertilizers for Sustainable Soil Management

expensive phosphate fertilizer (Mahanta et al. 2018; Rafique et al. 2017). In this regard, research
activities are being carried out worldwide to identify microbes that may be important in maintaining
agricultural sustainability. According to various researchers, bacterial strains such as Micrococcus,
Achromobactin, Erwinia, Pseudomonas, and Aerobacter, etc. play a prominent role in solubilization
of unavailable insoluble complexed forms of phosphate (Chen et al. 2006). Aerobic and anaerobic
microbes coexist in the rhizospheric soil. Bacterial strains or spores have different degrees of P
solubilization depending on the location from which they are collected, and among all, the spores
isolated from the rhizosphere have the highest P solubilization capacity. Phosphorus can bind with
Fe, Al, and K to generate complex compounds (Wahid et al. 2019). The entire conversion process
is made up of a series of biochemical processes involving the action of several enzymes caused by
bacterial strains. The conversion of strongly bound P into organic and inorganic acids takes place in
the first stage, which reduces the soil pH and maximizes the accessibility of phosphorus to growing
plants.

5.2.3 Rhizobium Bio-​fertilizers
In developing countries, critical nutrient deficiencies in food crops are more difficult to overcome
(Kumari et al. 2018). To solve these issues, there is a strong focus on the employment of micro-
bial consortia, particularly PGPR, for continuous plant growth and meeting food requirements in
the future (Khatoon et al. 2020). Rhizobium is a nitrogen-​fixing continually evolving member of
the Rhizobiaceae family. Rhizobium infects plant roots, causing the production of particular rhizo-
sphere soil (Gouda et al. 2018). According to Kumari et al. (2018), the more common Rhizobium
isolates BHU-​M and BHU-​B13-​398 were extracted from mung bean roots. These strains enhance
the shoot and root growth, and the height and yield of the plants as they are associated with plant
roots and capture nutrients for plant growth. Moreover, Rhizobium inoculation was reported to be
involved in the regulation of phytochelatin-​related gene expressions in Medicago sativa and protects
plants against excessive Cu-​stress (Chen et al. 2018). Their findings revealed that Rhizobium strain
inoculation enhanced plant growth through higher N uptake by the plants. When untreated and
Rhizobium-​inoculated treated plants were compared, a significant increase in Cu uptake was noted.
Several scientific studies have found that inoculating chickpeas with efficient microbial strains at the
time of planting increases the total grain yield (Funga et al. 2016).
Microorganisms in root nodules degrade molecular nitrogen to ammonia, which is then used by
the plant system to synthesize proteins, vitamins, and other N-​containing substances (Belhadi et al.
2018). The use of Rhizobium in particular legumes and other host plants aids in the maintenance of
major agricultural benefits (Sahu et al. 2019). These bacteria are harmless and have shown no nega-
tive environmental impact (Singh et al. 2011). Despite their occurrence in leguminous plant nodules,
several artificially created Rhizobium formulations are also available in the market.

5.2.4 Arbuscular Mycorrhizal Bio-​fertilizers

Natural resources are constantly subjected to abiotic stressors at various growth and development
phases which soil microbes are able cope with (Wahid et al. 2019). Plants begin manufacturing a
particular type of minor metabolites when stressed to combat the excessive production of reactive
oxygen species (Kaur et al. 2018a; Kaur et al. 2018b). To some extent, the creation of certain
ingredients aids the plant’s survival under severe conditions. One of the essential factors contributing
to crop plant health is the symbiosis interaction. Arbuscular mycorrhizal fungi (AMF) are essential
symbionts with roots that aid in nutrient uptake and numerous enzymatic activities in most plants
(Ortas et al. 2021; Yang et al. 2018). The AMF connections with plant rhizospheres give a variety
of growth-​promoting effects such as improved nutrition, increased resistance, drought tolerance,
and modified soil composition (Berruti et al. 2016; Rafique & Ortas 2018). Water-​soluble chemical
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Bio-fertilizers as a Substitute for Chemical Fertilizers 95

fertilizers are avoided in organic farming, and involve a variety of crop rotations. According to sci-
entific investigations, this increases AMF infection in soils with maximum nutrient uptake (Ortaş
et al. 2017). As a result, AMF may be a viable alternative to chemical fertilizers.

5.2.5 Azotobacter Bio-​fertilizers

Azotobacter are anaerobic bacteria from the family Azotobacteraceae (Sethi & Adhikary 2012).
They are non-​symbiont, Gram-​positive diazotrophs, that give numerous benefits to plants and their
interaction with growing crops enables them to maintain a stable growth with enhanced produc-
tion. The use of Azotobacter as a bio-​fertilizer to maximize the production and cropping yield has
been recommended by several researchers. It also helps in improving plant dry matter, yield, and
secondary metabolite synthesis (Damir et al. 2011). Azotobacter strains with imperative practical
qualities (enhancing the health of soil and N-​fixation, promoting growth and production of crops,
protecting plants against drought and pathogens) could be a boon for sustainable farming techniques
(Shirinbayan et al. 2019). In certain conditions, Azotobacter and related bacteria begin to develop
cysts –​a normal defensive mechanism against various environmental factors (Socolofsky & Wyss
1962). The strains commence the production of pigments from a deep brown to yellowish-​green and
purple colour throughout the N-​fixation process. The fundamental reason strains produce pigment
during the nitrogen fixation process is to shield nitrogenase from the destructive impact of oxygen
(Shivprasad & Page 1989). Azotobacter is now produced using a fermenter and mixer on a com-
mercial scale. The use of a fermenter is a scientific and automated method for the proliferation of
microbes. Specific nutritional media essential to maintain microorganism development are created
and pasteurized, and the pH of the medium may be controlled to commence appropriate microbial
populations. A mother culture (1–​2% of the total) may be employed to enhance growth. Other sig-
nificant needs include a constant supply of oxygen and the ability to maintain a constant tempera-
ture. Depending on the required demand, growth can be accelerated by utilizing a shaker, which
increases the rate of nutrient absorption in a brief period.

5.2.6 Azospirillum Bio-​fertilizers

Azospirillum is another type of bio-​fertilizer that aids crops in maintaining different biochemical
reactions essential for agricultural production (Llorente et al. 2016). It is an essential member of
the group Rhodospirillales and is closely associated with grasses and occasionally with monocots,
particularly rice and corn (Ruíz-​Sánchez et al. 2011). Their interaction is directly related to nitrifi-
cation, the release of particular fungicides, and plant hormones (Gonzalez et al. 2015). Azospirillum
is capable of producing phytohormones such as salicylic acid (Sahoo et al. 2014), auxins (Spaepen
& Vanderleyden 2015), and indole-​3-​acetic acid (Fukami et al. 2018). Azospirillum improves mois-
ture and nutrient retention by plants and protects plants against environmental stress, resulting in
higher total production (Fukami et al. 2018). Azospirillum inoculation in plants results in dramatic
morpho-​physiological alterations, including shoots and grains with increased nitrogen content.
When Azospirillum is used on the field, it requires less synthetic fertilizer than in fields without its
application (Cassán & Diaz-​Zorita 2016).

5.2.7 Azolla and Blue-​green Algae Bio-​fertilizers

Azolla is a member of the Salviniaceae family, which includes seven different species of duckweed
phototrophic ferns (Roger & Ladha 1992). Depending on numerous circumstances, including soil
properties, Azolla can develop to generate massive biomass in as little as 10 days. Azolla is a tiny
free-​floating plant having rough leaves and flowing roots. It is well known for its N-​fixing sym-
biotic relationship with Anabaena azollae in developing and underdeveloped nations (Emrooz
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96 Biofertilizers for Sustainable Soil Management

et al. 2018). Rice crops are widely recognized for their high-​water use, and growers use Azolla
to prevent extreme weed development. It can deliver up to 10 tons of proteins and other critical
nutrients to rice crops in cultivation (Yao et al. 2018). Blue-​green algae (BGA) are N-​fixing
microorganisms filamentous by nature and have a type of cell called a heterocyst (micronodules).
Heterocysts demonstrate nitrogen fixation process functioning. These microorganisms form sym-
biotic partnerships with fungal strains, ferns, and flowering plants for nitrogen fixation (Soma
et al. 2018). Blue-​green algae are particularly important in agriculture because of their fast activity
and effective nitrogen fixation. As well as N-​fixing, they also fix P, Zn, K, S, and other nutrients
(Adeniyi et al. 2018).

5.2.8 Silicon-​solubilizing Microbe Bio-​fertilizers

Disintegration of silica and silica-​based rocks and minerals can lead to a change in the soil layer
(Vasanthi et al. 2018). Microbial consortia of several types play a significant part in the decay, trans-
formation, and activation of silicon and its derivatives. A microbial consortia’s action is determined
by the availability of condensation, pH conditions, and growth regulators in soil. These are involved
in synthesizing various enzymes and metabolic products that may be useful in mineralization (Gadd
2010). Biological methods of converting tough silicon derivative products into the simplest eatable
forms have gained significance over chemical and physical methods. Biological methods include
microbial activities, which are self-​manageable and inexpensive and can result in conversion in a
small period. Thiobacillus thiooxidans and Bacillus globisporus showed the greatest ability to leach
silicon (Friedrich et al. 1991; Sheng et al. 2008).

5.3 BIO-​FERTILIZER EFFECT ON QUALITATIVE AND QUANTITATIVE

ATTRIBUTES: A CASE STUDY OF CUCUMBER
Several studies have been conducted on studying the effects of bio-​fertilizers on soils for different
crops and vegetables. When applied to various crops in combination with synthetic or other
fertilizers, it showed promising results in meeting the plant’s nutritional demand in an eco-​friendly
manner. A detailed study of the efficiency of bio-​fertilizers in cucurbits is available (Kumar et al.
2018). The useful insight and use of bio-​fertilizers and their effects on cucumber as a case study are
outlined in Table 5.1.

5.4 MARKET CHARACTERISTICS NEEDED FOR THE RELEASE OF

BIO-​FERTILIZER
Farmers’ use of bio-​fertilizers for increased crop production is one of the foremost constraints in
the farming sector. Although various bio-​fertilizers are now commercially available, their quality
and quantity may fluctuate based on the manufacturing division. Bio-​fertilizers should have the
following characteristics before being released to the market.

5.4.1 Availability
Bio-​fertilizers should be widely available in the marketplace. Farmers benefit from reduced trans-
portation costs and saved time.

5.4.2 Solubility and Mode of Action

The formulation should be water-​soluble to decrease costs and allow for spray application in larger
field areas.
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Bio-fertilizers as a Substitute for Chemical Fertilizers 97

TABLE 5.1
Use of Bio-​fertilizer and Integrated Nutrient Management (INM) Practices in
Cucumber Crop

S. no. Treatments Characters Enhanced in Cucumber References

Crop

1 Application of minerals (25%) and Increase in plant growth, yield, and Mahmoud et al. 2009
organic N (75%) quality
2 Use of bio-​fertilizers Increase in the fruit count, fruit length, Jilani et al. 2009
average fruit weight, and fruit yield
3 Use of FYM/​vermicompost An increase in the yield was observed Narayanamma et al. 2010
4 Use of bio-​fertilizers Enhanced yield and yield attributing Isfahani & Besharati 2012,
characters Saeed et al. 2015
5 Use of vermicompost An increase in yield and fruit weight Ghasem et al. 2014
was noted
6 Use of poultry manure with NPK A significant increase in the weight, Okoli & Nweke 2015,
number of leaves, fruit count, and Solaiman et al. 2020
size with quality and yield were
found
7 Use of bio-​fertilizers Significant increase in the fruit length Kanaujia & Daniel 2016
and diameter, fruit count, average
fruit weight, and yield
8 Use of poultry manure at 20 ton/​ha An increase in yield was noted Khan et al. 2017

5.4.3 Stability of Storage
Bio-​fertilizer formulations must be reliable in a broad range of climate circumstances. The strength
of the preparation must not deteriorate over time.

5.4.4 Effectiveness
Bio-​fertilizers should be used in small quantities in the field and must be successful in providing a
balanced mix of nutrients to plants. The formulation should provide crops with an immediate supply
of nutrients while causing no adverse effects. It should be simple to use and have no negative effects
on the health of growers. It must be affordable to growers, as this impacts crop prices. It should be
season-​independent and accessible to farmers throughout the year.

5.5 PAKISTAN AND BIO-​FERTILIZERS

Presently, Pakistan spends a significant amount of money importing and producing 8.41 million
nutrient tons of synthetic fertilizers. On the other hand, a significant opportunity exists for the
enhancement bio-​fertilizers use in sustainable agriculture. In Pakistan, saving of 10.0 billion rupees
annually is possible by the addition of a 10% contribution of bio-​fertilizers to the total fertilizer
consumption (Ali et al. 2012). Various groups/​organizations are engaged in bio-​fertilizers research
and innovation in Pakistan. They have stated substantial rises in yield and yield components of
important crops due to microorganism inoculation (Alam et al. 2007, Zahir et al. 2005) . The
extent to which this bio-​fertilizers benefit depends on their quantity and efficiency, which is ruled
by a diversity of environmental and soil elements. In comparison to chemical fertilizer plants, the
system used for bio-​fertilizers production is much simpler and the costs for its installation are very
negotiable. Furthermore, the use of bio-​fertilizers for a long time is efficient, more cost-​effective,
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98 Biofertilizers for Sustainable Soil Management

eco-​friendly, effective, and readily available to growers. A list of major problems, limitations, and
recommendations regarding the production of bio-​fertilizers on large-​scale and future technologies
in the county also have been discussed in detail.

5.5.1 History of Bio-​fertilizers in Pakistan

Rhizobium is the world’s oldest bio-​fertilizer for leguminous plants and soil quality enricher
“Theophrastus, 372 287 BC” as observed by (Danso 1992). J.B. Boussingault, a French chemist
and agronomist, proposed the classic concept of biological nitrogen fixation (BNF) in 1834 and
later Hellriegel and Wilfarth (1888) confirmed it. Beijerinck isolated N-​fixing Rhizobium organisms
in 1888, Azotobacter in 1901, and Azospirillum in 1925. Rhizobium is an N-​fixer, which was first
commercialized in the United States in 1895 under the trade name “Nitragin” and was developed by
Noble and Hiltner in 1896 (Chandra & Sharma 2017). Stalstrom (1903) was the first to report micro-
bial P-​solubilization and Pikovskaya isolated microbes in 1948 and Sperber in 1957.
Before the establishment of Pakistan, in early 1920, India’s first Agricultural College, named
the Punjab Agricultural College and Research Institute Lyallpur, began research on biological N
fixation. After six years, in 1926, these research activities were boosted when an independent post
of “Agricultural Bacteriologist” was established at the Institute. The microbiological centre was
developed in 1927 in Lyallpur (Naveed et al. 2015). The laboratories were developed, and field
trials were conducted at a larger scale in the Lyallpur and Gurdaspur areas to evaluate the effect-
iveness of synthetic inoculum on chickpea, Egyptian and Persian clover, alfalfa, sweet clover, mash
beans, mung bean, and cluster bean. It was concluded from the early research that seeds treated with
inoculum generated greater yields of higher quality than untreated seeds (Naveed et al. 2015). After
this, the commercial production of Rhizobium inocula began in 1956 in the region.

5.5.2 Bio-​fertilizer Research and Development in Pakistan

The uniqueness and capabilities of microbes, particularly in specific cultural and environmental
conditions, have shown that they have the potential to resolve the food security issues in agricul-
ture and other areas of life. Several organizations, research groups, and institutes in Pakistan are
working on the research and development of bio-​fertilizers to overcome food scarcity and increase
the country’s agricultural production. As summarized in the following sections, research and devel-
opment efforts are underway to expand the role of bio-​fertilizers in Pakistan.

5.5.3 AARI (Ayyub Agricultural Research Institute), Faisalabad

The Ayub Agricultural Research Institute in Faisalabad, formerly known as the Punjab Agricultural
Research Institute Lyallpur, is a parallel research institute of the Punjab Agricultural College.
Lyallpur was the country’s first and earliest BNF and bio-​fertilizers research institute. Work on
research and innovation began in the early 1920s and was focused on at various times. Since 1956,
AARI scientists have provided organic fertilizers with the trade name “Associative Diazotrophs”.
The fruitful and steady approval of legume Rhizobium cultures in the field (Naveed et al. 2015)
prompted the AARI’s “Soil Bacteriology” section to collaborate for useful microbial associations
prevalent in different crops in the early 1990s, and Azospirillum and Azotobacter inoculants were
introduced. Their consortia were released as a commercial product in the mid-​1990s under the trade
name “Fasloon ka jarasimi teeka”. This contains familiarized phosphate-​solubilizing microbes
(PSMs), which grabbed the attention of many growers who were struggling with P-​fertilizer scarcity
market prices. Data from the field experiments resulted in a 20% increase in the yield of leguminous
and non-​leguminous crops by applying rhizobial, diazotrophic, and PSM inoculants. On a limited
scale, the AARI’s Soil Bacteriology Section was manufacturing and providing 38,800 carrier-​based
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Bio-fertilizers as a Substitute for Chemical Fertilizers 99

250.0 g inoculum culture bags in the region. This was adequate for the inoculation of 14,000 ha of
arable land during 2000–​2011 (Naveed et al. 2015).

5.5.4 NIAB (Nuclear Institute of Agriculture and Biology) and NIBGE (National

Institute for Biotechnology and Genetic Engineering), Faisalabad
In 1972, NIAB established a very energetic BNF research centrr in the Department of Soil Biology,
having published work at the national and international levels. They have conducted research using
Azolla anabaena as N-​fixing BGA used as symbiont on rice bio-​fertilizers “Azolla” a water-​fern.
The severe hot environmental conditions of Punjab did not respond according to its potential on a
larger scale. In contrast, this technology provided the best in rice production in northern areas where
the environmental conditions were mild and humid (Malik et al. 2002).
With the foundation of NIBGE in 1992, Dr Kausar Abdullah Malik led the “Bio-​fertilizers
Division” by securing funding from several donor agencies such as the International Centre
for Nuclear Research, International Atomic Energy Agency, International Centre for Genetic
Engineering and Biotechnology, and the Islamic Development Bank for the development of a Bio-​
fertilizers Resource Centre in the South Asian region. In 1996, they successfully introduced the
commercial organic fertilizer “BioPower”. Rhizobium species were used for chickpea, mash bean,
soybean, mung bean, cowpea, and alfalfa as legume bio-​fertilizers, whereas implicit nitrogen-​fixing
and PGPR were used in crops such as wheat and maize. The research conducted revealed that bio-​
fertilizer use could meet 40–​70% of crop plant nitrogen requirements, which also improves crop
yield by 60–​80% (Hafeez et al. 1998). After pot and field trials, “BioPower” was used commercially
on an area of 11,000 ha with different testing crops in Punjab, and a 50–​70% reduction in nitrogen
fertilizer costs with a 20% increase in crop production was claimed by the research team (Naveed
et al. 2015). It was revealed that using half the recommended dose of NPK with “BioPower”
produced the same results as with the full recommended dose of NPK alone. NIBGE joined public
and private sector entrepreneurs to popularize the bio-​fertilizers, transfer manufacturing capabilities,
and provide proper training to farmers. Farmers were able to save a significant amount of money
(up to $292 USD ha–​1) by using “BioPower” in several crops, as per the benefit–​cost ratio of the
technology (Naveed et al. 2015). The NIBGE has a fully established bio-​fertilizers pilot production
unit to scale up bio-​fertilizer production to meet rising demand. “BioPower” has been supplied to
between 9000–​12,000 hectares (Naveed et al. 2015).

5.5.5 NARC (National Agricultural Research Centre), Islamabad

During the early 1980s, the Soil Biology and Biochemistry Department of NARC’s Land Resources
Research Program began investigating N-​fixation in legumes. They investigated the effects of
imported rhizobial strains (from NifTAL, Hawaii) on legume production in Pakistan. Later, a local
Rhizobium spp. was isolated and used to inoculate important crop legume crops (lentil, chickpea,
mash bean, mung bean, groundnut, soybean, Egyptian clover, pea, alfalfa, and sesbania). The
NARC “Rhizobium Gene Bank” contains over 200 isolates of various rhizobia. In 1990, the Centre
introduced “Biozote”, a bio-​fertilizer product. The efficacy of “Biozote” was tested commercially
during a combined project of PARC (Pakistan Agricultural Research Council), Islamabad and
Engro-​Chemical Pakistan Ltd. This project ran for three years to test different leguminous crops,
and approximately 60,000 packets of “Biozote” were provided to growers. Data from 300 growers’
fields revealed a 20–​50% improvement in crop production using “Biozote”. Additionally, it was
added that the benefit–​cost ratio of this technology was 30:1, and if applied to a 50% leguminous
area, it has the potential to improve the national economy by enhancing crop yield (Naveed et al.
2015). The Centre could produce 150,000 culture bags per year and currently, it is supplying about
2000 culture bags to growers each year.
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100 Biofertilizers for Sustainable Soil Management

5.5.6 ISES (Institute of Soil and Environmental Sciences), University of Agriculture,

Faisalabad
In 2003, the University of Agriculture, Faisalabad’s Department of Soil Science was upgraded to
the status of “Institute of Soil and Environmental Sciences”. The Institute is vigorously involved in
basic and applied research on soil microbiology and biotechnology etc., by isolating soil microbes
with various beneficial strains and using them as bio-​fertilizers. The researchers are not using
only living cells of inoculants but also proposed using microbial metabolites or plant growth
regulators, which could be the best approach to improve crop growth (Khalid et al. 2009). In
2002, they created a liquid preparatory work of microbial metabolite-​based bio-​fertilizers called
“Rice-​Biofert”. Data collected from multi-​location experimental fields for three years indicated an
increase of 20% in rice production (Zahir & Arshad 2004). The Soil Microbiology and Biochemistry
Group has also isolated various cultures of Azotobacter from various soils, and their performance
in increasing crop production has been extensively studied. Many PGPR, such as Burkholderia,
Pseudomonas, Serratia, Bacillus, and others, have been isolated and demonstrated their value as
plant-​growth promoters. ACC-​deaminase is an enzyme that hydrolyses ACC (ethylene precursor)
into ammonia and α-​ketobutyrate in various PGPR strains. The PGPR having ACC-​deaminase act
as ACC reservoirs when colonized with plant roots and lower the plant ethylene concentration.
This mechanism has the potential to inhibit the impact of high ethylene concentrations in plants
and promote stronger root structure and function. These plants also develop anti-​environmental
stressed qualities such as protecting against drought, salinity, heavy metals, etc. (Ahmad et al.
2011; Nadeem et al. 2010). A series of field trials were conducted by ISES on growers’ land to
demonstrate the potential impact of PGPR-​based bio-​fertilizer “Uni Grow” for the purpose of
encouraging the use of bio-​fertilizers in the farmer community and received highly encouraging
results (Shahzad et al. 2008). According to the literature, the combined application of chemical,
bio-​, and organic fertilizers has the potential to increase the crop yield and meet the food demands
of the country. Under field conditions, inoculation of rhizobia in leguminous as well as in non-​
leguminous crops produced excellent results (Hussain et al. 2009; Mehboob et al. 2011). The
ISES recently developed a combined culture of ACC-​deaminase containing PGPR and Rhizobium
called “Rhizogold” (RG), which enhanced 40–​45% yield of legumes. Another multi-​strain bio-​
fertilizer called “RhizogoldPlus” (RG+​) was obtained from effective strains of PGPR having ACC-​
deaminase with the purpose of mitigating salinity stress on cereal crops (Khan et al. 2013; Naveed
et al. 2015).

5.5.7 NFRDF (Nature Farming Research and Development Foundation)

The effective microorganism (EM) technique was introduced by a former scientist of the Soil Science
Department of the University of Agriculture, Faisalabad, who brought it from a Japanese scientist,
Dr. Teruo Higa, and used it as biological input for sustainable yield. After his tremendous work,
the foundation of the Nature Farming Research Centre was laid at UAF to work on this technology.
Further, the soil fertility and productivity were enhanced by using beneficial microorganisms (BM)
s in combination with manure, crop residues, waste from industries, green manures, and composts
from various sources. This technology reduced the costly application of chemical fertilizers. A new
type of BM fermenter/​superfermenter has been developed to achieve the minimum number of
organisms available and to use salt water to irrigate with BM technology (Hussain et al. 2009).
This centre has carried out many experimental projects in growers’ fields to assess the efficacy
of this technology for preserving soil fertility and productivity, encouraging the sustainable use
of soil, proliferating soil biological activities, reducing pollution, and recycling waste of plants
and animals. It was concluded that using the technology improved the soil’s biological activities,
increased crop yield and profit per hectare, and improved the quality of soil and water resources.
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Bio-fertilizers as a Substitute for Chemical Fertilizers 101

Many of the products of EM technology are in use by farmers in Punjab, e.g., for crop production
and fish farming, EM-​BIOAAB is used, whereas for animal and poultry production, EM-​BIOVET
is preferably used. EM-​BIOCONTROL, which is not a pesticide or insecticide, is used to control
insect/​pest diseases in crops, vegetables, and fruits (Hussain et al. 2009).

5.5.8 Bio-​fertilizer Study in Higher Education Institutes

At various higher education institutions across the nation, researchers are studying soil–​microbial
prospects and plant–​ microbe relations to understand how they affect the health of soil and
plants. Various higher institutes such as Quaid-​i-​Azam University Islamabad, Comsat University,
Islamabad; Karachi University, Karachi; PMAS-​Arid Agriculture University, Rawalpindi; Punjab
University, Lahore; The University of Agriculture, Peshawar; AJK University, University of Poonch,
Rawalakot; and many others have made outstanding contributions. An extensive list of research
work published in national and international journals has been documented. Relationships among
Rhizobium and leguminous and non-​leguminous crops, and the isolation and identification of various
microorganisms for disease management (Hussain et al. 2009; Mehboob et al. 2011) have been
studied in depth. Microflora supplying resistance to various stresses (Arshad et al. 2008; Saleem
et al. 2007), microorganism production of phytohormones (Qureshi et al. 2013; Shakir et al. 2012),
exploitation of bacterial and fungal populations for improved health of soil and plants, assessment of
variations and development of markers for maintaining and evaluating microbial efficacies (Malusà
et al. 2016), and phytoremediation of soil and environment (Naveed et al. 2015) are amongst the
research areas. Research is being conducted at various universities and higher education research
centres, providing applied research strategies. Interactions among research and educational institutes
can lead to the translation of scientific concepts into practice. Academia and industrial linkages for
the cheap, sustainable, and easy supply of the end product to the consumers (farmers) are the need
of the time.

5.6 PROBLEMS IN MASS PRODUCTION AND COMMERCIALIZATION OF

BIO-​FERTILIZERS IN PAKISTAN
Although microbe technology has shown its value when used in various agricultural and environ-
mental issues with remarkable success over the past 50 years, it has not been widely accepted. It is
often difficult to replicate its positive effects in different areas. These conditions are most common
in the upper and lower extremities. The following are the major barriers to mass production and
technological advancement in pakistan.

1. Lack of bio-​fertilizer regulation and standards. Regulations for the production and selling
of bio-​fertilizers have yet to be established at the national level in Pakistan. As a result, sub-
standard inoculants are among the significant limitations.
2. An insufficient community of growers that undestand microbial inoculants.
3. As most bio-​fertilizers are environment and ecology specific, they do not produce the
required results always, and so, eventually growers may lose faith in this technology.
4. The communication difference between marketing, extension work, and end-​users.
5. Lack of qualified labour and the excessive cost of making high-​quality organic fertilizers.
6. The country lacks transportation and storage facilities to prevent contamination.
7. Extreme climatic conditions frequently cause bio-​fertilizer results to be inconsistent.
8. A low amount of soil organic matter prevents beneficial microorganisms from surviving and
interacting positively with plants.
9. An insufficient supply of appropriate excipients for bio-​fertilizer production.
10. Poor labelling and packaging of bio-​fertilizers damage their reliability.
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102 Biofertilizers for Sustainable Soil Management

5.7 FUTURE SCENARIOS AND RECOMMENDATIONS

There are several concerns that need to be addressed by the government in future studies for more
comprehensive production and application of bio-​fertilizers.

1. Necessary legislation to monitor bio-​fertilizers, their quality, and any harmful effects on
humans and plant species. This serious concern must be evaluated and necessitates collabor-
ation among the government and private sectors.
2. The government should sponsor the production of bio-​fertilizers, or there should be the
availability of loans from the government to produce bio-​fertilizers on a small scale, e.g.,
seed money, agri-​preneur start ups, etc.
3. The country is in desperate need of microbial strain banks. All characterized microbes/​poten-
tial bio-​fertilizer candidates from various institutes and independent scientists should be
collected, conserved, molecular tagging internationally, and validated chemotaxonomically
if necessary.
4. Farmer communities and stakeholders should be trained by adopting intensive training and
extension workshops to use bio-​fertilizer technology to its full potential.
5. Development of bio-​fertilizers by using microbial consortia having active, competitive, and
stress-​tolerant microbial strains.
6. The ability of bio-​fertilizers to provide micronutrients and bio-​fortify food plants should be
investigated.
7. Phosphate-​ solubilizing microorganisms (PSMs) and P mobilizers such as vesicular-​
arbuscular mycorrhizae (VAM), which are less commonly used bio-​fertilizers, show prom-
ising results in providing P and other micronutrients. Therefore, the laboratory-​produced
strains of these symbionts will allow testing of their performance in the field. The genetic
basis for a competitive advantage must still be determined.
8. Selection of a low-​cost synthetic carrier capable of maintaining a high viable count and
developments in inoculation procedures to guarantee soil establishment and perseverance.
9. Creation of polymicrobial bio-​fertilizers such as PGPR, Rhizobium, PSM, and VAM.
10. Locally available organic wastes should be converted into value-​added bio-​fertilizers.
11. Endophyte molecular breeding is also required to improve endophyte–​ host plant
interactions. Endophytic bacteria genetic engineering should be a much simpler process
than crop genetic engineering. Endophytes that have been genetically modified by using
helpful genes will introduce new characteristics to host plants that have been inoculated
with these strains.
12. Synthetic fertilizers coated with promising microbial strains may mark the start of a new
understanding of synthetic/​natural sources of nutrition, potentially providing knowledge of
“microbial-​enhanced” fertilizer use efficiency.

5.8 CONCLUSIONS
Understanding the production and application of bio-​fertilizers is needed for a country’s eco-
nomic growth. Knowing the basic sustainability principles in agriculture requires understanding
the design, method of production, utilization, and storage conditions. Sustainability in agriculture
is extremely beneficial in resolving the actual problems in the agriculture sector with crop produc-
tion. Furthermore, marginal farmers in developing countries must be trained in the biotechnological
features of bio-​fertilizers in agricultural system planning. This chapter provides an in-​depth exam-
ination of the efficacy of bio-​fertilizers in achieving sustainable agriculture. Bio-​fertilizers can meet
agro-​industry challenges and create novel prospects for the benefit of growers in the agriculture
sector and business, and for the research, academia, and other government sectors.
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Bio-fertilizers as a Substitute for Chemical Fertilizers 103

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6 Phosphate-​mobilizing
Mycorrhizal-​based
Bio-​fertilizers
Muhammad Jawad, Ayesha Jabeen, Ali Raza Gurmani,
and Mazhar Rafique*
Department of Soil & Climate Sciences, The University of Haripur,
Khyber Pakhtunkhwa, Pakistan
Correspondence: [email protected]

CONTENTS
6.1 Introduction........................................................................................................................... 119
6.2 Classification of Bio-​fertilizers............................................................................................. 120
6.3 Importance of Bio-​fertilizers in Agriculture.......................................................................... 122
6.4 Limitations of Using Bio-​fertilizers...................................................................................... 123
6.5 Contribution of AMF in Nutrient Uptake.............................................................................. 123
6.6 Arbuscular Mycorrhizal Fungi Mechanism for Phosphorus in Soil...................................... 124
6.7 Commercially Available Mycorrhizal Bio-​fertilizers............................................................ 127
6.7.1 Oregonism XL.......................................................................................................... 127
6.7.2 Great White............................................................................................................... 127
6.7.3 Myco Madness.......................................................................................................... 127
6.7.4 Mycorrhizae (Soluble).............................................................................................. 127
6.7.5 MycoStim................................................................................................................. 127
6.7.6 MYKOS.................................................................................................................... 128
6.7.7 Piranha...................................................................................................................... 128
6.7.8 Plant Success (3-​1-​2)................................................................................................ 128
6.7.9 Root Maximizer........................................................................................................ 128
6.7.10 Root Rally (0-​3-​0).................................................................................................... 129
6.7.11 Rooters...................................................................................................................... 129
6.7.12 SubCulture-​M........................................................................................................... 129
6.7.13 White Widow............................................................................................................ 129
6.7.14 ZHO.......................................................................................................................... 129
References....................................................................................................................................... 129

6.1 INTRODUCTION
Fertilizers are compounds that are either natural or man-​made (Fuentes-​Ramirez & Caballero-​
Mellado 2005). These compounds stimulate crop development, and improve soil nutrients and
soil fertility when applied to soil and plants through irrigation water, or by fertigation (Benaffari
et al. 2022). Important macronutrients (phosphorus, nitrogen, potassium, sulphur, calcium, and
magnesium) as well as several micronutrients (boron, copper, zinc, iron, and molybdenum) are
provided to plants through various sources of chemical and bio-​fertilizers (Waqeel & Khan 2022;
Fahad et al. 2016; Fahad et al. 2015a,b; Fahad et al. 2020; Fahad et al. 2021a,,cb,d,e,f; Fahad et al.

DOI: 10.1201/9781003286233-6 119

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120 Biofertilizers for Sustainable Soil Management

2022a,b). Some fertilizers are in high demand such as nitrogen-​based urea (Singh 2018), calcium
ammonium nitrate (CAN), ammonium sulphate (AS), phosphorus based di-​ammonium phos-
phate (DAP), ammonium nitrate (AN), superphosphates, powdered rock phosphates, and potas-
sium (potassium chloride/​potash). Modern agriculture relies on synthetic chemical fertilizers,
which results in creating environmental challenges such as the greenhouse effect, soil degrad-
ation, and air and water pollution (Chen et al. 2018; Adnan et al. 2018a,b; Adnan et al. 2020;
Adnan et al. 2019; Atif et al. 2021). Furthermore, suitable agricultural practices are necessary
for appropriate cost-​effective food production to accommodate the increasing human popula-
tion, reduced energy, and upcoming environmental challenges (Williams et al. 2016; Khan et al.
2016; Fakhre et al. 2021; Muhammad et al. 2022; Shafi et al. 2020; Wahid et al. 2020; Zahida
et al. 2017).
Bio-​fertilizers are implemented in farming as an alternative to traditional fertilizers (Barragán-​
Ocaña & del Carmen del-​Valle-​Rivera 2016). Bio-​fertilizers include microorganisms such as bac-
teria, fungi, and algae that are natural, eco-​friendly, and cost-​effective, to sustain soil structure and
biodiversity (Fakhar et al. 2020). Microbial bio-​fertilizers improve plant development by enhancing
the effective absorption or uptake of nutrients for plants and inhibiting phytopathogenicity, in add-
ition to delivering nutrient enrichment to the soil (Amna et al. 2015; Rafique et al. 2017; Thomas
& Singh 2019). Organic fertilizers primarily augment nutrients by fixing atmospheric nitrogen,
solubilizing phosphorus, and manufacturing plant development chemicals (Rafique & Ortas 2018).
Since the green revolution, chemical fertilizers have reduced soil health by making the soil eco-
system unsuitable for soil microbiota, which are essential for maintaining soil health and supplying
crops with essential nutrients (John & Babu 2021).
Bio-​fertilizers are known as a product that include one or more species of microbial organism
(John & Babu 2021), that can mobilize soil nutrients and make them into an available form in soil.
They include compost, phosphorus-​solubilizing bacteria, nitrogen-​fixing bacteria, and release of
plant growth-​promoting substances (Umesha et al. 2018). Thus, bio-​fertilizers are inoculants of
bacteria, algae, and fungi, or natural fertilizers that increase the accessibility of nutrients to plants
(Kumar et al. 2017). Bio-​fertilizers are gaining increased significance in agriculture, specifically
with the escalating expense of conventional fertilizers and their possible detrimental effects on soil
fertility (Igiehon & Babalola 2017; Ortaş et al. 2017).

6.2 CLASSIFICATION OF BIO-​FERTILIZERS
Bio-​fertilizers have been proven to help soil fertility in a variety of ways such as enhancing crop
productivity and the provision of ecological benefits for maximizing crop yield (Kumar et al. 2017).
Bio-​fertilizers have several key functions or responsibilities in agriculture. In the development of
bio-​fertilizers, a diversity of microbes and their relationship with agricultural plants is of key import-
ance (John & Babu 2021). Bio-​fertilizers can be classified on the basis of their functions (Kaur &
Purewal 2019) (Table 6.1).

i. Rhizobium: This is a soil-​dwelling microorganism that take over the roots of legumes, pro-
viding atmospheric nitrogen in a symbiotic manner. Rhizobium comes in a variety of shapes
and physiologies, ranging from free-​living to nodular bacteroid.
ii. Azotobacter: The most common Azotobacter species is A. chroococcum. It may fix nitrogen
(2–​15 mg N2 fixed/​g of source of carbon) and is present in arable soils. These bacteria
produce slime, which aids in soil clumping. Because of the presence of hostile bacteria and
the scarcity of organic matter content in Asian soils, the population of A. chroococcum rarely
reaches 105 per gram of soil (Mishra et al. 2013).
Azospirillum lipoferum and A. brasilense are present in soil, rhizosphere, and root cortex
intercellular spaces (called Spirillum lipoferum in the earlier literature) of gramineous plants
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Phosphate-mobilizing Mycorrhizal-based Bio-fertilizers 121

TABLE 6.1
Types of Bio-​fertilizer

Group of Bio-​fertilizers Subgroup Example

Bio-​fertilizers for micronutrients Silicate and zinc solubilizers Bacillus sp.

Plant growth-​promoting Pseudomonas Pseudomonas fluorescens
rhizobacteria (PGPR)
P Mobilizing bio-​fertilizer Orchid mycorrhiza Rhizoctonia solani
mycorrhizae Ericoid Pezizellaericae
Ectomycorrhiza Amanita sp., Boletus sp., Pisolithus sp., Laccaria sp.
Arbuscular mycorrhizal fungi Sclerocystis sp., Gigaspora sp., Glomussp.,
(AMF) Acaulospora sp., Scutellospora sp.
Nitrogen fixing Free living Rhodopseudomonas, Klebsiella, Nostoc, Chromatium,
Bacillus polymyxa, Rhodospirillum, Desulfovibrio.
Derxia, Aulosira, Clostridium, Cylindrospermum
Anabaena, Azotobacter, Stigonema, Tolypothrix,
Associative Bacillus Herbaspirillum spp., Enterobacter,
Klebsiella, Azoarcus spp., Azospirillum spp.,
A. brasilense, A. halopraeferens, Acetobacter
diazotrophicus, A. lipoferum, A. irakense
A. amazonense, Pseudomonas.
Symbiotic Anabaena azollae, Trichodesmium Rhizobia
(Mesorhizobium Allorhizobium Rhizobium,
Bradyrhizobium, Sinorhizobium, Azorhizobium)
and Frankia.
Phosphorus (microphos) Phosphate solubilizing Burkholderia, Achromobacter, Microccocus,
B. subtilis, B. megaterium var. phosphaticum,
Achromobacter, Achromobacter, Agrobacterium,
B. circulans, B. polymyxa, Pseudomonas striata,
Penicillium spp., Aspergillus awamori, Erwinia

Source: Thomas & Singh (2019)

as they have a symbiotic relationship. Apart from nitrogen fixation, Azospirillum inoculation
has several additional benefits, including the production of growth-​promoting chemicals
such as indole acetic acid, mitigating drought stress, and providing disease resistance (Mishra
et al. 2013).
iii. Cyanobacteria: Rice cultivation in India has incorporated cyanobacteria, which can be
either free-​living or symbiotic. In India, cyanobacteria have been advertised as a rice bio-​
fertilizer but they have not attracted the attention of farmers. Under ideal conditions, the
advantages of algalization (a technique for mass cultivation of blue-​green algae to be used as
a bio-​fertilizer in paddy fields) could be as high as 20–​30 kg N/​ha, albeit the labour-​intensive
process for producing blue-​green algae bio-​fertilizer is in itself a restriction (Thomas &
Singh 2019).
iv. Azolla: Azolla is a free-​floating aquatic plant that fixes atmospheric nitrogen in collaboration
with a nitrogen-​fixing blue-​green algae, Anabaena azollae. It can be utilized as an alterna-
tive to conventional nitrogen fertilizers. Azolla is a wetland rice bio-​fertilizer that provides
40–​60 kg N/​ha to rice crop (Mishra et al. 2013).
v. Phosphate-​solubilizing microorganisms: Many fungi and soil bacteria, including
Penicillium, Bacillus, Aspergillus, Pseudomonas, and others, release organic acids that
lower the soil pH in their surrounding area, allowing binding phosphates in soil to dissolve.
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122 Biofertilizers for Sustainable Soil Management

Increased wheat and potato yields were obtained by inoculating Pseudomonas striata peat-​
based and Bacillus polymyxa cultures (Mishra et al. 2013).
vi. Arbuscular mycorrhiza fungi: This is an obligatory intracellular fungus endosymbiont of
the genera Acaulospora, Glomus, Endogone, Sclerocysts, and Gigaspora. The AMF have
vesicles for storing arbuscles and nutrients to funnel these nutrients into the root system,
and they oversee delivering nutrients from the soil to the root cortex cells. Glomus seems
to be the most common genus, with many species identified in the soil (Van Der Heijden
et al. 2006).
vii. Plant growth-​promoting rhizobacteria (PGPR): These are bacteria that form colonies in
plant roots or the soil’s rhizosphere that are beneficial to plants. The PGPR inoculant boosts
development by suppressing plant diseases (bio-​protectants), improving nutrient acquisi-
tion (bio-​fertilizers), or producing phytohormones (bio-​ stimulants). Growth regulators
or phytohormones produced by Pseudomonas and Bacillus species encourage plants to
develop more healthy roots, improving the absorptive superficial area of plant roots for
nutrient absorption and water. The phytohormones synthesized by such PGPR are called
bio-​stimulants. The bio-​stimulants include gibberellins, indole acetic acid, cytokinin, and
inhibitors of ethylene synthesis (Mishra et al. 2013).

6.3 IMPORTANCE OF BIO-​FERTILIZERS IN AGRICULTURE

Long-​term viability of agriculture in changing environmental and climatic conditions is an emer-
ging concern (Spicka et al. 2019). Based on the aforementioned data, it appears that long-​term use
of bio-​fertilizers over chemical fertilizers is cost-​effective, environmentally friendly, productively
viable, and easily accessible to the farming community (Itelima et al. 2018). Hence, the use of bio-​
fertilizers is generally required for two reasons. First, enhanced fertilizer use increases agricultural
output; secondly, a high amount of chemical fertilizer application destroys soil health and produces
numerous environmental problems (Khan et al. 2016; Thomas & Singh 2019). Bio-​fertilizers, com-
monly known as bio-​inoculants, are a type of fertilizer where nutrients are supplied by a living
organism (microbe) such as a bacterium for enhancing agricultural productivity in terms of N and
P (Itelima et al. 2018). A bio-​fertilizer is an organic substance that contains a large number of
efficient microbes isolated from the plant’s root or the rhizosphereic soil (Umesha et al. 2018).
They reproduce quickly and form a dense community in the rhizosphere. As far as their applica-
tion is concerned, they are used as a seed treatment, or soil application, or through seedling root
dip. Bio-​fertilizers fix atmospheric nitrogen, solubilize plant nutrients, and promote plant growth
and phosphates, by synthesizing growth-​promoting chemicals (Itelima et al. 2018). Bio-​fertilizers
are gaining attention in agriculture because they are non-​toxic, eco-​friendly, and non-​hazardous
(Itelima et al. 2018). Azolla, Azospirillum, P-​solubilizing microbes, blue-​green algae, Azotobacter,
Sinorhizobium, and mycorrhizae, are examples of bio-​fertilizers that promote crop yield (Mishra
et al. 2013).
Bio-​fertilizers have a lot of potential in terms of providing plant nutrients while reducing the use
of chemical fertilizers (Kaur & Purewal 2019). The bio-​inoculants are used as seed treatments or soil
treatments to improve plant nutrient accessibility, which leads to increased crop growth and produc-
tion (Chatterjee & Bandyopadhyay 2017; Ortas et al. 2017). Bio-​fertilizers can also protect plants
from soilborne diseases (Chatterjee & Bandyopadhyay 2017) and can help plants to increase crop
yield by 10–​25%. Plant pathogenic fungal activity is influenced by bio-​fertilizer agents. Rhizobium
culture applied to legume seeds controls seedborne fungi such as Colletotrichum, Ascochyta, and
Helminthosporium. When rhizobia multiply on the seed and in the rhizosphere, they generate a poi-
sonous toxin (Brahmaprakash et al. 2017).
Antibiotic compounds are produced by phosphate-​solubilizing fungi such as Aspergillus niger
and Penicilla, which kill plant pathogenic fungi (Devi et al. 2020). Plant pathogens are killed
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Phosphate-mobilizing Mycorrhizal-based Bio-fertilizers 123

indirectly by creating healthy seedlings and phytoalexins (Pedras & Abdoli 2017). The use of mycor-
rhizae results in stronger root systems that are more resistant to root rotting, collar rot diseases, and
soilborne pathogens. Bio-​fertilizer application in the soil has been shown to encourage and augment
the activity of saprophytic microorganisms in numerous studies (Naseer et al. 2016; Etesami et al.
2017). Under optimum agronomic and pest-​free conditions, they can reduce the use of chemical
fertilizers to no more than 40–​50 kg N/​ha (Asoegwu et al. 2020).
the benefits of employing bio-​fertilizers include root development, improved mineral and water
intake, vegetative growth, and nitrogen fixation (Ortaş et al. 2019; Singh 2018). Some bio-​fertilizers
(e.g., Rhizobium BGA, Azotobacter sp.) promote the formation of growth-​promoting substances
such as gibberellic acids, indole acetic acid (IAA), vitamin B complex, and other compounds
(Mishra et al. 2013).

6.4 LIMITATIONS OF USING BIO-​FERTILIZERS

1. As bio-​fertilizers are living entities, they require careful handling during storage for lengthy
periods of time (Itelima et al. 2018).
2. They have to be utilized before they expire or loose their activity (Vondeling et al. 2018).
3. They are less efficient if other microbes pollute the carrier medium or if an unwanted strain is
present (Vondeling et al. 2018).
4. For bio-​fertilizer organisms to flourish and function, the soil must have sufficient nutrients
(Anand et al. 2016).
5. Other fertilizers can be supplemented with bio-​fertilizers, but they cannot completely replace
them (Panhwar et al. 2019).
6. If the soil is excessively warm or dry, bio-​fertilizers lose their efficiency (Barati et al. 2017).
7. Inordinately acidic or alkaline soils can make it tough for helpful microbes to thrive.
Furthermore, if the soil includes an abundance of the microbe’s competition, they are much
less effective (Martínez-​Hidalgo et al. 2019).

6.5 CONTRIBUTION OF AMF IN NUTRIENT UPTAKE

Arbuscular mycorrhizal fungi (AMF) are a type of root-​bound biotroph that help roughly 80% of
plants by exchanging mutual benefits for plant growth and soil health improvement. In exchange
for photosynthetic products, they assist the host with essential requirements of nutrients and water
along with protection from pathogens. Consequently, AMF are an important organic component
of soil whose absence can cause ecosystems to function inefficiently. With an eye to sustainable
agriculture, the procedure of re-​creating the usual level of AMF is a viable approach to current
fertilization methods. The AMF association with maize plants results in osmolytes and aquaporin
deposition, as well as antioxidant activities. PGPR and AMF are promising strategies for increasing
P content at limited cost in plants, and their effects under low soil P supply are well understood
(Zahoor et al. 2016). By modulating critical plant processes, these bacteria can also enhance the P
content under varying stresses (Saia et al. 2015a, 2019a). This is especially true when both the plant
and microorganisms meet their N requirements (Saia et al. 2020). Although these microorganisms
can influence other plant strategies for receiving P by reducing direct absorption (and increasing
plant needs for P from bacteria) or influencing root morphological traits, they cannot change the
other plant mechanisms for acquiring P. Despite the importance of potassium to plants, less research
has been done on the involvement of AMF in the accession of potassium by host plants. Potassium
is abundant in soil, although it has low bioavailability to plants, and has been found in vesicles,
hyphae, and spores. Phosphorus is a macronutrient that plants require for growth and evolution.
Plants uptake P as orthophosphate (Pi) from the soil, but the amount of Pi in the soil solution rarely
surpasses 10 M (Schachtman et al. 1998), and the diffusive movement of Pi in the soil solution is
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124 Biofertilizers for Sustainable Soil Management

slow. When soil P levels are low, the symbiotic interaction between plants and AMF may be one
strategy to improve P uptake. Plants deliver carbon to the AMF, and the fungus feeds P and other
weakly accessible nutrients to the plant via an extensive hyphal network in this symbiotic interaction
(Drew et al. 2003; Ortaş et al. 2016). AMF has also been shown to boost the activities of phosphatase
enzymes and the solubilization of P (Tarafdar & Marschner 1994). However, an increase in P con-
centration in AMF-​infected plants is not always associated with increased growth; it can also be
associated with growth depression (Tran et al. 2019; Zhu et al. 2001), as AMF receive more C from
the plants than they would otherwise contribute to plant growth, or AMF colonization may suppress
direct P uptake by plant roots (Smith & Smith 2011; Zhu et al. 2001). Because of both envir-
onmental and genetic factors on colonization, plant susceptibility to AMF colonization is highly
varied. Researchers have been studying the nutrient uptake processes in AM relationships (Ortaş &
Rafique 2017). Plant genes are known to be stimulated by AM contact to make proteins that trans-
port Pi. The AMF not only improve Pi absorption in plants, but also help to form the structure and
shape of arbuscules and maintain symbiotic connections (Xie et al. 2013). It has been previously
shown MtPT4 and LjPT4 Pi transporters are found in the root tips of Medicago truncatula and Lotus
japonica plants not colonized by AMF because of AM interaction, and that PT4 genes serve as
elements of the Pi detecting machinery (Volpe et al. 2016). Plant transporter proteins can absorb and
transmit inorganic nutrients from the peri-​arbuscular apoplast to the cortical cell through the mem-
brane mentioned earlier (Bapaume & Reinhardt 2012; Shah et al. 2016b). Ammonium transporters
(enzymes that move ammonium from one place to another) produced by AMF have been found in
the peri-​arbuscular membrane of soybeans, indicating that they play a role in ammonium transfer to
the cortex cell (Breuillin-​Sessoms et al. 2015).

6.6 ARBUSCULAR MYCORRHIZAL FUNGI MECHANISM FOR PHOSPHORUS

IN SOIL
There is an increasing demand to boost food production to fulfill the needs of the growing global
population. This can be accomplished in one of two approaches: by expanding the cultivation area
or by increasing the production per unit area. Previous methods are not achievable in several coun-
tries throughout the world owing to an assortment of limitations, such as a paucity of soil or water
resources, drought, climate change, and soil salinization (Anukwonke et al. 2022). Alternatively,
improving the nutritional characteristics of the soil is another approach to boost the yield per
unit area. Phosphorus is necessary for energy generation, carbon metabolism, enzyme activation,
energy transmission, nitrogen (N2) fixation, and membrane development in plants (Lyu et al. 2022).
Phosphorus also contributes to the formation of important biological components such as nucleic
acids, ATP, and phospholipids (Park et al. 2022). Phosphorus scarcity is a critical limiting factor
for crop development and productivity, affecting nearly half of all agricultural ecosystems world-
wide (Etesami & Jeong 2021; Wu et al. 2022). To solve this issue, there has been a massive growth
in the usage of phosphorus fertilizers all over the world. The increasing agricultural P demand
has called into doubt the long-​term viability of P mining for fertilizer manufacturure (Lun et al.
2021). Phosphorus fertilizers frequently result in the addition of an excessive amount of P to agri-
cultural soils. Unfortunately, due to fixation and adsorption processes, more than 80% of P fertilizers
applied to soil are lost in precipitated form (Bindraban et al. 2020) or converted into organic forms,
which account for 40–​80% of soil total P (Zhu et al. 2018), with phytates as the most familiar
form (Menezes-​Blackburn et al. 2018). As a result, the accessibility of this additional P to plants is
restricted (almost 0.1% of the total P).
Plants can only absorb P in a limited range of soil conditions, such as HPO42–​ and H2PO4–​,
which have a pH of 6.5–​7. Inorganic phosphate (Pi) is immobilized and mineralized into calcium
phosphates when the pH of soil exceeds 7.0. At lower soil pH, soluble aluminium, manganese, iron,
or similar hydrous oxides are usually absorbed by P (Etesami & Jeong 2021). Inorganic phosphate
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Phosphate-mobilizing Mycorrhizal-based Bio-fertilizers 125

adsorbs to weathered silicates such as clay minerals at neutral pH, resulting in a 5–​15% reduction in
agricultural yield (Aziz et al. 2014; Hashizume et al. 2020).
The predicted improvement in plant development proficiency from the addition of chemical P
fertilizers has peaked, hence more fertilization of chemical P is unlikely to enhance plant production
(Yadav et al. 2022). According to the US Geological Survey, 22 million tons of P (3–​4% of total P
consumption) are removed yearly from natural sources (Etesami & Jeong 2021), as a result, nat-
ural P supplies are at risk from this reduction (Mnthambala et al. 2021). More effective use of P is
required, which includes optimizing P collection and usage efficiency (Han et al. 2022).
Some plants can effectively accumulate and utilize P to uphold development and metabolism
(Dissanayaka et al. 2021; Sarkar & Sadhukhan 2022). The following are some plant strategies for
enhancing P uptake efficiency: (1) growth rearrangement among root types; (2) topsoil foraging;
(3) soil investigation at a low metabolic rate; (4) root hair growth stimulation; (5) increased expres-
sion of high-​affinity P carriers; (6) increased root-​to-​shoot ratio; (7) increased organic acids secre-
tion (e.g., oxalate, malate, citrate,) from roots to the soil; and (8) improved phytase secretion or acid
phosphatase (Wang et al. 2006).
Plants also have improved biotic relations with a variety of soil microbes that boost plant devel-
opment. The most prevalent microorganisms are plant growth-​promoting bacteria (PGPB) and AMF.
PGPB and AMF, particularly the phosphate-​solubilizing bacteria (PSB) subgroup, is recognized
to assist plants in overcoming P deficiency (Khatoon et al. 2020; Rafique et al. 2017). AMF and
PSB are part of important biogeochemical cycling mechanisms (Gouda et al. 2018; Pathak et al.
2017). Endomycorrhizal communities in which fungal hyphae enter root cell walls and contact the
plasmalemma are known as AMF. They can be found in almost every plant habitat in the world
(Wang et al. 2021). The formation of AMF has allowed plants to live and thrive in their natural
settings for millions of years without the use of pesticides or fertilizers. AMF belong to the phylum
Glomeromycota and the subphylum Glomeromycotina, which comprise 340 species (Bano & Uzair
2021; Reinhardt et al. 2021).
AMF are obligate symbionts that depend entirely on their plant hosts for organic carbon. This
symbiosis is arguably the earliest kind of mycorrhiza, having originated 400–​450 million years
ago and involving a broad range of plants (Feijen et al. 2018). The symbiosis is typically mutual-
istic, relying on plant carbon exchange (4–​20% of photosynthetically fixed carbon) and fungus-​
provided P (Ravi et al. 2021; Vijayakumar 2018; Watkinson 2016). This mycorrhizal symbiosis
is thought to be capable of aiding more than 80% of the world’s plant species (Vasar et al. 2021).
The advantages of AMF in many plants (mainly crops) have been demonstrated (Chen et al.
2022). The AMF and bacterial species boost plant resilience to abiotic stressors, improve min-
eral absorption (especially of P), improve water relationships, and protect plants from soilborne
diseases, all of which promote plant development (Fakhar et al. 2022; Ortas et al. 2021; Rafique
et al. 2019; Sakthieaswari et al. 2022). AMF can support plants to acquire nutrients such as Mg,
Cu, Zn, K, and N, especially when they are present in less soluble forms in soils (Bhantana et al.
2021; Saboor et al. 2021). These fungi enter the root cortical cell walls and create arbuscules,
which resemble haustoria and mediate metabolite exchanges between the host cell and the fungi
(Sakthieaswari et al. 2022). Increasing the root zone absorption area by 10–​100%, AMF improve
the plant’s ability to use more soil resources. Because of the extraradical hyphae that enhance
nutrient absorption and transport, mycorrhizal roots can reach a larger soil volume than non-​
mycorrhizal roots (Beltrano et al. 2013). AMF increase nutrient absorption by expanding the root’s
absorption area and secreting compounds such as glomalin, a glycoprotein produced by AMF
hyphae and spores. Glomalin in the soil aids in the absorption of difficult-​to-​break-​down nutrients
such as Fe and P (Herath et al. 2021; Pal & Pandey 2014). Phosphorus is quickly absorbed from
soil particles, and Pi-​free zones form around the roots with ease. Mycorrhizal roots’ extraradical
hyphae reach beyond these P-​depleted zones, absorbing bioavailable Pi that would otherwise be
unavailable to plants.
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126 Biofertilizers for Sustainable Soil Management

Arbuscular mycorrhizal plant roots absorb P via two mechanisms. The first mechanism is shared
by both non-​arbuscular mycorrhizal and arbuscular mycorrhizal plants, and entails P absorption
directly from the root epidermis and hairs. Phosphorus enters the root cortical cells in the second
mechanism (intraradical mycelium) (González-​González et al. 2020), and arbuscules or hyphal coils
offer symbiotic interactions between fungal hyphae (cell-​specific Pi transporter gene expression
in mycorrhizal roots transports P from the interfacial apoplast) (Balestrini et al. 2007; Balestrini
et al. 2021; Fochi et al. 2017). This is a fast P translocation that extends several centimetres. Recent
molecular and physiological data show that the mycorrhizal pathway is active for P, regardless of
plant growth responses (Smith & Smith 2011). The role of carriers has been carefully investigated,
as has the translocation of Pi in fungi and the transfer of Pi to host plants (Chan et al. 2021; Ezawa &
Saito 2018; Johri et al. 2015).
As previously stated, poor solubility of P in alkaline and acidic soils (e.g., less than 10 M)
leads to very limited mobility (Seshadri et al. 2020). As a result, when roots absorb P, it takes a
long time for it to be replaced in the bulk soil, leading to the formation of P-​depleted zones, where
all the available P has been rapidly removed from the environment around the roots, lowering P
uptake by the root epidermal hairs (the first method of P absorption) (De Parseval et al. 2017;
Smith et al. 2011). Plants should move beyond these deficient areas and demonstrate root activity
in other parts of soil to increase their P acquisition. The success of this effort to absorb P (and
other normally immobile soil nutrients) is defined by the root system surface area. The highly
essential function of AMF hyphae is to enhance root surface area (depletion is reduced near small-​
diameter AMF hyphae) (Mei et al. 2014; Okiobe 2019). Furthermore, mycorrhizal plants can
exhale organic acids like citrate and malate, which chelate Al3+​ and Ca2+​, and dissolve calcium and
aluminium phosphates (Seguel et al. 2017). With the help of AMF hyphae, plants gain improved
access to orthophosphates and Pi in the soil solution by expanding their soil contact area (Rubin &
Görres 2021). AMF hyphae allow the roots to immediately absorb released Pi because AMF
roots lack a fungal coat, and they can potentially use both routes of nutrition absorption. The
two nutrient absorption mechanisms in AMF symbiosis are thought to work in tandem (Xie et al.
2021). However, it is believed that AMF supply around 80% of P absorption in a mycorrhizal plant
(Andrino et al. 2021). The AMF boosts legumes’ ability to fix nitrogen and minimizes the quantity
of inorganic N that percolates (Meena et al. 2018). Chlorophyll contains N2, which is required for
photosynthesis. Soil microorganisms such as AMF and PSB become more active when photosyn-
thetic resources are transported to the roots.
The AMF can help P-​deficient soils absorb more P by increasing the rate of P absorption (P
input) per unit of AMF root. The enhanced P absorption rate with AMF is responsible for the
improved efficiency with which hyphal surfaces absorb P from soil as compared to cylindrical root
surfaces (Hammer et al. 2014). Absorbing Pi through fungal AMF hyphae, increasing the mycor-
rhizal hyphal network beyond the rhizosphere Pi transporters, which transfer Pi to internal fungal
structures in root cortical cells, can be found up to 25 cm from the roots (Mbodj et al. 2018; Smith &
Smith 2011). By successfully transporting P from soil to plant-​based hyphae via appressoria and
from extraradical mycelium to intraradical mycelium, the fungus can maintain low internal Pi
levels (Preeti & Panwar 2013). Hyphae with a small diameter (2–​20 m) allow the fungus to pene-
trate in small soil cores in search of phosphate and attain higher P inflow rates for a given sur-
face area (Bitterlich et al. 2018); lowering the depletion zone around roots or hyphae (reduced
rhizospheric Pi depletion) (Smith & Smith 2011). Phosphorus depletion surrounding the roots of
Capsicum annuum L. or the hyphae of Rhizophagus mossea was just 0.06 cm in a study, meaning
that only 7% of the soil P was available to the roots. The hyphae had complete access to soil since
half the distance between close hyphae was barely 0.01 cm. In most cases, the high ability of
hyphal surfaces to absorb P from soils may be sufficient to explain how AMF enhances accessible
P absorption in soil (Sharif & Claassen 2011).
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Phosphate-mobilizing Mycorrhizal-based Bio-fertilizers 127

6.7 COMMERCIALLY AVAILABLE MYCORRHIZAL BIO-​FERTILIZERS

6.7.1 Oregonism XL
Soluble root growth enhancer
Oregonism XL was particularly chosen for fruiting and blooming plants that can withstand intense
fertilization. It improves nutrient availability and absorption, promotes strong root systems, and aids
plant resistance to stress.
Ingredients
Oregonism XL is a proprietary mix of AMF, Bacillus, and Pseudomonas microorganisms along with
humic acid. It works well in both soil and hydroponics. (www.planet​natu​ral.com/​prod​uct/​roots-​
organ​ics-​oregon​ism-​xl/​)

6.7.2 Great White
The Great White mycorrhizae are a blend of carefully selected mycorrhizal fungus and beneficial
bacteria that are well adapted to a wide range of soils, climates, and plants. These bacteria flourish
in and on plant roots in nature, considerably enhancing plant growth and vitality.
Ingredients and features
• There are seven species of Endomycorrhizae and 11 species of Ectomycorrhizae
• There are 67,000 endo-​and 1.5 billion ecto-​propagules per pound.
• There are 19 species of bacteria and two species of Trichoderma.
• It is a biostimulant and vitamin package. (www.planet​natu​ral.com/​prod​uct/​great-​white-​myco​
rrhi​zae/​)

6.7.3 Myco Madness
Humboldt Nutrients’ Myco Madness contains a biologically active powerhouse of nine mycorrhizal
species, 15 helpful bacteria, and two Trichoderma species. This soluble powder, which is well suited
to a variety of climates, soils, and plants, can assist plants in boosting nutrient and water absorption
for improved plant performance. (www.planet​natu​ral.com/​prod​uct/​myco-​madn​ess/​)

6.7.4 Mycorrhizae (Soluble)
Mycorrhizae (Soluble) is a mixture of humic acid, vitamins, and 12 helpful bacteria (seven strains of
endo-​and five strains of ectomycorrhizae). It reproduces quickly once applied and acts to provide a
favourable environment for the growth of seeds and transplants. It reduces transplant losses by redu-
cing plant stress and boosting new root formation. It can be used in tank mixes containing organic
fertilizers. Its addition aids in the preservation and expansion of beneficial bacteria and fungi in the
soil. This increases biomass energy, which promotes soil tilth and nutrient absorption. Excessive
application is not detrimental. (www.planet​natu​ral.com/​prod​uct/​solu​ble-​myco​rrhi​zae/​)

6.7.5 MycoStim
Organic Research Laboratories’ MycoStim is a potent soil inoculant that mixes eight super-​strains
of mycorrhizae and two species of Trichoderma with amino acids, kelp extracts, and humic acid.
These plant-​friendly fungi promote root development, which provides:

• Increased growth and nutrient intake

• Resistance to transplant shock
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128 Biofertilizers for Sustainable Soil Management

• Vigorous plants
• Resistance to stress
• Increased plant quality, yield, and vigor.
• (www.planet​natu​ral.com/​prod​uct/​mycos​tim-​myco​rrhi​zae/​)

6.7.6 MYKOS
MYKOS (mycorrhizae) is a soil fungus that aids in the breakdown and transfer of nutrients to plant
roots. MYKOS boosts the availability of both moisture and nutrients essential for plant growth, as
well as connecting numerous microorganisms in healthy soil to host plants. MYKOS may change
an average landscape into something with hugely improved performance with just one treatment.
(www.planet​natu​ral.com/​prod​uct/​mykos-​myco​rrhi​zal-​inocul​ant/​)

6.7.7 Piranha
Pirhana is a 100% organic product. Advanced Nutrients’ Piranha maximizes plant root mass by up to
700%. More roots guarantee that plants get the most nourishment, blooming, and harvest. It contains
a unique blend of ecto-​and endomycorrhizae to optimize the potential of vegetation.

• Helps roots to develop surface mass for maximum plant growth

• Increases the creation of essential oils
• Increases the production of plant aromatics
• Beneficial fungus increases root absorption of oxygen, nutrients, and water
• Increases the survival rate of clones and seedlings.
• (www.planet​natu​ral.com/​prod​uct/​liq​uid-​pira​nha/​)

6.7.8 Plant Success (3-​1-​2)

This product improves soil and roots. Plant Prosperity Granular includes a variety of beneficial
fungi (11 mycorrhizae and two Trichoderma species) that are well adapted to a wide range of soils,
climates, and plants. Kelp meal, humus, vitamins, and amino acids are also used to increase mycor-
rhizal germination and efficacy.

• Contains both endo-​and ectomycorrhizae

• Generates strong root systems and healthy plants
• Increases water and nutrient absorption
• Granular recipe is simple to use
• Contains a high-​quality nutrition packet
• (www.planet​natu​ral.com/​prod​uct/​plant-​succ​ess-​granu​lar/​)

6.7.9 Root Maximizer
Beneficial fungi may work their magic in any growing environment. Clonex Root Maximizer
enables an endo-​mycorrhizae network to promote optimal plant health in soil or indoor systems
such as hydroponic or aeroponic setups.

• Mycorrhizal fungi, beneficial bacteria, and Trichoderma increase nutrition and water
absorption
• Increases plant root surface area
• Protects plants from environmental stress and diseases
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Phosphate-mobilizing Mycorrhizal-based Bio-fertilizers 129

• Can be used dry or in a liquid solution

• Developed by Clonex, a pioneer in plant propagation resources for more than 30 years.
• (www.planet​natu​ral.com/​prod​uct/​clo​nex-​root-​maximi​zer/​)

6.7.10 Root Rally (0-​3-​0)

This product is totally organic. Age Old Root Rally contains endo-​and ectomycorrhizae spores as
well as vitamins, minerals, and nutrients. The inoculants encourage the growth of mycorrhizae in
trees, shrubs, flowers, fruits, and vegetables. When applied to the plant’s root system, this 100%
organic combination reduces transplant shock, promotes root development, and increases water and
nutrient intake. (www.planet​natu​ral.com/​prod​uct/​root-​rally/​)

6.7.11 Rooters
This is a customized combination of eight mycorrhizal fungi chosen for a diverse range of plants,
media, and habitats. Rooters of Earth Juice Mycorrhizae works with the plant’s root system to
increase the surface area of the root mass. It improves most plants’ growth and vigor by increasing
nutrient and water absorption. For use in soil and hydroponic systems. (www.planet​natu​ral.com/​
prod​uct/​root​ers-​myco​rrhi​zae/​)

6.7.12 SubCulture-​M
This product increases root mass significantly to boost growth and yield. Subculture-​M is a mycor-
rhizal root inoculant containing a diverse range of endo-​and ectomycorrhizal fungi that colonize
plant roots. These helpful fungi generate a thin network of fibrous strands that extend the plant’s root
system, increasing the root area and assisting plants in absorbing water and nutrients.
From germination or propagation through harvest, this product can be sued. The quantity of
fertilizer used is reduced while still achieving exceptional growth and enormous harvests. When
combined with Subculture-​B, it creates a diversified microbial population that is favourable to plant
health. (www.planet​natu​ral.com/​prod​uct/​sub​cult​ure-​m-​myco​rrhi​zal-​root-​inocul​ant/​)

6.7.13 White Widow
The mycorrhizal fungus in Humboldt Nutrients’ White Widow can assist plant roots to have better
access to water and nutrients in the soil. When applying this mycorrhizal powder, it is normal to see
huge, bright white roots come out of the bottom of pots barely a week after transplanting. (www.
planet​natu​ral.com/​prod​uct/​white-​widow/​)

6.7.14 ZHO
This is an inoculant for the rhizosphere. Botanicare ZHO is a patented combination of mycorrhizae
and Trichoderma fungus that establishes a natural microbial system in and around plant roots, sig-
nificantly boosting plant and root growth, vigor, and production organically. (www.planet​natu​ral.
com/​prod​uct/​zho-​root-​inocul​ant/​)

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and NPK fertilizer on potato yield and soil fertility. Pure Appl Biol 5(3), 439–​445.
Zhu J, Li M, Whelan M (2018) Phosphorus activators contribute to legacy phosphorus availability in agricul-
tural soils: a review. Sci Total Environ 612, 522–​537.
Zhu Y, Christie P, Laidlaw AS (2001) Uptake of Zn by arbuscular mycorrhizal white clover from Zn-​
contaminated soil. Chemosphere 42, 193–​199.
137

7 Quality Standards for

Production and Marketing
of Biofertilizers
Adeel Ahmad,1* Amir Aziz,2 Muhammad Irfan,3
Mukkram Ali Tahir,2 Faiza Rawish,4 and Noman Younas1
1
Institute of Soil and Environmental Sciences, University of Agriculture,
Faisalabad, Pakistan
2
Department of Soil and Environmental Sciences, College of Agriculture,
University of Sargodha, Sargodha, Pakistan
3
School of Environmental Science and Engineering, Tianjin University,
Tianjin, PR China
4
Superior College of Commerce Sat Sira Chowk, Mandi Bahauddin,
Punjab, Pakistan
* Correspondence: [email protected]

CONTENTS
7.1 Background to Biofertilizers................................................................................................. 137
7.2 Types of Biofertilizers........................................................................................................... 138
7.3 Production Processes for Biofertilizers................................................................................. 138
7.4 Biofertilizer Production Protocols......................................................................................... 141
7.5 Prevention of Contamination................................................................................................. 142
7.6 Ways to Improve Production Systems................................................................................... 143
7.7 Limitations in Marketing....................................................................................................... 143
7.7.1 Edaphic and Environmental Conditions................................................................... 143
7.7.2 Plant-​related Factors................................................................................................. 144
7.7.3 Inoculant-​related Factors.......................................................................................... 145
7.8 Strategies to Improve the Marketing Process........................................................................ 147
7.9 Conclusions and Recommendations...................................................................................... 147
References....................................................................................................................................... 148

7.1 BACKGROUND TO BIOFERTILIZERS
In recent decades, the term “biofertilizer” has been defined in a variety of ways, owing to an
improved understanding of rhizosphere–​microorganism–​plant interactions. Soil nutrient utilization
is improved with the use of biofertilizers such as mycorrhizal fungi and plant growth-​promoting
rhizobacteria (PGPR), which were first proposed by Okon and Labandera-​Gonzalez (1994). Vessey
(2003) describes the term “biofertilizer” as “a substance which contains living microorganisms”.
When biofertilizer is applied to seeds, plant surfaces, or soil, it colonizes the rhizosphere of the plant
or the interior of the plant and promotes growth by increasing the supply or availability of primary
nutrients to the host plant. Biological fertilizer was shortened to biofertilizer, which implies the use

DOI: 10.1201/9781003286233-7 137

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138 Biofertilizers for Sustainable Soil Management

of living organisms. Biofertilizer is “a product that contains living microorganisms that exert direct
or indirect beneficial effects on plant growth and crop yield through different mechanisms”, as later
defined by Fuentes-​Ramirez and Caballero-​Mellado (2005). Biological control of plant pathogens
was included in the expanded definition. But microorganisms that promote plant growth by control-
ling harmful organisms, such as biofungicides, bioinsecticides, bionematocides, or other products
with similar activities favouring plant health, are generally defined as biopesticides, not biofertilizers
(Siddiqui and Mahmood 1999; Vessey 2003).
A new term has been proposed to describe a microorganism’s primary mechanism of action in
promoting plant growth: biofertilizers. Rhizoremediators are those that degrade organic pollutants
that can harm plant growth. In contrast, bioenhancers or phytostimulators are those that produce
phytohormones to boost plant growth (Somers et al. 2004; Ali et al. 2018). A species’ primary mech-
anism of action may be used to characterize its classification. However, according to Kloepper (1993)
and Vessey (2003), even a single microorganism in the field will often reveal multiple mechanisms
of action, making further sub-​classification a purely theoretical endeavour.
Descriptions that could lead to incorrect classifications are also a factor in the need for a legal
definition of biofertilizers. Gianinazzi and Vosatka (2004) classified mycorrhizal fungi as “plant
natural parts” rather than microorganisms, which could save time and money by avoiding lengthy
registration processes. However, it also opens the market to fraudulent products.

7.2 TYPES OF BIOFERTILIZERS
Biofertilizers are classified into four categories:

• Nitrogen biofertilizer (N-​BF)

■ Rhizobium –​symbiotic BF with legumes of all varieties
■ Azotobacter –​non-​symbiotic BF for cereals, vegetables, and horticultural crops.
■ Azospirillum –​the associative BF for grains such as millets (blue-​green algae, azolla,
Gluconoacetobacter diazotrophicus is also N-​BF, but is yet to be included in FCO)
• Phosphorus biofertilizers (P-​BF)
■ P-​solubilizes –​PSB (Bacillus, Pseudomonas etc., for all crops)
■ P-​mobilizers –​mycorrhizae (Glomus, Gigaspora etc. for all crops)
• Potash biofertilizers –​K-​BF (microbes like B. mucilaginosus and F. aurantia for all crops).
• Zinc solubilizers –​Z-​BF (Bacillus microbes are capable of zinc solubilization).

If you’re looking for a biofertilizer, you’ll typically find it in either solid form (like peat and other
types of lignite) or liquid form (like broths with polyvinyl pyrrolidone or biosurfactants). Single or
multiple strains of microorganisms can be used to produce biofertilizers, which can also be frozen,
polyacrylamide-​encased, or granular. It has yet to be developed with the best production technology
and packaging.

7.3 PRODUCTION PROCESSES FOR BIOFERTILIZERS

Biofertilizer production involves a wide range of considerations, including the microbes’ growth
profiles, the types and ideal conditions of organisms, and the formulation of inoculum. The formu-
lation of the inoculant, the method of application, and the storage of the product are all crucial to
the success of a biological product. Making biofertilizer involves five major steps. These include the
isolation and selection of target microbes, the selection of method and carrier material, the selection
of propagation method, prototype testing, and large-​scale testing. Figure 7.1 elaborates the steps in
biofertilizer production.
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Quality Standards for Production and Marketing of Biofertilizers 139

FIGURE 7.1 Step-​by-​step production process for biofertilizers.

• Production flow
1. The materials can be composted and fermented. Composting and fermentation are required
steps in the production of a quality organic fertilizer.
2. Crushing and blending. Hammer crusher, chain crusher, and semi-​wet material crusher are
some of the machines used for crushing. Single-​shaft continuous mixer, double-​shaft hori-
zontal mixer, and so on are some of the mixing machines available.
3. Organic fertilizer production relies heavily on the granulating process. Today, the granula-
tion industry employs various techniques such as double-​roller extrusion granulation, disc,
and rotary drum.
4. Granulating produces a large number of particles, but the strength and water content of
these particles are lower than expected. This is why drying is so important.
5. A conveyor belt transfers the materials from the drying machine to the cooling machine,
where they cool. Using this machine, the particle strengths can be improved even further,
and the water content can be reduced.
6. After cooling, a powdery substance is left behind. The fine powders and large particles can
be screened out by this machine. The fine powder is then re-​involved in the granulation
process.
7. Afterwards, a protective film is applied to the finished products, insulating the dust particles
from the ambient air.
8. Packaging. The automatic packing machine takes care of the packaging of qualified
products.

When compared to chemical fertilizers, the production of biofertilizers is more cost-​effective and
simpler. However, the production of biofertilizers must take into account a number of factors,
including field applications, microbial strains, formulation type, and carrier materials (Malusa et al.
2012; Mohammadi & Sohrabi 2012; Arif et al. 2015). Standardizing the commercial production of
biofertilizers necessitates the consideration of six key steps (Figure 7.2). In the first step, microbes that
may be beneficial to plant growth are isolated, identified, and functionally characterized. Common
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140 Biofertilizers for Sustainable Soil Management

FIGURE 7.2 Standardization process for commercial biofertilizer production. In order to produce biofertilizer,
microbes must be present in the rhizosphere and plant tissues, as well as the surrounding environment.
A fermenter is used to cultivate and multiply candidate microbes in the presence of optimal growth factors.
After that, the effectiveness of the product is put to the test in the field. Biofertilizers need to be tested for
quality at each stage of standardization and formulated with appropriate carriers in order to produce a high-​
quality product.

sources of microbial strains are the plant tissues (flowers, leaves, stems, roots, and seeds), bulk soil,
or rhizosphere (Thomas & Singh 2019; Gupta et al. 2014). Microbial strains are characterized using
a variety of laboratory techniques, such as qualitative testing and differential culture media. Because
functional characteristics can be checked quickly and cheaply, they are an inoculant’s main way to
make sure it’s good.
When selecting a biofertilizer, it’s important to keep in mind how it will be used in the field, such
as for nitrogen fixation, nutrient solubilization, or phytohormone production. In vitro testing: selected
strains are then subjected to additional processes (Figure 7.2), including growth on specific media and
potency quantification (Deaker et al. 2011). In greenhouse experiments, the strains are also tested as
part of the selection process before going into the field for field trials and applications (Bhattacharjee
& Dey 2014; Murphy et al. 2018). Analysis of plant growth-​promoting mechanisms and pathways
is also part of this step. Genomic, metabolomics, proteomic, and transcriptomic techniques, along
with culture-​based methods, have made it possible to compare the ability of inoculant strains to pro-
mote plant growth (Bilqees et al. 2019; Krishnamoorthy et al. 2020; Luziatelli et al. 2020; Kumari &
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Quality Standards for Production and Marketing of Biofertilizers 141

Thakur 2018; Terra et al. 2020). Using this information, biofertilizer formulations can be developed
that can thrive in a wide range of agricultural ecosystems. Molybdenum, for example, could be used
to make N-​fixing inoculants if the soil doesn’t have enough of it (Deaker et al. 2011).
In the third step, to determine the final product form, it is crucial to select the right formulation
materials, which include liquid or carrier-​based, such as granules, powder, or slurry. The carrier is
essential in order to keep the microbes in a viable state and in the proper quantity. Propagation methods
must be chosen for the cultivation and multiplication of the selected strains in a laboratory under
ideal conditions to preserve their inherent properties for effective field performance in the fourth step.
Monitoring the microbial growth profile under various conditions helps to determine the best conditions
for microbial growth. Typically, strains are multiplied using a conventional system of fermenters
(Suthar et al. 2017; Khan et al. 2016). For biofertilizer production, solid-​state and submerged fermen-
tation are two of the most common methods. The fifth step is prototyping, which entails testing a var-
iety of different products. This step ensures that the most efficient product is chosen for use in the field.
Before setting up a standard commercial production process (Figure 7.2), the products must be tested
in the field on a large scale to find out how well and how well they work in different ecological areas
and conditions (step six) (Mohammadi & Sohrabi 2012; Bhattacharjee & Dey 2014).
Because AM fungi are obligate symbionts, their recovery and multiplication strategies differ
from those of other microbes, such as bacteria and fungi. Using monogenic culture, AM fungi can
be multiplied in a controlled environment where they are grown on a host plant (Parnell et al.
2016). A pot-​house is used for commercial production because of the controlled conditions. This
method can be used to monitor the spores and mycelia of inoculum attached to the roots of host
plants. Obtaining high levels of synergy between the host organism and the AM fungi inoculum
requires careful attention to soil nutrient conditions. There should be good affinity for AM fungi
colonization and rapid growth of the host plant with significant root development (Treseder 2013).
A contaminant-​free biofertilizer can be made in an in vitro monogenic culture (Mukhongo et al.
2016) if certain rules are followed.

7.4 BIOFERTILIZER PRODUCTION PROTOCOLS

a. Safety measures
• No smoking
• No eating
• No drinking
• Good washing facilities
• Good disposal facilities
• Responsibilities

The laboratory supervisor/​manager must ensure that:

1. The lab is a safe place to work.

2. All of the equipment is in working order.
3. It is important that all technicians be aware of the risks.
4. All technicians are properly trained to carry out their duties.
5. Be responsible for chemicals.

Technicians are responsible for:

1. Maintaining the cleanliness of the benches.

2. Keep the floors neat and tidy.
3. Glassware cleaning and proper storage.
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142 Biofertilizers for Sustainable Soil Management

4. Chemicals should be stored in the proper locations (cupboards, stores).

5. The upkeep of machinery.
6. Notifying the supervisor of any problems or potential problems.

b. Laboratory requirements
A microbiology lab must have the following items as a bare minimum for effective operation:

• Equipment
1. Low, high, and oil-​immersion lenses, as well as micrometres and haemocytometres, are
included in a compound biological microscope for microscopic examination, sizing, and
counting of vegetative and spore/​budding/​cyst specimens
2. An autoclave is used to sterilize media,
3. Hot-​air oven for glassware sterilization.
4. Incubator for cell growth (temperature range of 25–​40°C).
5. Colony counter (cfu) for counting colonies in an agar plate.
6. Agar media melting by using a water bath.
7. Refrigerator in order to store strains.
8. pH meter for measurement of media acidity and alkalinity.
9. Weighing balance for weighing of media ingredients.
10. Bunsen burner/​spirit lamp.

• Glassware and other necessary materials

1. Petri dishes for storing molten media.
2. Conical flasks ranging in size from 50 ml to 1000 ml.
3. Culture tubes with caps or test tubes without rims (Biju bottles).
4. Funnels for pouring media.
5. Pipettes of various sizes, from capillary to 10 ml.
6. Cylinders to take measurements.
7. 50–​1000 ml beakers.
8. Glass slides and cover slips.
9. Bent glass rods (spreaders).
10. Needles and loops for inoculation.
11. Dropping stain and reagent bottles.
12. Saucepans for boiling media.
13. Wire baskets for Petri dishes and tubes.
14. Filtering papers.
15. Wax markers and pencils.
16. Steel pipette and Petri dish storage container.
17. Cheese cloth and cotton wool.
18. Forceps and a scalpel.
19. Disinfectant-​filled bottles.
20. Cleansing agent in powder form.
21. Necessary chemicals.

7.5 PREVENTION OF CONTAMINATION
In agricultural systems, the use of biofertilizers instead of chemical fertilizers holds great
promise. However, technical reliability and accessibility for farmers are two major challenges
that must be overcome. Aside from the potential impact on human and ecosystem health, it
is important to consider the long-​term consequences. For these reasons, we believe that all
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Quality Standards for Production and Marketing of Biofertilizers 143

inoculant research and development should be evaluated using a “One Health” approach that
considers the health of plants, animals, people, and ecosystems as a whole (Van Bruggen et al.
2019). Because we don’t yet have a complete picture of how a product interacts with the soil
and other microorganisms, we can’t yet determine how dangerous it is. A variety of inoculants
have been studied in an attempt to close knowledge gaps and develop guidelines for evaluating
the risks associated with each one. An accurate assessment of the risks posed by the large-​scale
introduction of microorganisms into agricultural systems depends on an understanding of the
mechanisms underlying successful PGP.

7.6 WAYS TO IMPROVE PRODUCTION SYSTEMS

In addition to selecting specific microorganisms or functions, the success of biofertilizers is
dependent on developing new formulations to ensure the survival of inoculated strains. In this chapter,
we reviewed a variety of techniques for improving bioformulations, including the use of biofilm-​
producing strains, microencapsulation with alginate, and fluidized bed dryer (FBD) processes. In an
ideal world, new technologies would focus on carriers and additives that are both inexpensive and
simple to use, but also capable of storing and applying a greater number of viable cells. In addition,
a “One Health” approach should be used to evaluate the biosafety of microbes that are inoculated.
In order to ensure the safety of the product, proper screening tests (e.g., toxicity and pathogen-
icity testing) must be carried out. Research into ecological interactions and how plants shape their
microbiomes in agricultural systems is still vital. Soil microorganisms play a critical role in many
biogeochemical processes, and these processes could be negatively impacted by climate change.
Due to the genetic diversity and complexity of soil and plant microorganisms, not all fields will
benefit from a single formulation. However, designing biofertilizers for every single field is impos-
sible and unrealistic. Thus, we agree with Bell et al. (2019) that the design, refinement, and valid-
ation of microbial products should be oriented toward optimal and suboptimal environmental ranges
(i.e., agricultural practices, crop, climate, and soil type). Public and private funding are essential to
filling critical knowledge gaps in this field. This effort needs to be supported by regulatory agencies
and policymakers who support biofertilizers and sustainable practices.

7.7 LIMITATIONS IN MARKETING
Before they can have the desired effect on plant growth and fitness, introduced microbes have to deal
with a number of big issues (Figure 7.3), like how they interact with plants, how long they last, and
how symbiotic or parasitic they are (e.g., PGP performance, symbiotic/​parasitic behaviour). During
the lab-​to-​field transition, a microbial strain that performs well in the lab may perform poorly in
greenhouse and field trials (Uddin et al. 2016; Keswani et al. 2019; Parnell et al. 2016). It is diffi-
cult to predict the outcome of an inoculation because we only take into account and control for a
small number of variables, often without taking into account the complex interactions between them
(Busby et al. 2017; Moutia et al. 2010; Sasaki et al. 2010). Plant-​related factors (e.g., concentration,
viability) and edaphic/​environmental factors (e.g., additives) are among the most important factors
influencing inoculation success (Malusà et al. 2016).

7.7.1 Edaphic and Environmental Conditions

Both edaphic and environmental factors play a significant role in the variability and low reproduci-
bility of biofertilizer results in field trials. Initially, biofertilizer testing is done in an aseptic
environment, which allows for an unbiased evaluation of the microorganism under study. There
are many uncontrolled biotic and abiotic factors that can interfere with inoculation success when
testing in growth chambers, greenhouses, and even in the field (Bacilio et al. 2017; Nkebiwe et al.
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144 Biofertilizers for Sustainable Soil Management

FIGURE 7.3 Constraints in successful utilization of biofertilizers.

2016). An important consideration in inoculation outcomes is whether or not there are any antagon-
istic microorganisms in the resident microbiome that can act as competitors, predators, or other
antagonists. Climate and edaphic factors can also affect the efficiency and yield of biofertilization on
crop nutrient use (Al-​Zahrani et al. 2022; Rajesh et al. 2022; Anam et al. 2021; Deepranjan et al. 2021;
Haider et al. 2021; Amjad et al. 2021; Sajjad et al. 2021a,b; Fakhre et al. 2021; Khatun et al. 2021;
Ibrar et al. 2021; Bukhari et al. 2021; Haoliang et al. 2021; Sana et al. 2022; Abid et al. 2021; Zaman
et al. 2021; Rehana et al. 2021; Yang et al. 2022; Ahmad et al. 2022; Shah et al. 2022). Schütz et al.
(2018) conducted a meta-​analysis and found that biofertilizers perform better in dry climates, with
high levels of soil P (especially for N2-​fixers), and, for AMF, lower soil organic matter content and
neutral or slightly acidic pH. Higher growth and yield responses to biofertilization can be expected
when the plant is able to fully benefit from the interaction with the introduced microbe. According
to a number of studies, there is a negative correlation between biofertilization and soil nutrient con-
tent, both for AMF colonization (Jansa et al. 2009a,b; Kaeppler et al. 2000; Ullah et al. 2016) and
rhizobia nodulation with soil N.
Additional inoculation success variability can be caused by edaphic properties that are par-
ticularly sensitive to agricultural practices. Inoculation of microbes with chemical fertilizers,
organic amendments, pesticides, and tillage have all been shown to interact so far (Da Costa
et al. 2014; Ozturk et al. 2003; Jansa et al. 2009a,b), but there is still more research to be done
(Manandhar et al. 2017; Mulas et al. 2015; Miller et al. 1995; Muhammad et al. 2022; Wiqar et al.
2022; Farhat et al. 2022; Niaz et al. 2022; Ihsan et al. 2022; Chao et al. 2022, Qin et al. 2022;
Xue et al. 2022; Ali et al. 2022; Mehmood et al. 2022; El Sabagh et al. 2022; Ibad et al. 2022).
Many different things can affect how well biofertilization works. Crop rotations and cover crops, for
example, can change the microbial communities in the soil (Buysens et al. 2016).

7.7.2 Plant-​related Factors
The results of biofertilization can vary depending on the type of crop that is being grown. Genetic
markers (such as quantitative trait loci or QTLs) have been identified in some studies, but the
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Quality Standards for Production and Marketing of Biofertilizers 145

underlying plant factors have not yet been fully understood (Remans et al. 2008; Deepranjan et al.
2021; Haider et al. 2021; Huang Li et al. 2021; Ikram et al. 2021; Jabborova et al. 2021; Khadim
et al. 2021a,b; Manzer et al. 2021; Muzammal et al. 2021; Abdul et al. 2021a,b; Ashfaq et al. 2021;
Amjad et al. 2021; Atif et al. 2021; Athar et al. 2021; Adnan et al. 2018a,b; Adnan et al. 2019;
Akram et al. 2018a,b; Aziz et al. 2017a,b; Chang et al. 2021; Chen et al. 2021; Emre et al. 2021).
Through rhizodeposits and root architecture changes, plants are widely accepted to have an impact
on the rhizosphere environment (Saleem et al. 2018; Somers et al. 2004; Habib et al. 2017; Hafiz
et al. 2016; Hafiz et al. 2019; Ghulam et al. 2021; Guofu et al. 2021; Hafeez et al. 2021; Khan et al.
2021; Kamaran et al. 2017; Muhmmad et al. 2019; Safi et al. 2021; Sajjad et al. 2019; Saud et al.
2013; Saud et al. 2014; Saud et al. 2017; Saud et al. 2016; Shah et al. 2013; Saud et al. 2020; Saud
et al. 2022a,b; Qamar et al. 2017; Hamza et al. 2021; Irfan et al. 2021;Wajid et al. 2017; Yang et al.
2017; Zahida et al. 2017; Depeng et al. 2018; Hussain et al. 2020; Hafiz et al. 2020 a,b). Plant roots
secrete secondary metabolites, such as antibiotics, flavonoids, and hormones, which are recognized
and interacted with by PGP microbes in their numerous rhizodeposits (Bais et al. 2004). Rhizobia–​
legume symbioses, in which the compatibility of microorganisms with host plants is critical to col-
onization success, can have a high degree of specificity in this signalling process (Thilakarathna and
Raizada 2017; Hirsch et al. 2003). Arbuscular mycorrhiza may not require as much host specificity
as previously thought (Koyama et al. 2017), but the genotype of the plant can still influence colon-
ization and PGP (Hoeksema et al. 2010; Yao et al. 2001; Linderman and Davis 2004). AMF–​host
plant compatibility has been explained by differences in root architecture, aerial architecture, P
utilization, and uptake efficiency (Declerck et al. 1995; Kaeppler et al. 2000; Yao et al. 2001). Also,
PGPRs such as Azotobacter sp. and Pseudomonas sp. were found to have a high degree of specificity
for plant genotypes, as were endophytes (Anbi et al. 2020; Yoon et al. 2016; Vujanovic and Germida
2017; Mezei et al. 1997). Most plant breeding programmes do not take into account interactions
between plants and microbes, resulting in a wide range of biofertilization outcomes. Because of
this, the impact of genotypes isn’t just based on how old and healthy the plants are, but also how the
environment is and when the plants were inoculated (Dennis et al. 2010; Shafi et al. 2020; Wahid
et al. 2020; Subhan et al. 2020; Zafar-​ul-​Hye et al. 2020a,b; Zafar et al. 2021; Adnan et al. 2020;
Ilyas et al. 2020; Saleem et al. 2020a,b,c; Rehman 2020; Farhat et al. 2020; Wu et al. 2020; Mubeen
et al. 2020; Farhana 2020; Jan et al. 2020; Wu et al. 2019; Ahmad et al. 2019; Baseer et al. 2019;
Hafiz et al. 2018; Tariq et al. 2018).

7.7.3 Inoculant-​related Factors
Selecting a microbial genotype that is compatible with the plant host genotype is just as important
as determining PGP functions (Vargas et al. 2012; Ehinger et al. 2014; Linderman and Davis 2004).
Among the many processes influenced by bacterial characteristics are inoculation, establishment,
colonization, and persistence. Genetic and PGP traits, rather than ecological traits, are typically the
focus of inoculant development. However, inoculation success is ultimately determined by eco-
logical traits (Hart et al. 2018; Kaminsky et al. 2019). Osmotic tolerance and psychotolerance, for
example, are two traits that can be used to select strains that are better suited to the environment
in which they are grown (Garca et al. 2017; Rawat et al. 2019). Another option is to isolate native
strains that have been proven to improve biofertilization performance (Ahmed et al. 2013; Maltz and
Treseder 2015; Melchiorre et al. 2011). When developing an inoculant, there is a trade-​off between
the ability to establish and survive as well as the PGP traits (Kaminsky et al. 2019; Parnell et al.
2016). An important question arises as a result of this trade-​off: should we pursue highly targeted
biofertilizers or broad-​spectrum versatile ones, given the high degree of microbial strain specificity
to environment and genotype (Tosi et al. 2020; Parnell et al. 2016; Bell et al. 2019)? A mixed inocu-
lant with a wide range of ecological adaptability would be the preferred method of achieving this
goal (i.e., functionally redundant strains that encompass a wider range of environmental adaptation).
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146 Biofertilizers for Sustainable Soil Management

FIGURE 7.4 Factors governing the efficiency of biofertilizers.

If a mixed inoculant interferes with other parts of the inoculant, there is a lot of room for this
inoculant to work, even if there are some challenges (Ballesteros-​Almanza et al. 2010; Xavier
and Germida 2003; Remans et al. 2008). Major factors limiting the efficiency of biofertilizers are
described in Figure 7.4.
For the microbes to be able to withstand and protect themselves from environmental constraints,
it is important to know how the biofertilizer is formulated and delivered. For a plant to respond
effectively, the inoculant formulation must support microbial growth while protecting a sufficient
number of viable cells (Bashan et al. 2014; Herrmann and Lesueur 2013). It’s not uncommon for
the strain to undergo genetic or physiological changes as a result of scaling up and commercial-
ization, and there’s also the risk of viability loss (especially due to desiccation) and contamination
(Greffe and Michiels 2020; Parnell et al. 2016; Glick 2020). There has been an increase in the
variety of biofertilizer formulations as a result of the growth of the industry, including the devel-
opment of new carriers (solids, liquids, and slurries), additives (such as nutrients and stimulants),
and preservatives (such as adhesives and surfactants) to improve the inoculants’ physical prop-
erties (Preininger et al. 2018; Bashan et al. 2014). In the distribution, storage, and application of
microbes, these additives allow them to withstand the fluctuating and suboptimal conditions. Some
think that adding biofertilizer carriers and other additives is a last-​minute thing, but they can be very
important for obtaining good results (Lee et al. 2016; Gomez et al. 1997; Herrmann and Lesueur
2013; Gopakumar et al. 2020; Zia-​ur-​Rehman 2020; Ayman et al. 2020; Mohammad I. Al-​Wabel
et al. 2020a,b; Senol 2020; Amjad et al. 2020; Ibrar et al. 2020; Sajid et al. 2020; Muhammad et al.
2021; Sidra et al. 2021; Zahir et al. 2021; Sahrish et al. 2022).
Similar to delivery methods (such as seed or soil), timing and frequency of application can be
crucial for inoculation success (Parnell et al. 2016). It has been found that soil applications of a
granular Rhizobium inoculant on Pisum sativa resulted in higher PGP than seed applications of a
liquid formulation inoculant (Clayton et al. 2004). Endophytic and phyllosphere microorganisms
could also be delivered via foliar and flower applications, which were considered safer and more
effective (Preininger et al. 2018; Mitter et al. 2016; Iqra et al. 2020; Akbar et al. 2020; Mahar et al.
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Quality Standards for Production and Marketing of Biofertilizers 147

2020; Noor et al. 2020; Bayram et al. 2020; Amanullah, Fahad S 2018a,b; Amanullah, Fahad S 2017;
Amanullah et al. 2020; Amanullah et al. 2021; Rashid et al. 2020; Arif et al. 2020; Amir et al. 2020;
Saman et al. 2020; Muhammad Tahir et al. 2020; Md Jakirand Allah 2020; Mahmood et al. 2021;
Farah et al. 2020; Sadam et al. 2020; Unsar et al. 2020; Fazli et al. 2020; Md. Enamul et al. 2020).
Consideration should also be given to other agricultural practices, such as fertilization and trans-
plant (Pii et al. 2019; Fahad and Bano 2012; Fahad et al. 2017; Fahad et al. 2013; Fahad et al.
2014a,b; Fahad et al. 2016a,b,c,d; Fahad et al. 2015a,b; Fahad et al. 2018a,b; Fahad et al. 2019a,b;
Fahad et al. 2020; Fahad et al. 2021a,b,c,d,e,f; Fahad et al. 2022a,b; Hesham and Fahad 2020; Sajjad
et al. 2021a,b), timing and frequency of applications, as well as plant growth stages (Sohn et al.
2003; Bashan 1986; Fallik et al. 1988; Linderman & Davis 2004). After the microbe is applied, it
needs time to establish itself before it can provide any benefit to a plant. This time period is critical.

7.8 STRATEGIES TO IMPROVE THE MARKETING PROCESS

On the other hand, there are a number of practical considerations inherent in biofertilizer develop-
ment that should not be overlooked in light of the technical constraints we’ve already discussed in
this chapter. Farmers and manufacturers alike have expressed concerns about the ease of use and
accessibility of inoculants in comparison to chemical fertilizers. Accessibility to biofertilizers is
influenced by a number of factors, including price/​benefit ratios, versatility, robustness and reprodu-
cibility, shelf life and storage requirements, user-​friendliness (in terms of handling and application),
adaptability to agricultural practices and machinery, and biosecurity (Bell et al. 2019; Bashan et al.
2014; Parnell et al. 2016). If biofertilizers are to have a long-​term positive impact on the environment,
farmers need to be educated about the long-​term benefits of using biofertilizers and encouraged to
use them. More rarely discussed are intellectual property rights and patent development, which may
help transfer technology between the academic and industrial sectors, but may also limit researchers’
ability to freely share ideas and materials (Glick 2020). Proper regulations, including standard and
universal testing protocols and guidelines for risk evaluation, are also required for the commercial-
ization of new products (Timmusk et al. 2017; Sessitsch et al. 2019; Kaminsky et al. 2019). For the
most part, there aren’t any standard procedures for evaluating how well microbes are inoculated and
how to monitor them after they’ve been used in the field.

7.9 CONCLUSIONS AND RECOMMENDATIONS

Solubilization of nutrients, increased absorption of nutrients, or decomposition of organic residues
are some of the ways in which biofertilizers can increase crop productivity. Reducing chemical fer-
tilizer use by substituting with biofertilizers will save money while also reducing pollution caused
by excessive chemical fertilizer use. A low-​input farming system can be achieved with the use of
biological and organic fertilizers. This helps ensure the long-​term sustainability of farms.
Sustainable agriculture relies heavily on the use of biofertilizers. Biofertilizers are still being
produced in small quantities, with little public awareness of their existence, and few people are using
them. To combat food insecurity and soil depletion, biofertilizers should be used more frequently.
An effective extension programme, public–​private partnerships, and adequate training can help
increase the use of biofertilizers. Cooperative efforts between government agencies and biofertilizer
technology companies should be encouraged. These strategies will help high-​quality biofertilizers
to be manufactured that are widely used and valued.
In addition, research should focus on how to increase the efficiency of biofertilizers in the field.
These new findings could help us better understand how beneficial microbes promote plant growth,
as well as how to better utilize soil and plant microbiomes in general. Plant-​associated microbes
have been established as having key roles in the growth and development of plants. As a result,
improving plant productivity and health by modifying these functions is clearly an area of research
148

148 Biofertilizers for Sustainable Soil Management

that merits further investigation. If researchers want to make new biofertilizers, they need to find
and make inoculant strains that can work well in a wide range of field conditions and with a wide
range of plant species.
Developing agencies should work together to define the quality of biofertilizers and develop spe-
cific standards for different biofertilizers. Biofertilizers and their quality testing methods should also
be re-​evaluated in the light of changing market demands and feedback from farmers. Development
agencies should take the initiative to train various production units, state government extension staff,
and farmers about quality control in biofertilizers.

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8 Limitations of Using
Biofertilizers as an Alternative
to Chemical Fertilizers
Muhammad Haroon,1 Fazli Wahid,1 Rafi Ullah,1
Muhammad Adnan,*1 Mukhtar Alam,1 Hidayat Ullah,1
Muhammad Saeed,1 Muhammad Saeed,2 Shah Fahad,3*
Muhammad Romman,4 Nazeer Ahmed,1 Taufiq Nawaz,5
Anas Iqbal,6 Zia Ur Rehman,1 Ayman El Sabagh,7
Shah Saud,8 and Shah Hassan9
1
Department of Agriculture, University of Swabi, Swabi, Pakistan
2
Department of Weed Science and botany, University of Agriculture,
Peshawar, Pakistan
3
Department of Agriculture, Abdul Wali Khan University Mardan, Khyber
Pakhtunkhwa, Pakistan
4
Department of Botany, University of Chitral, Khyber Pakhtunkhwa,
Pakistan
5
Department of Food Science and Technology, The University of
Agriculture, Peshawar, Pakistan
6
College of Agriculture, Guangxi University, Nanning, China
7
Department of Agronomy, Faculty of Agriculture, University of
Kafrelsheikh, Kafr El-​Sheikh, Egypt
8
College of Life Science, Linyi University, Linyi, Shandong, China
9
Department of Agricultural Extension Education and Communication,
The University of Agriculture, Peshawar, Pakistan
* Correspondence: [email protected]; shah_​[email protected]

CONTENTS
8.1 Introduction........................................................................................................................... 164
8.2 Advantages of Biofertilizers.................................................................................................. 164
8.3 Sources of Biofertilizers........................................................................................................ 165
8.3.1 Nitrogen-​based Biofertilizer..................................................................................... 165
8.3.2 Phosphorus-​based Biofertilizer................................................................................ 165
8.3.3 Compost-​based Biofertilizer..................................................................................... 165
8.4 Crop Response to Biofertilizer.............................................................................................. 165
8.5 Response of Soil Fertility to Biofertilizer............................................................................. 166
8.6 Limitations of Biofertilizers.................................................................................................. 166
8.6.1 Agri-​climatic Conditions.......................................................................................... 166

DOI: 10.1201/9781003286233-8 163

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164 Biofertilizers for Sustainable Soil Management

8.6.2 Inconsistent Nature................................................................................................... 167

8.6.3 Formulation and Storage Problems.......................................................................... 167
8.6.4 Nutrient Availability Constraints.............................................................................. 167
8.6.5 Extension and Market Constraints............................................................................ 167
8.6.6 Farmer Awareness..................................................................................................... 168
8.7 Future Prospects for Biofertilizers........................................................................................ 168
References....................................................................................................................................... 168

8.1 INTRODUCTION
The agriculture sector plays an important role in improving the standard of living in a country.
Hence, it is necessary to ensure food security and access for the global population. The agriculture
sector has experienced a massive change (Ajmal et al. 2016). Today, the agriculture sector not only
improves food supply but also improves the living standard of all. To fulfil this demand, farmers
have used an excessive amount of agrochemicals to enhance crop growth and productivity (Aktar
et al. 2009; Santos et al. 2012). Agrochemicals are synthetic products that contain essential elem-
ents, e.g. nitrogen, potassium, and phosphorus. Nowadays, to increase soil fertility, farmers mostly
use synthetic fertilizers, which cause numerous environmental issues, e.g. health problems, air, soil,
and water pollution, climate change, loss of beneficial biodiversity, etc. In long term, they may
destroy soil fertility (Bhardwaj et al. 2014; Khan et al. 2016). According to Chun-​Li (2014), agro-
chemical applications increase soil acidity and result in environmental pollution, with plants also
becoming susceptible to many diseases (Khosro and Yousef 2012). Researchers have developed a
new approach in the formulation of biofertilizers, which keep the soil rich in all nutrients and protect
the soil from pollution.
Biofertilizers are the most essential part of integrated nutrients management that play a vital
role in productivity and soil sustainability when added to soil. Biofertilizers do not supply nutrients
directly to the plant, they use strains of efficient beneficial microbes added to soil, seeds, and com-
post to accelerate the process and to enhance nutrient availability to plants that help in plant growth
(Yadav and Sarkar 2019). Biofertilizers include nitrogen-​fixing bacteria (Rhizobium, Azospirillum,
cyanobacteria, Clostridium, azotobacter, and Bacillus polymyxin), phosphate-​solubilizing bacteria
(Aspergillus, Bacillus, Fusarium, etc.), potassium-​solubilizing bacteria (Bacillus edaphicus, Bacillus
mucilagenosus), sulphur-​solubilizing bacteria (chemolithotrophs, Thiobacillus denitrificans, etc.)
and arbuscular mycorrhiza (Gautam et al. 2021). These microbes are used in different combinations
for the preparation of biofertilizer formulations for enhancing crop productivity. Biofertilizers are
an eco-​friendly approach that help to enhance soil fertility, sustainability, and plant growth and
productivity (Mohapatra et al. 2013). The above-​mentioned aspects aid farmer incomes through
a noticeable reduction in the cost of agrochemicals (Fundases 2005). Biofertilizer application can
be a feasible option with many social, economic, and environmental benefits (Carvajal-​Munoz
and Carmona-​García 2012). However, biofertilizer implementation needs more practice, studies
of environmental variables, assets, and time in research (Vanegas 2003; Fresco 2003). In order to
achieve sustainability, it is necessary to follow an implementation plan, business plan, project plan,
and production plan, and to implement sustainable technology toward the minimization of environ-
mental impacts and improved subsequent profits for growers.

8.2 ADVANTAGES OF BIOFERTILIZERS
The advantages of biofertilizer use include the following:

1. Enhances crop yield and soil health.

2. Safe to the environment and health.
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Limitations of Using Biofertilizers 165

3. Provides basic nutrients, e.g. NPK to plants.

4. Maintains soil’s natural fertility and microbial biodiversity.
5. Helps to control plant and soil pathogens.
6. Helps to increase resistance against harsh environmental conditions.
7. Easy to apply, cost-​effective, and affordable to farmers.
8. Increases plant growth and productivity.

8.3 SOURCES OF BIOFERTILIZERS
8.3.1 Nitrogen-​based Biofertilizer
Nitrogen is an important crop nutrient. However, it is deficient in most soils. To meet crop demand,
nitrogen-​based biofertilizer is a new approach that is used worldwide. It contains beneficial microbes
such as Azotobactor, Azospirillum, Actinobacteria, and Rhizobium (El-​Sirafi et al. 2005). It helps to
lower the requirement for nitrogen fertilizer by up to 35% (Favilli et al. 1987).

8.3.2 Phosphorus-​based Biofertilizer
Phosphorus is an important plant growth-​limiting factor. Its deficiency may disturb plant growth
and other biological process (Azziz et al. 2012; Tak et al. 2012; Ilyas et al. 2016). Phosphorus-​
solubilizing microbes help to improve the soil nutrition status and also increase its availability
to plants through biological processes. Phosphorus biofertilizer is a possible way to increase the
availability of phosphorus to plants. In an ecosystem many beneficial microbes are involved in
releasing phosphorus, these are called phosphorus-​solubilizing microorganisms (Bhattacharyya and
Jha 2012). Bacteria and fungi have been found to be effective in phosphorus solubilization, with
bacteria contributing up to 50%; while the fungal potential is very low, i.e. 0.1 to 0.5% (Alam et al.
2002; Chen et al. 2006). Phosphate-​solubilizing bacteria are a group of plant growth promotors
reported in many crops including rice, tomato, etc. (Rodriguez and Fraga 1999; Hafeez et al. 2004;
Hilda and Fraga 2000).

8.3.3 Compost-​based Biofertilizer
Compost biofertilizers are those containing different microorganisms that have the potential to con-
vert organic waste material from unavailable to available forms to enrich soil with important elem-
ents through biological activities (Vessey 2003; Ebrahimi et al. 2010). The application of compost
improves soil porosity, fertility, cation exchange capacity, water retention capacity, and microbial
activities (Ahmad et al. 2008; Fiorentino and Fagnano 2011).

8.4 CROP RESPONSE TO BIOFERTILIZER

Biofertilizer is a sustainable tool used to enhance soil health and crop yield (Igiehon and Babalola
2017; Bhattacharrya et al. 2020). The incorporation of biofertilizer is a crop management practice
that helps to improve yield-​attributing characteristics and soil fertility. Biofertilizers impart better
health to soil and plants. Azolla is a common biofertilizer used in paddy fields as it is quickly
decomposed in soil and enables efficient nitrogen availability to rice crop. Azospirillum lipoferum
produces chemicals which stimulate plant growth and enhance productivity (Yadav and Gautam
2019). Likewise, Park et al. (2015) reported that PGPR has a significant effect on crop aerial growth,
i.e., number of leaves, plant height, shoot biomass, and stem width. Also, Adiprasetyo et al. (2014)
observed that the application of multi-​microbial biofertilizer was able to increase the growth-​related
parameters of oil palm plants. Yang et al. (2014) detected increased water use efficiency in arbuscular
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166 Biofertilizers for Sustainable Soil Management

mycorrhiza-​treated seedlings. Some beneficial microorganisms (Bacillus megaterium and Frateuria

aurantia) have been reported as effective phosphorus-​and potassium-​mobilizing bacteria in crop
cultivation (Subhashini 2014; Ghaffari et al. 2018). It has also been reported that adding biofertilizer
can efficiently increase Angelica dahurica growth and yield (Doan and Cao 2020).

8.5 RESPONSE OF SOIL FERTILITY TO BIOFERTILIZER

Soil fertility is an important function of soil that provides the essential nutrients to soil. Crop growth
is mainly dependent on soil fertility status. As decreasing of soil fertility occurrs continuously, to
overcome this problem, it is necessary to boost the soil condition and fertility. Among the soil man-
agement practices, biofertilizer is a good approach to manage soil nutrients. Moreover, biofertilizers
are an ecofriendly approach that helps to increase the soil properties, i.e. macro-​and micronutrients,
increase soil pH, ion exchange capacity, and biological properties (Kariada et al. 2003). In addition,
microbial activities help in the availability and recovery of nutrients, hence they increase soil quality
(Upadhyay et al. 2012a; Arafat et al. 2016; Yadav and Sarkar 2019). Biofertilizers have the ability to
improve soil fertility by delaying the nitrification process for a long time.

8.6 LIMITATIONS OF BIOFERTILIZERS
Biofertilizers are nutrient-​based organic fertilizers that are mostly used as a supplement to syn-
thetic fertilizers. However, they need improvement to replace synthetic fertilizers totally. There are
many limitations of biofertilizer use that need studies and research to find a better solution for the
future.

8.6.1 Agri-​climatic Conditions
Many factors affect agriculture worldwide. The use of synthetic fertilizers hardens the soil, decreases
soil productivity, increases salinity, with irregularity in soil nutrients and loss of water-​holding cap-
acity (Savci 2012). To stop the use of synthetic fertilizers, biofertilizers have emerged as alternative
and effective fertilizers. Biofertilzers can be a good option for basic inputs of nutrients for sus-
tainable production with more ecofriendly approaches (Al-​Taey 2018). However, the performances
of biofertilizers are inconsistent due to many factors. Among these factors, the most limiting are
the agro-​environmental conditions (Mathenge et al. 2019). Agroclimatic conditions are the essen-
tial factor that influences the performance of a biofertilizer. Results have been reported that agro-​
climatic conditions greatly affect the soil pH, and reduce phosphorus and the nodulation process
(Rengel 2002). Likewise, the maximum phosphorus availability was reported to be very low in
mineral soil (Wolf 1999). The reaction of phosphate with other elements like iron, calcium, and alu-
minium may reduce its availability to plants (Gyaneshwar et al. 2002; Ali et al. 2013). Although the
application of biofertilizers may increase the input by up to 35% in saline and acidic soil, it is less
effective in neutral and calcareous soil (Hashem 2001). A recent report has shown that may benefi-
cial microbes such as Bacillus, Azotobacter, Enterobacter, and Paenibacillus help in minimizing the
agrochemical toxicity (Shahid et al. 2019).
However, its efficacy depends on whether the strain can survive and also sufficient knowledge
about the soil pH for each type of biofertilizer is needed.
Agrochemicals are widely used in agriculture for managing different pests and disease. The
high use of agrochemicals damages the agroecosystem in terms of soil microbial biodiversity. The
massive use of fertilizers also disturbs microbial activity and biodiversity (Stolte 2016). Likewise
herbicides also cause everlasting changes to the microflora of soil, also affecting soil health and
crop growth by inhibiting the activity of nitrogen-​fixing bacteria (Sachin 2009) and interfering with
ammonification (Reinhardt et al. 2008).
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Limitations of Using Biofertilizers 167

8.6.2 Inconsistent Nature
Over the past few years, significant success has been recorded for biofertilizers which offer a
successful and natural option in the agriculture sector. Biofertilizers are gaining increased awareness
each day with their role in improving plant health, productivity, and soil fertility. However, they are
not widely accepted due to difficulty in reproducing their useful outcomes on natural ecosystem
swhere there are variations in the environmental conditions.
Extreme environmental conditions often cause a loss of their effectiveness. Highly alkaline and
acidic soils also affect the growth of useful microbes, which has a great influence if the soil contains
excessive natural enemies. It also affects its efficiency in soil, which depends on the particular strain
availability, growing medium, and specific environment.

8.6.3 Formulation and Storage Problems

Formulation is an important step in maintaining microorganism viability, to provide a long shelf life
and ensure that microorganisms establish themselves (Soumare et al. 2019). Formulation is a major
step that provides optimal conditions to enhance the activity and persistence of microorganisms in
soil to obtain maximum benefits for the host plants (McQuilken et al. 1998). A good bioformulation
should be non-​toxic, inexpensive, easily available, with good adhesion to seeds and have a good pH
to be recommended as a carriers (Hassan and Bano 2016). A short shelf life and inadequate formula-
tion discourage sellers and farmers because they cannot store the product for a long time. A problem
for the production of biofertilizers is the low demand in the market and slow adoption rate. Thus it
is important to improve the formulation and shelf life of locally formulated biofertilizers in various
condition to ensure their significance, activity, persistence, and product viability over a period time.

8.6.4 Nutrient Availability Constraints

Plants are continuously exposed to many microorganisms and interact with plants differently (Shah
et al. 2017; Glick and Gamalaro 2021). These plant–​microbe interactions are important and have
been used to improve crop yield and production. These activities help to improve soil physical prop-
erties, structure, access to nutrients, and resistance against pathogens (Yadav et al. 2011). Beneficial
bacteria provide important nutrients to improve crop growth (Basu et al. 2021). Any nutrient defi-
ciencies may lead to malfunction and growth imbalance in plant growth. Kumar et al. (2019) reported
that zinc deficiency in wheat crop causes yellowing of leaves and stunted growth. Many beneficial
microbes are involved in fixing zinc availability to plants (Sindhu et al. 2019a). Nutrient deficien-
cies such as phosphorus and molybdenum negatively affect the growth and population of rhizobia
(Giller 2001). Many environmental factors are involved that affect nutrient availability, composition,
and microbial diversity (Hartmann et al. 2017; Ahmad et al. 2016). Biofertilizers must be kept at
room temperature or cold storage away from sunlight and heat (Mishra and Dadluck 2010). Abiotic
stress can change the rhizosphere and may affect the potential, survival, and diversity of these bene-
ficial microbes (Etesami 2020). For any biofertilizer product to produce the desired effects on crop
growth and production, it must be well formulated and a suitable source of all the essential nutrients
to support plant growth.

8.6.5 Extension and Market Constraints

Biofertilizers are an emerging industry and market growth is extremely important. However,
biofertilizers face many challenges due high market competition. Therefore, it is necessary to
develop the right marketing strategy to maintain and increase the demand for biofertilizers.
Therefore, the growth prospects of the biofertilizer market and its product availability are equally
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168 Biofertilizers for Sustainable Soil Management

important (Giazinnazi and Vosatka 2004; Tariq et al. 2013). A poorly developed marketing strategy
will certainly have a negative impact with limited involvement of customers and the private sector.
A proper marketing plan is necessary to get consumer satisfaction. Consumers can gain awareness
of biofertilizers through active marketing plans, research, and the availability of products (Banayo
et al. 2012).

8.6.6 Farmer Awareness
Challenges common to biofertilizers include inadequate biofertilizer awareness among farmers. It
is necessary to explore the potential of biofertilizers for crops and the environment. It is need of
the day that farmers should be aware of their sources and bioinoculants. Inadequate awareness may
lead to low adoption of biofertilizers by farmers. Therefore, it is necessary to educate farmers on
the societal and ecological benefits of biofertilizers and to improve the application of biofertilizers
by growers. Inadequate information and poor awareness may lead to low development and poor
acceptance of biofertilizers among growers.

8.7 FUTURE PROSPECTS FOR BIOFERTILIZERS

Growing crops sustainably allows growers to protect the soil and increase crop productivity in a
way that is less harmful to the environment. Sustainable crop management approaches help to min-
imize the impact on the natural habitat, improve soil health, and protect water resources. This can
be achieved only by implementing sustainable management methods in agriculture. Biofertilizer
application is the most important part of sustainable agriculture and is a good option as an alterna-
tive to chemical fertilizers. However, there are many challenges in the preparation and development
of biofertilizers, and additional research is needed to support the promotion of biofertilizer use. In
addition, public and private sectors, and commercial investors have the potential to improve the
formulation and sale of biofertilizer substitutes to replace synthetic fertilizers. Thus, biofertilizer
development faces many challenges that pose many threats.
New interest in biofertilizers to increase productivity and reduce health, environmental and
commercial pressures for agrochemicals have caused significant advances in the research into
biofertilizers. Using modern approaches, the efficacy of biofertilizers can be improved. Moreover,
advances in production technology lead to a resuction in the cost of products. However, there remain
many challenges that need to be solved to reflect the full potential for commercial biofertilizer
production.
It is very important to develop integrated nutrient programs that can offer a good option and
economic choices to favour the use of biofertilizers. Synthetic fertilizers should be used at a
minimal level to reduce environmental pollution and support the use of biofertilizers. Different
approaches should be developed to protect soil fertility, and to avoid health and environmental risks.
Biofertilizer efficiency can be enhanced by using different approaches such as advances in tech-
nology and formulation.

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173

9 Phosphorus-​solubilizing
Bio-​fertilizers
Kinza Iqbal,1 Sajid Masood,1 Qaiser Hussain,1*
Rabia Khalid,1 Khalid Saifullah Khan,1
Muhammad Akmal,1 Shahzada Sohail Ijaz,1
Muhammad Jamil,2 Muhammad Azeem,1 and Servat Jehan1
1
Institute of Soil & Environmental Sciences, PMAS-​Arid Agriculture
University, Rawalpindi, Pakistan
2
Soil and Water Testing Laboratory, Sahiwal, Pakistan
Correspondence: [email protected]; [email protected]

CONTENTS
9.1 Introduction........................................................................................................................... 173
9.2 Phosphorus-​solubilizing Microorganisms (PSMs)................................................................ 174
9.3 Mechanism and Processes of Inorganic P-​solubilization by PSMs...................................... 175
9.3.1 Non-​specific Acid Phosphatases (NSAPs)............................................................... 176
9.3.2 Phytases.................................................................................................................... 177
9.3.3 PSM-​derived P Desorption from Clay Minerals...................................................... 177
9.3.4 Siderphore Production.............................................................................................. 178
9.3.5 Solubilization via Exopolysaccharides (EPS).......................................................... 178
9.4 Factors Responsible for P-​solubilization............................................................................... 178
9.4.1 Temperature.............................................................................................................. 178
9.4.2 Soil pH...................................................................................................................... 178
9.4.3 Chelation................................................................................................................... 179
9.4.4 Mineralization........................................................................................................... 179
References....................................................................................................................................... 179

9.1 INTRODUCTION
Due to its low soil availability, phosphorus (P) is an important macronutrient for optimal
plant growth, especially in tropical environments (Santana et al. 2016). Plant phosphorus
concentrations range from 0.2% to 0.8% (Sharma et al. 2013), and it is, after nitrogen, the second
most important plant nutrient (Aziz et al. 2017a,b). Because of the formation of insoluble phos-
phorus complexes in soil, phosphorus availability in soil–​plant systems is a problem. Insoluble
forms of P, they if get precipitated, are unavailable to plants (Rengel and Marschner 2005; Adnan
et al. 2018a,b; Adnan et al. 2020; Adnan et al. 2019; Atif et al. 2021; Fahad et al. 2016; Fahad
et al. 2015a,b; Fahad et al. 2020; Fahad et al. 2021a,b,c,d,e,f; Fahad et al. 2022a,b). This means
that, in most cases, the phosphorus concentration in the soil is 0.05% (w/​w), with only 0.1% of
that being available to plants (Zhou et al. 1992). Phosphorus fixation is a term used to describe
the process of removing readily available phosphate from soil solutions and storing it in the solid
soil phase (Barber 1995; Fakhre et al. 2021; Muhammad et al. 2022; Shafi et al. 2020; Wahid
et al. 2020; Zahida et al. 2017).

DOI: 10.1201/9781003286233-9 173

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174 Biofertilizers for Sustainable Soil Management

FIGURE 9.1 A schematic diagram of P sources and forms in soil.

Phosphorus-​solubilizing microbes (PSMs) not only promote phosphorus cycling by mineralizing

organic compounds, but also dissolve insoluble phosphorus complexes and increase phosphorus
bioaccumulation in soil–​plant systems. It is possible for PSM to convert soil P into plant-​available P
(mainly PO43–​, HPO42–​, and H2PO4–​) through exudations, root acidification, and enhanced enzymatic
metal chelation (Billah et al. 2019). Because of this, it is critical to investigate management strategies
that can increase agricultural yields, reduce pollution caused by phosphorus loss in soil, and
improve the efficiency of phosphorus fertilizers. Numerous soil and rhizosphere microorganisms
have been found to improve phosphorus availability and plant phosphorus uptake (Bhattacharyya
and Jha 2012; Ahmad et al. 2017). It is even possible for these PSMs to mineralize organic phos-
phorus, and dissolve inorganic phosphorus, including in the presence of salt (Zhu et al. 2011).
As a highly active element, phosphorus does not exist in its elemental state in the soil. It’s pos-
sible to find soil solutions that contain both organic phosphorus (Po) and inorganic phosphorus
(Pi) (Walpola and Yoon 2012). Sedimentary circulation of P is possible because, unlike nitrogen, it
is not exchanged with any form of P in the atmosphere and there is no major atmospheric P stock
available for biological use. As a result, phosphorus deficiency leads to a reduction of plant growth
and yield (Rodriguez and Fraga 1999). Most of this (95–​99%) is in the form of insoluble phosphate,
despite the fact that soil test results are usually much higher (Pradhan and Sukla 2005). There is a
wide range of soluble phosphorus concentrations within soil solutions, ranging from ppb to 01 mg/​
L in highly fertilized soils (Khan, M.S. et al. 2009b). Organisms and plant residues that have not
yet decomposed, as well as organophosphorus compounds, make up the bulk of the soil’s organo-
phosphorus compounds. Organophosphorus mineralization and immobilization can be affected by
soil microbial activity. Cations such as Ca2+​ immobilize P to form Ca3(PO4)2 (calcium phosphate)
in normal and calcareous soils, while in acidic soils, Al3+​ and Fe3+​ immobilize P to produce AlPO
and FePO (Satyaprakash et al. 2017). A variety of P forms can be taken up by plant cells, but the
majority of phosphorus is taken up as phosphate anions, primarily HPO42–​ or H2PO4–​, depending on
the pH level of the soil (Mahidi et al. 2011). As a result, PSM’s strategy could mobilize the P and
transform it into soluble P and plant-​required P forms. Figure 9.1 depicts soil P transformations and
soil–​plant P availability in a comprehensive way.

9.2 PHOSPHORUS-​SOLUBILIZING MICROORGANISMS (PSMS)

Phosphorus-​solubilizing microorganisms (PSMs) play important roles in the soil phosphorus cycle
by mineralizing organic phosphorus, dissolving inorganic phosphorus minerals, and storing vast
quantities of phosphorus in biomass (Liang et al. 2020). Many microorganisms have the ability to
lyse and mineralize, including bacteria, fungi, actinomycetes, and algae. Plant growth-​promoting
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Phosphorus-solubilizing Bio-fertilizers 175

TABLE 9.1
PSM Types

Bacteria Alcaligenes sp., Achromobacter sp., Aerobactor aerogenes, Actinomadura oligospora, Azospirillum
brasilense, Agrobacterium sp., B. mycoides, Bacillus circulans, B. fusiformis, Bacillus sp.,
B. megaterium, B. pumils, B. cereus, B. polymyxa, B. coagulans, B. chitinolyticus, Bradyrhizobium
sp., B. subtilis, Brevibacterium sp., Pseudomonas sp., P. striata, P. putida, Enterobacter asburiae,
Escherichia intermedia, Nitrobacter sp., T. thioxidans, Thiobacillus ferroxidans, Rhizobium meliloti,
Xanthomonas sp.
Fungi Aspergillus awamori, A. tereus, A. wentii, A. niger, A. nidulans, A. flavus, A. foetidus, Alternaria
teneius, Achrothcium sp. Fusarium oxysporum, P. lilacinium, Penicillium digitatum, P. funicolosum,
P. balaji, Cladosprium sp., Cephalosporium sp., etc
Actinomycetes Actinomyces, Streptomyces
Cyanobacteria Anabena sp., Calothrix braunii, Nostoc sp., Scytonema sp.,
VAM Glomus fasciculatum

bacteria (PGPB) and other microorganisms have been shown to increase P availability to plants
via solubilization and mineralization (Adnan et al. 2018c). Many fungi have also been shown to
increase the availability of phosphorus in soil–​plant systems. To dissolve inorganic phosphorus,
Actinomyces, Micromonospora, and Streptomyces can produce acids such as gluconic, lactic, citric,
2-​ketogluconic, tartaric, and oxalic. It has also been discovered that algae such as cyanobacteria are
capable of dissolving phosphorus (Sharma et al. 2013). PSMs that live in alkaline, acidic, or organic
environments frequently form metal complexes (Bashan et al. 2013a,b) (Table 9.1).

9.3 MECHANISM AND PROCESSES OF INORGANIC P-​SOLUBILIZATION

BY PSMS
PSMs primarily solubilize P by releasing complex or inorganic solvent compounds (organic acid
anions and siderophores), CO2, protons, hydroxyl ions, and the release of P during substrate deg-
radation by extracellular enzymes (Sharma et al. 2013). The direct oxidation pathway, which
produces organic acids in the periplasmic space, results in lower soil pH and increases P availability
(Seshachala and Tallapragada 2012). Lower pH (less than 7) of microbial cells and the adjacent
environment occurs when H+​ replaces Ca2+​ during organic acid excretion, leading to the release of
P ions (Goldstein 1994). Illmer and Schinner (1995) proposed a H+​ acidification mechanism. They
described that H+​ excretion and NH4+​ uptake are related to the release of H+​, which increases P solu-
bilization. Krishnaraj et al. (1998) suggest that protons ejected from cells are the key to P solubil-
ization. When H+​ is released into the rhizosphere in response to cation uptake or with the help of H+​
ATP, organic acids are produced and inorganic P is solubilized (Rodriguez and Fraga 1999; Ali et al.
2018). Phosphorus is solubilized by microorganisms without the formation of organic acids via the
protons released during the absorption of NH4+​ (Sharma et al. 2013). Gram-​negative bacteria, for
example, can aid in the solubilization of inorganic P by oxidizing gluconic acid (Goldstein 2000).
Pyrroloquinoline quinone (PQQ) functions as a redox cofactor in glucose dehydrogenase (GDH),
resulting in phosphate solubilization (Rodriguez et al. 2006). In addition, hydrogen sulfide (H2S)
is formed, which reacts with ferric phosphate to form ferrous sulphate and phosphate (Swaby and
Sperber 1958).
Organic acid excretion by PSMs, which then chelates Fe and Al ions to release P, can make mod-
erately labile Pi (partly from Fe/​Al–​P and sesquioxide surface) available for use by soil organisms
(Baumann et al. 2018). Carboxyl groups found in organic acids have the ability to bind phosphorus
through either the substitution of cations or the competition for phosphorus adsorption sites. This
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176 Biofertilizers for Sustainable Soil Management

FIGURE 9.2 Phosphorus solubilization mechanisms/​processes by PSMs in soil.

results in an increase in the soil’s capacity to take up PO43–​ and the solubilization of phosphorus.
Phosphorus-​solubilizing bacteria (PSBs) are known to be rich in exopolysaccharides (EPSs), which
are necessary polymers made up of carbohydrates and play an important part in the process of
Pi solubilization (Sharma et al. 2013). EPS has the potential to cause a disruption in the homeostasis
of organic acids or H+​ that are involved in the process of Pi solubilization by keeping free
P in the medium. This could result in an increased release of P from Pi minerals (Yi et al. 2008).
Both the Streptomyces species and the Micromonospora species have been demonstrated in the past
to be capable of dissolving phosphate rock while simultaneously producing calcium chelators or
siderophores (Hamdali et al. 2008; Yandigeri et al. 2011). In addition to the mechanisms that have
already been described, the production of chelators (such as calcium, aluminium, and iron chelators)
by PSB, phosphorus-​solubilizing actinomycetes (PSA), and blue-​green algae (BGA) is another way
that phosphorus can be released from Pi crystals (Figure 9.2).

9.3.1 Non-​specific Acid Phosphatases (NSAPs)

An enzyme known as non-​specific acid phosphatase (NSAPs) is responsible for dephosphorylation
of phosphoester or phosphoanhydride bonds present in organic matter. Phosphomonoesterases,
also known as phosphatases, are the most common and have been the most researched of all the
different types of phosphatase enzymes generated by PSMs (Nannipieri et al. 2011). Depending
on the optimum pH at which they operate, these enzymes are classified as either acidic or alkaline
phosphomonoesterases. Depending on environmental conditions, both types can be generated by
PSMs (Jorquera et al. 2008; Basir et al. 2016). The presence of alkaline phosphatases is more preva-
lent in neutral and alkaline soils, while acid phosphatases are more prevalent in acidic soils (Renella
et al. 2006).
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Phosphorus-solubilizing Bio-fertilizers 177

Although plant roots can produce acid phosphatase, alkaline phosphatase is not very often produced
in significant amounts, suggesting that this could be a potential niche for PSMs (Criquet et al. 2004).
It is also difficult to distinguish between PSM-​produced and root-​produced phosphatases, but some
evidence proposes that microbial-​derived phosphatases have a greater affinity for Po compounds
than phosphatases of plant origin (Tarafdar et al. 2001). At present, it is unclear whether or not there
is a link between phosphatase activity, PSM that was injected into the soil, and future Po mineral-
ization (Chen et al. 2003).

9.3.2 Phytases
The consequent breakdown of the phytate leads to liberation of the element phosphorus. This is
the primary source of phosphate and the most common form of phosphorus stored in pollen and
plant seeds, and phytate is an essential component of the organic phosphorus that is found in soil
(Richardson 1994). When fed phytate, Arabidopsis plants that had been genetically transformed
with the phytase (phyA) gene obtained from Aspergillus niger showed significant improvements in
P growth and nutrition. This was the case regardless of the fact that plants have a limited capacity
to obtain phosphorus directly from phytate (Richardson 2001). Because of this, plant nutritional P
increased to the point where plant growth and P content were comparable to control plants that had
been given inorganic P. This occurred because it led to an increase in plant nutritional P. This is due
to an increase in the amount of nutrition provided by phosphorus.

9.3.3 PSM-​derived P Desorption from Clay Minerals

Adsorption reactions speed up the removal of soluble P forms from soil solutions, allowing for their
sorption in the solid phase. Volcanic ash and highly weathered tropical soils are rich in aluminium
hydroxides and amorphous iron and are particularly vulnerable to these chemical reactions (Osorio
and Habte 2012). Compared to their variable charge counterparts, permanently charged minerals
have a greater adsorption capacity and binding energy for phosphorus. Iron hydroxides and alu-
minium hydroxides are the most significant minerals with a variable charge (He and Zu 1998). Most
of the phosphorus in clay minerals in fertilized soil is bound to aluminium hydroxides, whereas iron-​
bound phosphorus is the most common form in unfertilized soils (Eriksson et al. 2015). Hydroxides
of Fe–​P (Al–​P) are formed because of the close relationship between iron (Al) and orthophosphate
(P). The high adsorption capacity of these hydroxides causes phosphate to be deposited in the soil
(Wu et al. 2019). Orthophosphate can be adsorbed by soil organic matter or immobilized by it,
resulting in relatively stable Po molecules and lowering the concentration of dissolved phosphate in
the soil (Qiao et al. 2017).
The P desorption ability of PSM depends on the clay mineral surfaces, saturation of Pi adsorbent
sites, and adsorption capacity. This is the case despite the fact that allophane has the greatest ability
to desorb P. Moreover, since it causes higher levels of absorbed phosphorus in a wide variety of
soils, inoculation with Mortiella sp. is able to significantly increase the phosphorus solution of the
soil in situ by desorption. It can be observed that the ability of PSM to increase soluble Pi in soil is
proportional to the amount of P taken up by soil and clay minerals, while the extent of Pi desorp-
tion by PSM is inversely proportional to the Pi-​absorptive capacity of soil and clay minerals. This
is something to keep in mind (Osrio and Habte 2012). Desorption of phosphorus from PSM usually
occurs in conjunction with a decrease in pH. The desorption of P is mainly caused by the increased
solubility of Fe and Al due to the potential complexation of Fe and Al with low-​molecular-​weight
organic acids (Hoberg et al. 2005; Iqbal et al. 2016). During the process of phosphorus mineral (Pi)
solubilization, plant roots and PSM may be responsible for releasing a variety of low-​molecular-​
weight organic acids. Some of these organic acids, such as citrate, malate, and oxalate are well known
for their ability to increase Pi availability in soil through desorption (Dodd and Sharopely 2015).
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178 Biofertilizers for Sustainable Soil Management

Pi can be displaced from adsorption sites by organic acids and anions due to ligand exchange
caused by microbial activity and temporary blockage of Pi adsorption sites (Andrade et al. 2013).
Depending on their dissociation characteristics and the carboxylic groups they contain, organic acids
can have a variety of different negative charges that help promote Pi desorption (Hoberg et al. 2005).

9.3.4 Siderphore Production
Almost all microorganisms produce siderophores, which are complexing agents with a strong affinity
for iron. Solubilizers of iron from mineral or organic molecules are provided by siderophores in the
case of iron deficiency. About 500 known siderophores are used by a wide range of microbes and
plants, while some are used only by microbial species and the strains that produce them (Crowley
2007). Aside from one report (Hamdali et al. 2008a; Zahoor et al. 2016) that siderophores are
released by PSM, the production of siderophores has not been consistently linked to P-​solubilization.
Mineral dissolution dominates the P-​solubilizing role of organic acid anions, so the potential role of
siderophores in increasing P availability should be obvious.

9.3.5 Solubilization via Exopolysaccharides (EPS)

Polysaccharides have been shown to have a significant impact on phosphorus solubilization by
microbes (Yi et al. 2008). Microbial exopolysaccharides (EPS) are polymers secreted by bacteria
and fungi that are known as microorganism exopolysaccharides. Some of these polysaccharides
are homopolysaccharides, while others are heteropolysaccharides, with a variety of organic and
inorganic substituents in between. Their structure and composition can differ greatly from one
another (Sutherland 2001). Yi et al. (2008) reported that tricalcium phosphate (TCP) was solubilized
using four different bacterial strains. Among the strains tested were Artrobacter sp. (ArHy-​505),
Enterobacter sp. (EnHy-​401 and EnHy-​402), and Azotobacter sp. (AzHy-​510). Bacteria with high
phosphate solubility were able to produce large amounts of exopolysaccharides (EPS). More research
is needed to better understand the relationship between EPS synthesis and phosphate solubilization.

9.4 FACTORS RESPONSIBLE FOR P-​SOLUBILIZATION

The ability of PSM to modify organic and insoluble inorganic phosphorus is dependent on the
nutrient content of the soil as well as the organism’s physiological and growth state. Compared to
PSMs residing in temperate soil conditions, PSMs from extreme soil conditions, such as heavy-​
metal-​contaminated soils, high nutrient-​deficient soils, saline soils, or soils subject to extreme
temperatures, are more likely to dissolve phosphate (Zhu et al. 2011).

9.4.1 Temperature
Temperature has been discussed in relation to phosphorus solubilization by bacteria. Varsha (2002)
discovered that 28°C was the optimum temperature to use for maximum microbial phosphorus solu-
bilization, whereas White et al. (1997) found that 20–​25°C was the best temperature to use. Others
have reported that 30°C is the optimal temperature for solubilizing P. Nautiyal et al. (2000) found
maximum P solubilization in desert soil at 45°C, whereas Johri et al. (1999) discovered P solubil-
ization in the same medium at 10°C.

9.4.2 Soil pH
The main mechanism responsible for phosphorus solubilization in soil is the reduction of soil pH
caused by the release of protons or microbial synthesis of organic acids (Pradhan and Sukla 2005).
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Phosphorus-solubilizing Bio-fertilizers 179

In alkaline soils, calcium ions react with phosphate to form calcium phosphates, which are insoluble
in soil and include rock phosphate. After that, the calcium phosphates can precipitate (fluorapatite
and francolite). As the pH of the soil decreases, so does its soluble capacity. PSMs produce organic
acids, which lower soil pH and make more phosphorus available to plants (Satyaprakash et al.
2017). Furthermore, aliphatic acids are more effective than phenolic, citric, and fumaric acids in
solubilizing phosphate, while tri-​and dicarboxylic acids outperform monobasic and aromatic acids
(Mahidi et al. 2011; Rehman et al. 2016). Oxalic, citric, acetic, gluconic, lactic, 2-​ketogluconic,
fumaric, adipic, malic, tartaric, succinic, malonic, glutaric, butyric, propionic, and glyoxalic acids
are the most common organic acids that can solubilize phosphates (Kumar et al. 2018).

9.4.3 Chelation
PSMs are responsible for the dissolution of insoluble soil phosphates because they produce both
organic and inorganic acids. They accomplish this by chelating cations and engaging in competition
with phosphate in the soil for adsorption sites (Khan, A. et al. 2009). The hydroxyl and carboxylic
groups that are present in the acids are responsible for the chelation of the cations that are linked
to the phosphate, as well as the transformation of those cations into forms that are soluble. These
acids are able to serve as attachment sites for the insoluble oxides of aluminium and iron because,
following their reaction with the insoluble oxides of those elements, the resulting compound is
stabilized. 2-​Ketogluconic acid is one of these, and it is a calcium chelator that is exceptionally
effective (Walpola and Yoon 2012). It has been observed that inorganic acids such as sulfuric nitric
acid and carbonic acid are produced as a result of the observations that have been made. As a result
of a reaction involving nitric and sulphuric acids, calcium phosphate will have a greater degree of
solubility (Khan et al. 2007).

9.4.4 Mineralization
Another method for releasing phosphorus from soil is mineralization. PSMs convert organic
phosphate into usable form through the mineralization process. This transformation occurs in
the soil and consumes a variety of organic phosphorus compounds, including phosphonates,
phospholipids, nucleic acids, sugar phosphates, polyphosphates, and phytic acid (Khan, A. et al.
2009). Organic phosphorus mineralization and immobilization in agricultural soil is a critical step
in the phosphorus cycling process. PSMs are responsible for mineralizing organic phosphorus
in the soil by producing phosphatases such as phytase (Aseri et al. 2009). These phosphatases
hydrolyse organic phosphate compounds, releasing inorganic phosphorus for use by plants. Both
acid and alkaline phosphatases use organic phosphate as a substrate in the chemical reaction to
convert it to phosphate.
Extracellular enzymes such as phosphoesterases, phosphodiesterases, phytases, and
phospholipases are produced in the soil by Bacillus and Streptomyces spp. Bacteria can mineralize
complex organic phosphates thanks to these enzymes (Walpola and Yoon 2012). The most effi-
cient method of mineralizing organic phosphate is to use a phosphorus-​solubilizing bio-​fertilizer
composed of mixed cultures of PSM (Bacillus, Streptomyces, and Pseudomonas). According to
Dodor and Tabatabai (2003), some PSMs can produce siderophores, which hydrolyse soil organic
phosphorus and make it more accessible.

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185

10 Potential Applications of
Algae-​based Bio-​fertilizers
Hafiz Muhammad Rashad Javeed,1* Mazhar Ali,1
Rafi Qamar,2 Fahim Nawaz,3 Humaira Yasmin,4
Koushik Chakraborty,5 Zainul Abideen,6
Muhammad Zahid Ihsan,7 and Muhammad Adnan Bukhari8
1
Department of Environmental Sciences, COMSATS University
Islamabad, Punjab, Pakistan
2
Department of Agronomy, College of Agriculture, University of
Sargodha, Sargodha, Punjab, Pakistan
3
Department of Agronomy, Muhammad Nawaz Shareef University of
Agriculture, Multan, Punjab, Pakistan
4
Department of Biosciences, COMSATS University Islamabad,
Islamabad, Pakistan
5
Department of Crop Physiology & Biochemistry, ICAR-​National Rice
Research Institute, Bidyadharpur, Cuttack, Odisha, India
6
Dr. Muhammad Ajmal Khan Institute of Sustainable Halophyte
Utilization, University of Karachi, Sindh, Pakistan
7
The Cholistan Institute of Desert Studies, Faculty of Agriculture
and Environment Sciences, The Islamia University of Bahawalpur,
Bahawalpur, Punjab, Pakistan
8
Department of Agronomy, Faculty of Agriculture and Environment,
The Islamia University of Bahawalpur, Bahawalpur, Punjab, Pakistan
Correspondence: [email protected]

CONTENTS
10.1 Alternative Fertilizer Sources: A Need of the Day.............................................................. 186
10.2 Introduction of Algae........................................................................................................... 186
10.3 Potential Applications of Algae........................................................................................... 187
10.4 Application of Algae-​based Bio-​fertilizers in Agriculture................................................... 187
10.5 Industrial Applications of Algae.......................................................................................... 188
10.6 Application of Algal Bio-​fertilizer....................................................................................... 188
10.7 Slow-​release Bio-​fertilizers................................................................................................. 188
10.8 Cyanobacteria Nitrogen Fixation......................................................................................... 189
10.9 Liquid Bio-​fertilizers........................................................................................................... 189
10.10 Algae-​based Bio-​fertilizers.................................................................................................. 189
10.10.1 Soil Fertility......................................................................................................... 189
10.10.2 Nitrogen Fixation................................................................................................. 190
10.10.3 Production of Plant Growth Stimulants............................................................... 190
10.10.4 Bio-​pesticidal Substances.................................................................................... 190
10.10.5 Algal Toleration and Mutation............................................................................. 190
10.10.5.1 Tolerance to Extreme Environmental Conditions............................... 190

DOI: 10.1201/9781003286233-10 185

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10.10.5.2  Mutation of Algae for Better Bio-​fertilizers................................... 190

10.10.5.3  Large-​scale Algal Growth............................................................... 191
10.11 Formulation of Algal Bio-​fertilizers.................................................................................. 191
10.12 Challenges and Measures for Commercialization............................................................. 192
10.12.1 New Pollutant Factors Discovered in Wastewater............................................. 192
10.12.2 High Cost of Algal Biomass.............................................................................. 192
10.12.3 Contamination of Algal Biomass...................................................................... 192
10.12.4 Water Consumption and Water Loss................................................................. 193
10.12.5 Potential Threats of Cyanobacteria in the Environment.................................... 193
10.13 Conclusion......................................................................................................................... 193
References....................................................................................................................................... 193

10.1 ALTERNATIVE FERTILIZER SOURCES: A NEED OF THE DAY

The main challenge in meeting rising energy demands and avoiding the negative environmental
impact of industrial waste is to overcome fossil fuel mismanagement and the global industrial revo-
lution. Industrial waste, in addition to agricultural waste, is the primary source of water contam-
ination (Hussain et al. 2021). Water scarcity or pollution, as a severe environmental concern, has
drawn a large number of people to seek solutions. In the last 120 years, global power generation
has been steadily expanding, accounting for more than 39% of global electricity generation. Even
though burning coal in nuclear reactors emits CO2, the most environmentally harmful greenhouse
gas, and a major contributor to observed global climate change (GCC), numerous regional and
global efforts to reduce coal use for electricity generation and CO2 emissions have been made in
the last 30 years (Iyovo et al. 2010). Other than hydroelectric, renewable energies increased from
0.7 percent in 1980 to 6.8 percent in 2015. Replacement of coal power plants has taken place with
widely available renewable energy sources (Zou et al. 2021). At the same time, as fossil fuel supplies
become depleted, the need for renewable energy is growing. In comparison to other physicochemical
treatment approaches, microalgae are the most appealing biological agents for dealing with both
energy emergencies and wastewater treatment (Zhao and Magoulès 2012). Synthetic fertilizer, pesti-
cide, and other chemical manufacturers and users have proliferated to fulfil the growing need for
food, polluting the planet and posing a serious threat to all living things. Moreover, as a result of
intensive farming operations and climate change, agricultural land is losing its fertility (Besser and
Hamed 2021). Pathogens, cyanobacteria, microbes, and other organisms are gaining popularity as
long-​term substitutes for synthetic chemicals due to their capacity to increase soil fertility, fix atmos-
pheric nitrogen for plant availability, manufacture plant anabolic steroids and biotoxins, and more
(Lee and Ryu 2021).

10.2 INTRODUCTION OF ALGAE
Algae are a varied collection of microorganisms that can perform photosynthesis by absorbing energy
from the sun. Algae are widely employed in agriculture as bio-​fertilizers and soil stabilizers (Park
and Kim 2018). Algae, especially seaweeds, are employed as fertilizers, resulting in less nitrogen
and phosphorus discharge than when cattle dung is used. As a result, the quality of water flowing
into rivers and seas improves. These organisms are grown and utilized as human food supplements
all over the world. They can also generate clean and carbon-​neutral food, and can be produced on
abandoned sites and arid desert regions with little freshwater requirement. Iodine may be found
in abundance in seaweed (Arshad et al. 2016; Kulikova et al. 2018). The amount of iodine in milk
is determined by the diet of the cow that produced it. According to Fuzhou Wonderful Biological
Technology, feeding seaweeds to milk calves can improve the amount of iodine in milk. Algae feed
additives also boost the egg-​laying rate of hens (Bhuyar et al. 2018).
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Potential Applications of Algae-based Bio-fertilizers 187

10.3 POTENTIAL APPLICATIONS OF ALGAE

It is unknown if algae can be employed in large-​scale farming. Although large-​scale algal production
has been optimized for developing treatments, there is no evidence that algae can be used for large-​
scale agricultural cultivation. Algal determinants that enhance plant development and immunity
have been identified and categorized as secretory products. Plants that were inoculated with cell wall
components like glucans grew faster and their defensive mechanisms were triggered (Adnan et al.
2018; Kumar et al. 2020). When algae are cultivated on liquid medium, their released products may
be gathered in enormous quantities (Lin et al. 2020). These defensive hormones greatly boost plant
defence systems when given exogenously. Items released by algae in liquid culture have a lot of
promise for use in the field. In general, large-​scale algae production, usually by heterotrophic culture,
is done to collect algal cells. The non-​cellular components are regarded as waste products that must
be detoxified (Reddy et al. 2019). The potential uses of cell-​free extracts will substantially expand
if they can be utilized for plants. However, before applying algae to crop plants, a number of issues
must be addressed. First and foremost, the potential risks of cell-​free algal extracts must be analysed
and eliminated. Algae frequently emit dangerous compounds during cultivation. As a result, quality
monitoring of algal liquid culture is crucial. Secondly, the development of algae determinants must
be improved for a complex programme. Third, consideration should be given to the preparation
of cell-​free extracts (Reddy et al. 2019). An immersion or drip irrigation method could be used
to give the cell-​free extract. It is, however, crucial to obtain a significant volume of extracts. As a
result, the extract should be evaporated and purified using chemical and physical methods, with the
completed substance demonstrating great efficacy and being employed in agricultural (Arun et al.
2020). Furthermore, determinants are granulated similarly to other agricultural goods like fertilizers
and agrochemicals. Finally, in the near future, a specialized process for isolating beneficial algae
for plant health enhancement must be created. The extract should be evaporated and purified using
chemical and physical methods, with the completed substance demonstrating great efficacy and
being employed in agriculture (Das et al. 2019). Despite the fact that microorganisms have been
employed to increase production fitness, a new study shows that phytoplankton may also improve
crop output and function as biocontrol agents against illnesses by directly blocking pathogen devel-
opment and activating plant immune systems. As a result, cyanobacteria represent a unique bio-
chemical supply that may be used as bio-​fertilizers and plant protectants, implying that microalgae
are a naturally occurring reactive ecosystem constituent (Win et al. 2018).

10.4 APPLICATION OF ALGAE-​BASED BIO-​FERTILIZERS IN AGRICULTURE

Fertilizers are used extensively in agricultural production operations to boost crop output, however
the widespread use of synthetic and pesticide fertilizers presently available could pose a severe
environmental threat (Khan et al. 2016; Godlewska et al. 2019). As a result, fertilizer research is
concentrating on employing microorganisms as a more environmentally friendly method for sus-
tainable agriculture. Microorganisms that may coexist with higher plants are included in the micro-
biological toolbox (Vishwakarma et al. 2018).
Bio-​stimulants are inorganic substances produced by diverse microorganisms that help seeds,
plants, and soil bacterial consortia thrive by supplying vital micronutrients, phosphorus, copper, as
well as other nutrients (Zahra et al. 2020; Saud et al. 2022b). Bio-​fertilizers provide contemporary
agriculture with renewable and environmentally friendly solutions, in specific, that may assist to
enable productivity expansion. Fertilizers, on either hand, face a slew of issues, such as a short shelf
life (3–​4 weeks) and stringent storage requirements (Stella et al. 2019).
Different species have been shown to be useful as bio-​fertilizers and soil conditioners in recent
research. Chrysophyta are the major groups of microalgae. The only photosynthetic prokaryotes
capable of producing oxygen are cyanobacteria (Stella et al. 2019).
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188 Biofertilizers for Sustainable Soil Management

There are over 1 million kinds of both unicellular microalgae and more sophisticated eukary-
otic eukaryotes. Microalgae are divided into 150 genera and roughly 2000 species. Eukaryotic
microalgae and bacterial cyanobacteria are both classified as algae (Schaap et al. 2012).

10.5 INDUSTRIAL APPLICATIONS OF ALGAE

The capacity of algae to flourish with low water resources and to use locations that would otherwise
be unavailable to agriculture provides economic advantages. Microalgae are commonly assumed
to have a large effect on important ecosystem processes as they can recover extra nutrients and
recycle water for reuse and may be created in wastewater and agricultural runoff (Priyadarshani
and Rath 2012). They can also help to reduce greenhouse gas emissions by capturing nitrous oxide
from industrial sources. The most basic photosynthetic plants on the Earth are microalgae. These
bacteria can make sustenance out of inorganic substances and can be found in large numbers in
liquid environments (Hussain et al. 2016; Zahra et al. 2020). Microbes have various unique traits
that make them a desirable bio-​fertilizer supplier for enhancing soil physio-​chemical properties,
including moisture capacity, rapid generation time, ability to fix atmospheric N2, and resistance to
hostile settings. Microbes can also produce biologically active compounds that improve soil chem-
ical characteristics, facilitate nutrient movement from the ground to crops, and cause soil aggrega-
tion (Wijffels et al. 2013).

10.6 APPLICATION OF ALGAL BIO-​FERTILIZER

Recent studies, both in fundamental and practical research, have demonstrated that algal bio-​
fertilizers have good effects on plants, crop output, soil microorganisms, fruit nutritional value, and
seed germination. Researchers from academia and industry have created three fundamental models
for employing algae in crops, motivated by the environmental and economic benefits (Altaf et al.
2016; Das et al. 2019). To commence with, biofuel is employed as a slow-​release fertilizer that may
be digested by soil bacteria and which delivers nutrients to plants on a continuous basis. Second, live
algae cells could be used to control soil moisture, improve soil fertility, and regulate the microbial
community. Finally, as a solvent plant, an algae extract containing amino acids and minerals could
be applied to the surfaces of plant leaves. Gradual bio-​fertilizers, ammonia microalgae, and aqueous
plant all have distinct manufacturing techniques and action mechanisms, but they all have the poten-
tial to accelerate plant growth and improve soil conditions (Iqbal et al. 2021).

10.7 SLOW-​RELEASE BIO-​FERTILIZERS
Microbial activity accelerates biomass degradation, which extends the life of algal bio-​fertilizers. As
a consequence, an algal fertilizer with a delayed release performe better than an ordinary chemical
fertilizer. Slow-​release algal fertilizer is made by combining it with an algal culture (Ramli 2019).
Dehydration is used to evaporate the water of microalgae biomass and keep the bio-​fertilizer from
decaying too quickly during storage. When paired with algae growth and biomass collection, this
technique can be successful, resulting in a high algae spore cost. Gradual algal fertilizer is used in
agriculture for a variety of reasons, including stimulating plant growth and improving soil health (Ali
et al. 2016; Chen et al. 2020). To begin with, plants may ingest algal fertilizer nutrients and minerals,
phosphate, hydrocarbons, and micronutrients. According to Coppens et al. (2016), applying algal
bio-​fertilizer to tomato plants boosted leaf elongation and fresh and dry weight, as well as increasing
the accumulation of sugar, sucrose, and antioxidants in tomatoes. Several more researchers have
confirmed the beneficial benefits of algal bio-​fertilizer on plant development (Yao et al. 2013).
Second, the nutrients produced by algal fertilizers have the potential to boost soil fertility.
Microalgae have been shown to enhance the amount of nitrogen, phosphorus, and potash accessible
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Potential Applications of Algae-based Bio-fertilizers 189

in topsoil, supplying more nutrition to plants. Furthermore, the amounts of ammonium, phosphate,
and potash that remained in algal biomass-​fertilized soil at harvest were greater than the amounts of
soil quality fertilized by other treatments. As a result, long-​term use of an algal bio-​fertilizer may aid
in soil health preservation (Cole et al. 2016).

10.8 CYANOBACTERIA NITROGEN FIXATION

Cyanobacteria that fix nitrogen are beneficial to agriculture in five ways (Cole et al. 2016).
Agricultural crops have been reported to obtain up to 20–​30 kg nitrogen ha–​1 using cyanobacteria
fertilizer. As a consequence, the amount of chemical nitrogen fertilizer used in farming could be
lowered (Agawin et al. 2007). As a conclusion, broad adoption of nitrogen-​fixing cyanobacteria
could pave the way for long-​term agricultural viability. In practice, cyanobacteria can be employed
as a bacterial biofilm or as a microbial inoculant. To begin with, it is the most straightforward tech-
nique for microbiological inoculating of soil with cyanobacteria organisms. Photosynthetic bacteria
have been shown in the past to improve soil quality and aid plant growth. In certain circumstances,
microbial inoculants may struggle to survive in soil, and their PGP powers are reliant on their ability
to thrive in this environment. Second, the cyanobacteria biofilm can be created before organisms are
inoculated into the soil. Photosynthetic bacteria can be coupled with other microbes to improve the
efficacy of the biofilm in soil protection in the real world (Meeks 1998).

10.9 LIQUID BIO-​FERTILIZERS
Aqueous plants are algae extracts that are filled with nutritional requirements for plant development.
When algal extracts are sprayed om leaves, nutrients may reach plants or crops through leaf pores.
A liquid bio-​fertilizer is more efficient than a slow-​release fertilizer in terms of plant and crop use. As a
response, eco-​sustainable farming is increasingly turning to liquid bio-​fertilizers made from microalgae
(Maheswari and Elakkiya 2014). High-​pressure gas treatment, bead grinding, and ultrasonic therapy
are examples of physical techniques that alter the structure of algal cells by physical hits or shocks
(Dey 2021). It is worth noting, however, that certain physical approaches consume a lot of energy and
require specialized equipment. Hydrolytic enzymes involve the process of using enzymes like cellu-
lase, mannanase, xylanase, or pectinase to break down particular components in cell walls. Enzymatic
hydrolysis, as opposed to physical and chemical methods, may take place in a much softer micro-
environment, decreasing the damage caused by wall breaking of value-​added biological composites
(Sivamurugan et al. 2018). In some cases, the aforementioned procedures for breaking down algal cell
walls can be combined to ensure the most effective efficiency (Sivamurugan et al. 2018).

10.10 ALGAE-​BASED BIO-​FERTILIZERS
Cyanobacteria provide the most remarkable potential because they can thrive in the midst of highly
concentrated pollutants in a range of pollution flows that are poisonous to living organisms. For agri-
cultural production to become more effective and efficient, this is crucial (Mahapatra et al. 2018).

10.10.1 Soil Fertility
Despite the lack of information on how algal biomass development from wastewater treatment
influences soil nitrogen kinetics, it has potential as a soil amendment. In studies concentrating on
indigenous Anabaena species, this strain’s ability to enhance soil fertility while reducing soil density
was established (Bhardwaj et al. 2014).
The study of microalgae and microbial communities in bio-​fertilizer applications is another fas-
cinating research topic. In fact, it may be more efficient than employing individual microbes, not
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190 Biofertilizers for Sustainable Soil Management

just for contaminant detoxification and nutrient removal from wastewaters, but also for maximizing
the utilization of available nitrogen, phosphorus, and K in the soil. If algae and bacteria could min-
imize pollution, consortium engineering would be a triumph. Moreover, data suggest that microalgal
partnerships have a lot of potential for soil amendment on marginal lands, which might help with the
conversion of the soil in these areas (Singh et al. 2020).

10.10.2 Nitrogen Fixation
The capacity of phytoplankton to fix ammonium is one of the reasons they are used as bio-​fertilizers.
Microbes transform inorganic nitrogen (N2) from the air into organic nitrogen that higher plants may
utilize. Microalgae have been used to boost rice output in India and Chile. In Chile, local cyanobac-
terial strains have been demonstrated to increase the efficiency of nitrogen absorption in rice paddies
(Bhat et al. 2015).
Inoculants were employed in another investigation to produce colonies in chickpea produc-
tion. Furthermore, when utilized as a bio-​stimulant, bacterial association (21 species including
proteobacteria, bacteriodetes, chlorophyta, and others) was demonstrated to get a higher potential
for nitrification (10,294 nmol ethylene/​g dry weight/​h) (Mohammadi et al. 2010).

10.10.3 Production of Plant Growth Stimulants

Several algal compounds have been found to boost root development, either actively or passively, or
vegetation symbiotic involving soil microbes, hence enhancing bioavailability. To test this theory,
plant growth-​stimulating chemicals were employed to cultivate Lupinus termis utilizing cyanobac-
teria and bacteria (Win et al. 2018).

10.10.4 Bio-​pesticidal Substances
In vitro studies have shown that cyanobacteria extracts and exudates impede hatching and cause
immobilization and fatality in adolescent plant parasite microbes, suggesting that microalgae might be
employed as nematocidal biocontrol agents (Kalra and Khanuja 2007). The antifungal and antibacterial
characteristics of culture filtrate were studied when it showed hydrolytic action against phytopathogens
(Morales-​Borrell et al. 2020). A most commercially significant fungal disease is Fusarium sp., although
other fungal diseases have been managed under the same conditions. According to studies, the biocidal
capabilities of algae have revealed significant promise for creating novel pest control systems. Further
study is required to establish their frequency and economic effect (Abbey et al. 2019).

10.10.5 Algal Toleration and Mutation

10.10.5.1 Tolerance to Extreme Environmental Conditions
Microalgae can resist a number of pressures in the ecosystem. Ningthoujam et al. observed that
Anabaena variabilis could survive 100 lg/​mL malathion in terms of pesticide tolerance. Jha et al.
demonstrated that Mn and sodium may negatively affect cyanobacteria, which is an example of sal-
inity tolerance ability (Na, –​30.19 percent) (Shehata and Whitton 1982).
Sinha and Hader looked at cyanobacteria’s UV-​B photosensitizing process, which might be
effective in bio-​stimulants for field crops. The ability of active microorganisms to adapt to their
environment is a major practical issue with commercial soil amendment (Lee et al. 2017).

10.10.5.2 Mutation of Algae for Better Bio-​fertilizers

As a result, novel methods for creating cyanobacterial mutants are appropriate for investigating
cyanobacteria’s potential (Win et al. 2018). To establish a gene transfer mechanism in Oscillatoria
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Potential Applications of Algae-based Bio-fertilizers 191

MKU 277, Ravindran et al. produced the plasmid pRL489 and electroporated it into the cyanobac-
terium. As a result of this research, the methods for producing more potent and viable bio-​fertilizer
strains have improved (Shah et al. 2016c; Chakraborty and Akhtar 2021). For nitrate shortage, this
mutant is employed in paddy fields as a soil amendment alongside commercial nitrate fertilizer
without impacting N2-​fixation. Singh et al. also improved an A. variabilis mutant that was grown in
a weedkiller environment. Advancements in synthetic biology have produced a new study area in
algal biotech which may provide a stronger solution to these challenges (Irisarri 2006).

10.10.5.3 Large-​scale Algal Growth

Biomass production for farming production, particularly from waste streams, has become a prof-
itable industry. Several research groups have linked the generation of microalgae biomass with
the bioremediation of industrial effluent. It is reported that using cyanobacteria will help only to
improve soil quality but it will not reduce the use of pesticides (Gupta et al. 2015). Its potential to
increase soil’s lateral rigidity, availability of nutrients, and crop growth has been further exploited
under a water-​constrained environment, which is critical for extended crop growth. Indigenous algae
strains are superior for fertilizer applications, according to the findings of previous investigations
(Schreiber et al. 2017).
Algae’s role in removing toxins from various ecosystems is presently undervalued. Several
studies have emphasized the importance of economically viable algal production. Algal biomass
can be utilized for grazing, bioenergy fuel, or organic fertilizers after harvesting (Chisti 2016).
On hydrocarbon discharge, municipal wastewater, slaughter house effluent, municipal wastewater,
residential sewerage, industrial discharges, farmed effluent, and milk raw sewage, phytoplankton
varieties were effectively produced. Barminski et al. used tank techniques, dump approach, outdoor
process, and nurseries combining algal methods of production to produce medium compositions for
continuous development of N-​fixing cyanobacteria (Lian et al. 2018).

10.11 FORMULATION OF ALGAL BIO-​FERTILIZERS

A commercially feasible algal bio-​fertilizer composition has been created and tested. Particulate
inoculants, for example, were used by Dubey et al. for longer-​term strain inoculation in soil. The
algal population in the soil was 10–​70 times higher in the injected regions and after 4 months.
Mishra et al. supplied technologies to growers after they received a soil-​based bacterial strain,
allowing them to produce bio-​fertilizer themselves with minimal additional imports. Tripathi et al.
used a technology known as fly-​ash (FA) to improve rice plant growth rate and yield by combining
cyanobacteria with nitrogen fertilizers. As a result of this method, the demand for nitrogen fertilizers
has decreased (Saif et al. 2021).
Using transparent containers, dry blending, and agricultural residues as a carrier can extend the
shelf life of macroalgal soil amendments. The use of phytoplankton as a carrier boosted inocula-
tion absorption and longevity. To reduce the risk of methanotrophic illness after preservation, it
was observed that tobacco waste outperformed its competitors. Moreover, because akinetes may be
dried for at least several weeks, it was suggested that they be used as a bio-​fertilizer after drying.
The effects of temperature and pH on algal growth have been studied (Prasanna et al. 2020). Wet
blue-​green algae (BGA) can be air dried or baked for 24 hours at 35–​40°C in the dark to preserve
cyanobacteria. This method is simple to use and ensures a high germination rate. Grzesik et al.
studied foliar bio-​fertilization by algae on willow monocultures and discovered that it improved
yield potential (Prasanna et al. 2015). Foliar applications of aqueous extracts of Acutodesmus
dimorphus, as well as the formulation of the nutrient broth, have been proven to have a positive
impact on germination and seedling growth in studies. A. dimorphus cellular extract and dry bio-
mass were shown to be effective as a bio-​stimulant and bio-​fertilizer in Roma seedlings, promoting
faster germination, plant growth, and flower production. The carrier-​based formulation is better for
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192 Biofertilizers for Sustainable Soil Management

N2-​fixing fertilizers and soil preparation, whereas the intervening factors composition is superior for
germinating boosting impacts (Holajjer et al. 2013).

10.12 CHALLENGES AND MEASURES FOR COMMERCIALIZATION

Finding an appropriate carrier for each algae, as well as soil and climatic conditions, and biotic
and abiotic stresses in the field, are all major obstacles to commercial use of microalgae as a bio-​
fertilizer. Because of their habitat and growth needs, cyanobacteria-​based organic fertilizers have
been frequently employed in improving paddy field productivity (Zou et al. 2021).
According to various studies, macroalgal bio-​fertilizers are limited in places where pesticide
wastes, herbicide by-​ products, toxic substances (including aluminium and copper), or highly
saline soils are prevalent. Algal development, cellular photosynthesis, and nitrogen fixation could
all be hampered by these chemicals. All four cyanobacterial cultures have been utilized as tools
for researching intracellular metabolic processes involved in the creation of medicinal and eco-
nomically useful compounds for a long time. Sharma et al. suggested integrating multidisciplinary
methodologies with a multiproduct process (biorefinery) strategy in order to get the most out of
cyanobacteria (Sampathkumar et al. 2019).

10.12.1 New Pollutant Factors Discovered in Wastewater

Microalgae have been shown to be able to absorb nutrients from wastewater, including ammonia,
phosphate, and hydrocarbons. Algal blooms, on the other hand, are not a good strategy for effluent
treatment since they might bring more polluting components into the aqueous phase. Algal devel-
opment, for example, can raise the acidity of effluent, therefore acidic effluent cannot be released
immediately (Molinuevo-​Salces et al. 2019). After algal cultivation, the pH of wastewater reached
9.32 in Khan et al (2019)’s research. Spirulina sp., a kind of cyanobacteria commonly used as a
bio-​fertilizer feedstock, elevated the pH of the culture medium to 10.60, according to Cardoso et al.
(2021). Second, a variety of prokaryotic cyanobacteria may emit poisonous components, resulting
in ecological disasters in waterbodies (Win et al. 2018). Finally, because most technologies cannot
guarantee 100% system throughput, some live algae may persist in the water phase after wastewater
treatment. Living creatures will penetrate streams as a result of effluent discharged, promoting algae
growth and disturbing ecological equilibrium. The functions of algae in treating wastewater should
be thoroughly researched due to the different pollution elements supplied by phytoplankton, not-
ably cyanobacteria. These new contamination components, especially bacterial toxins and buffering
capacity, may be more detrimental to the ecosystem than eutrophic wastewater, in our opinion (Win
et al. 2018).

10.12.2 High Cost of Algal Biomass

One of the most significant barriers to commercializing algal biomass is the excessive cost of manu-
facturing. Choosing algal varieties, developing plankton, collecting algae, drying biomass, and pre-
paring fertilizer are all steps in the manufacturing of algal bio-​fertilizers, which all result in excessive
organic matter and algae bio-​fertilizer prices (Sharma et al. 2021). Owing to the reduced economics
of conventional agriculture, utilizing algal bio-​fertilizer to replace chemical fertilizer in large-​scale
agricultural output is not the best option for farmers in underdeveloped countries (Iqbal et al. 2021).

10.12.3 Contamination of Algal Biomass

Toxic pollutants in sewage, especially heavy metals, may contaminate algal biomass, resulting in
toxic pollutants building up in the soil and crops. Furthermore, algae with hydroxyls and carboxyl
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Potential Applications of Algae-based Bio-fertilizers 193

on their cell surfaces are charged negatively and function well in the adsorption of heavy metals. As
a result, algae cultivation entails solubilizing and accumulating heavy metals while also devouring
minerals from growth materials (Han et al. 2020). Constant irrigation with wastewater may result
in heavy metal accumulation in the soil and, as a result, increased heavy metal absorption by crops
and plants. As a result, soil irrigation with sewage is strictly prohibited in many countries, as it has
the potential to cause huge ecological disasters and pollution. If heavy metal ions in sewage are not
properly cleaned, employing algae for soil fertilization is another method of irrigating fields with
wastewater, since algae may convey dangerous compounds into the surface (Tripathi et al. 2008).

10.12.4 Water Consumption and Water Loss

Algae cultivation is a water-​intensive process that takes a huge amount of water and produces
effluent containing leftover nutrients and/​or other pollutants (Zou et al. 2021).

10.12.5 Potential Threats of Cyanobacteria in the Environment

Microcystins have been seen to accumulate in salad lettuce and clover, posing a threat to food safety.
Secondly, live cyanobacteria used as bio-​fertilizers can spread to neighboring bodies of water, gen-
erating an algal bloom and biological invasions. This problem poses a serious threat to the modern
agricultural environment, which frequently involves both crop farming and fish aquaculture. Other
factors preventing algal bio-​fertilizer use in agricultural production include residueal nutrition in
sewage after algal growth, difficult storage and transportation of solvent bio-​fertilizers, unregulated
removal efficiency of microalgae biomass for use as a slow-​release bio-​fertilizer, and reduced con-
sumer acceptance of bio-​fertilizers (Win et al. 2018).

10.13 CONCLUSION
Algae-​based bio-​fertilizers have proven to be useful in the growth of green agriculture. In addition
to N2 fixation, they can improve soil fertility and enable the growth of crops that PGPR benefit.
Carrier technologies are widely available. Furthermore, supplying farmers with the technology they
require for their individual demands can provide value and assist in the development of small-​scale
bio-​fertilizer production in their area.
Other advantages of algal fertilizers include the expansion of organic agriculture without the need
for a large amount of land or even the efficient use of land areas. Finally, algal organic fertilizers can
be employed to meet the needs of sustainable agriculture, with three primary aims in mind: environ-
mental wellness, economic welfare, and social justice (Win et al. 2018).

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11 Ectomycorrhizal Fungi
Role as Bio-​fertilizers in Forestry
Hafiz Muhammad Rashad Javeed,1* Mazhar Ali,1
Muhammad Shahid Ibni Zamir,2 Rafi Qamar,3 Muhammad
Mubeen,1 Atique-​ur-​Rehman,4 Muhammad Shahzad,5
Samina Khalid,1 and Ayman EL Sabagh6
1
Department of Environmental Sciences, COMSATS University
Islamabad, Punjab, Pakistan
2
Department of Agronomy, University of Agriculture, Faisalabad, Punjab,
Pakistan
3
Department of Agronomy, College of Agriculture, University of
Sargodha, Sargodha, Punjab, Pakistan
4
Department of Agronomy, Bahauddin Zakariya University, Multan,
Punjab, Pakistan
5
Department of Agronomy, University of Poonch Rawalakot, AJK,
Pakistan
6
Department of Agronomy, Faculty of Agriculture, University of
Kafrel-​sheikh, Kafrel-​Sheikh, Egypt
Correspondence: [email protected]

CONTENTS
11.1 Introduction......................................................................................................................... 197
11.2 Analysing the Diversity of Ectomycorrhizal Fungi in Forest Ecosystems.......................... 198
11.3 Selection of ECMF for Sustainable Development.............................................................. 199
11.4 Ecological Functions of ECMF........................................................................................... 199
11.5 Evaluation of ECM Fungi................................................................................................... 199
11.6 ECMF and Forestry............................................................................................................. 200
11.6.1 Applications of ECMF in Forest Nurseries.......................................................... 200
11.6.2 Application of ECMF in Forest Management...................................................... 201
11.6.3 Role of Mycorrhizal Networks in Functioning of a Forest Ecosystem................. 202
11.6.4 Impacts of the Interaction between CMNs and Trees........................................... 203
11.6.5 Services of CMNs in the Composition and Functioning of Forest Plant
Communities......................................................................................................... 203
11.7 Conclusion........................................................................................................................... 204
References....................................................................................................................................... 204

11.1 INTRODUCTION
Fungi have a great role in land colonization, and the colonization of soil by plants without fungi
would probably not have been possible (Jansa et al. 2008). Phototrophs face many difficulties while
moving from an aquatic environment to an aerial habitat such as due to water shortages and/​or
a limited supply of soluble minerals, especially phosphorus (Mullineaux and Liu 2020). These

DOI: 10.1201/9781003286233-11 197

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198 Biofertilizers for Sustainable Soil Management

difficulties are overcome by photosynthetic organisms by making a mutualistic relationship with

the fungi that are called mycorrhizas (Hoffland et al. 2004). Fungi which have a symbiotic and
mildly pathogenic association with the roots of plants are called mycorrhizae (Goltapeh et al. 2008).
Mycorrhizae are divided into two classifications: ectomycorrhizae and endomycorrhizae (Bonfante
and Anca 2009; Shah et al. 2016). Ectomycorrhizae have an interaction with plant roots that is called
symbiosis. Intracellular hyphae make the Hartig and mantle network from the ectomycorrhizae
fungi (ECMF) (Mosse et al. 1981). Arbuscular mycorrhizae fungi (AMF) made from the arbuscules,
vesicles which variate as compared to ECMF and make association with trees and herbaceous
plants. Endomycorrhizae are also subdivided into different classes such as orchid mycorrhizae,
monotropoid mycorrhizae, arbutoid mycorrhizae, arbuscular mycorrhizae, and ectendo mycor-
rhizae (Dickie et al. 2013). These classes are categorized according to the attack of fungal hyphae
on plant roots and varies according to the intracellular development of fungal hyphae (Brundrett
2004). Mainly ectomycorrhizal are Basidiomycetes and some ectomycorrhizal are Ascomycetes
(Sharma 2017). In this symbiotic association, the Hartig network is the main one which provides
the metabolic exchange between the roots of plants and fungi. The mycorrhizal mantle is linked
to the fibres of fungi and spread over the soil. The mycorrhizal mantle performs functions such as
captivation, conducting the nutrients from one place to another, helping in mobilization, and pro-
viding water to the roots of plants (Horton and Bruns 2001). Ectomycorrhizae include more than
7000 species of fungi, and include significant profitable trees such as oak, pine, and poplar (Rinaldi
et al. 2008). Macromycetes are called mushrooms when the reproductive structures are produced on
soil, while they are called truffles when they are produced underground (Suz et al. 2012). It is very
important to identify a mutualistic partner and its functions to comprehend the biological signifi-
cance of symbiosis. ECMF were originally classified on the basis of their fruiting bodies but are now
divided based on direct identification (Fitter 2005). Mostly fungi are edible, such as saprophytes,
and some are ECMF. Some ECMF which have been studied are truffles, chanterelles (Cantharellus
spp.), and matsutake mushroom. The largely cultivable fungi are tuber melanosporum and black
truffle or perigord, however other mushroom species are not cultivated, including white truffles
(T. magnatum), an expensive Italian fungus (Savoie and Largeteau 2011).
ECMF provide many services in terrestrial ecosystems, especially in forest ecosystems, such as
nutrient cycling, etc. The global interest in ECMF is mainly in regard to eatable fruiting bodies and
the production of biomass, moreover, restoration of ecosystems and reforestation are also of great
interest (Requena 2005). Recently, scientists have been focusing on the application of ECMF in bio-
remediation, bio-​fertilization, and controlling pathogens. In this regard, a novel and innovative step
is the valuation of ECMF in forest management. This will ultimately be very helpful in sustainable
development (Alizadeh 2011).

11.2 ANALYSING THE DIVERSITY OF ECTOMYCORRHIZAL FUNGI IN FOREST

ECOSYSTEMS
To analyse the functional structure of ECMF, describing their biodiversity in forest ecosystems is
very important. Firstly, it can be checked by numbering the fruiting fungal entities of recognized
ECMF (Buscot et al. 2000). Taxonomic identification can be done by using different mycological
identification methods, but most of the fungi forming ECMs do not fulfil this requirement with
most forming many unavailable fruitbodies (Agerer 2001). If described morphologically, the root
tips of ECMF can be divided into anatomotypes and morphotypes. However, usually, the root tips
of ECM are morphologically and anatomically not identified at the fungal species level (Dahlberg
2001). For the past 20 years, PCR-​based techniques and methods have been used for these types of
analyses. Amplification of the internal transcribed spacer (ITS) area of the ribosomal genes is the
most applicable method for species level identification. DNA extraction from only a single mycor-
rhizal tip allows identification of ECM, then PCR amplification and sequencing are followed by its
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comparison with the sequences of databases, which has enabled the discovery of many novel ECM
fungi such as Tomentella sp., Clavulina sp., etc. (Courty et al. 2010a,b). The assessment of ECM in
many forest ecosystems has been boosted by advancements in sequencing technologies and success
in database searches. The analysis of the 16S rNA gene enables scientists to understand and elab-
orate the bacterial community and ectomycorrhizosphere diversity (Uroz et al. 2007).

11.3 SELECTION OF ECMF FOR SUSTAINABLE DEVELOPMENT

Most preferred ECMF are selected on the basis of the following biotic criteria: isolation, weather or
climatic conditions, high or low temperature, insolation, moisture content, properties of soil, such as
quality, texture and pore spaces, ability to tolerate the abiotic stress of soil, alleviation of soil con-
tamination, mobility of metal in soil, and/​or nutrient cycling (Azul et al. 2014; Khan et al. 2016).
ECMF can also be selected on the basis of increased biomass of plants, growth and development
of plants, and specification of plant or plant health. There are also different criteria relating to fungi
which include fungus availability, effectiveness, ability to grow, and ability to be eaten. Other cri-
teria on which fungus selection is based involve functioning of the environment, human well-​being,
food availability, and nutraceutical value (Suz et al. 2012).

11.4 ECOLOGICAL FUNCTIONS OF ECMF

ECMF perform many functions such as increasing nutrient and water supply, and increasing the land
availability for plants. ECMF increase the availability of those nutrients which are not easily avail-
able to plants (Claridge 2002). They also improve the absorption mechanism of P which includes the
following: postponement of extrametrical hyphae and Pi (inorganic) transfer, mobility of organic P,
release of phosphatases, Pi transporters on the soil edge, and ectomycorrhizal fungi. In forestry, they
act as bio-​fertilizers and help in the mobilization of insoluble mineral Pi, and release organic acids
with low molecular weights. They also improve the N absorption mechanism in the nitrogen cycle
(NH4+​ NO3) and acclimatization of organic nitrogen (releasing chitinases and protease) (Nara 2008;
Ibrahim et al. 2016). Colonies of ECMF around roots provide resistance to soil pathogens. Plants
also obtain many non-​nutritive benefits due to acclimatization of carbon, variation in the water
relation, and the level of phytochromones. ECMF transfer carbon through the fungal mycelium
through which different species of plants are connected. Therefore, this improves the stability and
variety of ecologies and decreases competition among them (Mello and Balestrini 2018). Placental
and marsupial mammals use epigeous and hypogeal sporocarps of ECMF as a food source. The
roots of mycorrhizae, their mycelium, and the fruiting bodies provide homes for invertebrates and
are a significant food source. The microbial population present in soil is affected by mycorrhizae
and the exudates in the mycorrhizosphere and hyphosphere. Soil structure is also improved by the
hyphal network formed by ECM (Koide et al. 2014). Mycorrhizal fungi also alter the quality and
quantity of soil organic content and subsidize the carbon in soil. They also increase the resistance of
plants against biotic and abiotic stresses. There are two types of fungal species, hydrophilic, which
are drought sensitive, and hydrophobic, which are drought tolerant. These different species have
varying reactions depending upon the hydraulic redistribution pattern (Prieto et al. 2016).

11.5 EVALUATION OF ECM FUNGI

Biodiversity, climatic stability, biogeochemical cycles, and economic growth are regulated and
boosted by mycelial chains of ECMF in forest ecosystems. ECMF regulate and enhance the growth
of host plants and also create great metabolism networks that play an important role in the functions
of an ecosystem. This is ultimately very good for the health of plants (Smith and Read 2008). These
fungi are vital drivers for sustainability, innovation, and research in biotechnology, food industry,
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200 Biofertilizers for Sustainable Soil Management

agroforestry, biomedicine, etc. (Courty et al. 2010a,b). Globally, native forests are under threat from
lack of management, pollution, fire, contamination of soil, spread of diseases, and water scarcity. In
forest management strategies, the interactions among ECMF and native species are considered to
be very important (Dahlberg et al. 2010). However, in a bio-​industrial innovation, the contribution
of ECMF has been underestimated. The most fundamental services of ECM fungi to mankind are
as food, indicators of quality of the environment, enhancement of the value of landscape, ecology
tourism, and multifunctionality. In addition, many compounds that are bioactive have been collected
from ECMF, and these have many biological activities, properties, and applications. Some of these
are organic compounds with low molecular weight that can be utilized in the food production indus-
tries to enhance the flavours of mushrooms (Mizuno and Kwai 1992). Some are compounds with
antioxidant and anticancer properties (Arshad et al. 2016; Kreisel et al. 1990). The polysaccharides
in these compounds could be employed in the diets of diabetic patients and show immunosuppres-
sive action (Huijuan et al. 1994). Some of the lipids and fatty acids from them also have antioxidant,
anticancer, immunosuppressive, and anti-​inflammatory properties (Reis et al. 2011). Finally, the
enzymes have roles in the pollution control, detergent, textile and paper, food, and cosmetic indus-
tries (Casieri et al. 2010; Gupta et al. 2003).

11.6 ECMF AND FORESTRY

ECMF have had applications in forestry since the late 1950s, as they are considered as bio-​fertilizers.
These fungi have the ability to increase the up-​take of P, N, water, and some other nutrients by plants,
and ultimately boost and nourish plant growth (Feldmann et al. 2009). Studies are being carried out
to understand the functions of ECMF and their practical use in forestry. Nursery inoculation and
successful inoculation of tree seedlings have been described. Before outplanting, those seedlings
which are inoculated in the nursery can form a great system of ECMF (Domínguez-​Núñez et al.
2019). The ECMF’s interaction with symbiotic fungi expands to larger fungal communities that also
have saprotrophic species in rich forest soils. In addition, oxidation of different phenolic substrates
may be catalysed by various other soil prokaryotes. All this illustrates the functional complexity of
the ectomycorrhizosphere microflora (Frey-​Klett et al. 2007).

11.6.1 Applications of ECMF in Forest Nurseries

Producing a good-​quality ECMF plant that is inhabited by a specific fungus is a huge challenge
in the synthesis of ECMF symbiosis. The essential steps of the production procedure are the
accurate inspection of the employed inoculum and limiting adulteration during the development
of inoculated plants. These steps can prevent the mix-​up of genetic material of unwanted species
with the indigenous species and can stop the attack of unwanted species (Murat and Martin 2008).
The most important step for successful mycorrhization is proper selection of the appropriate plant-​
host species. For this purpose, generally, fast-​growing fungi are selected as they have a short incu-
bation period (Olivier 2000; Fatima et al. 2016). However, some undesirable kinds of ECMF are
found. Therefore, it is preferred to use fresh cultures, rather than repeatedly transferring and storing
cultures (Marx 1980). It is suggested that best practice is to pass essential fungus cultures from
a host inoculation and mycorrhiza development and then its re-​isolation to maintain the fungus-​
forming capacity (Amaranthus 1998). In addition, these fungi are superior in the uptake of minerals
and soil exploration that make large hyphal stands of rhizomorphs in soil cultures, as compared to
those that lack rhizomorphic growth. On the other hand, the mycorrhizal state of the seedlings alone
does not determine the fruiting of the ECMF species (Hortal et al. 2009). After planting, the spread
and persistence of the cultivated fungus is influenced by the already-​present native competitors and
physiochemical or biotic properties of that soil. The progress of a mycorrhizal inoculation pro-
gramme is influenced by the kind of ECMF material employed for inoculation (Rossi et al. 2007).
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Ectomycorrhizal Fungi 201

The sources of fungal inoculum include utilizing the spores of fruiting bodies taken from the field.
This is a suitable method as the spores do not need the aseptic culture’s extension, moreover, the
spore inoculum is not heavy (Sharma 2017). The disadvantages of this method are the process’s
success is determined by the viability of the spores, and a sufficient amount of fruiting entities are
needed that might not be available every year, and also, the genetic definition may not be sufficient.
Before inoculating in soil, the spores should be mixed with some sort of physical supports such
as being soaked in water, suspended in water, sprayed and sprinkled on the ground, embedded in
hydrocolloid chips, or coated on the seeds. Their viability can be maintained by freezing and storing
them at low temperature in a dark place (Marx and Cordell 1989). The second method is the use
of humus or soil obtained from the soil in which mycorrhizae seedlings are going to be embedded.
The disadvantage of this method is that there is no control over the species of ECMF present in the
soil or of other microbes and harmful pathogens. This method is discarded in the mycorrhization
programmes but is still widely used in developing countries. Moreover, a “nurse seedling” of mycor-
rhizae is planted or may be a chopped ECMF root host that is incorporated into the nursery beds as a
sources of fungi for the adjacent young seedlings (Sim and Eom 2006). The third method is the use
of hyphae in a substrate, liquid, or solid medium as an inoculum. The success rate of fungal hyphae
from mycorrhizae is very low, at about 5–​20%. Therefore, it is mainly cultivated from the sterile
portions of the fruiting bodies rather than mycorrhizae (Molina 1982). Very often, fungal hyphae are
taken from sexual spores or less frequently from sclerotia (Fries and Birraux 1980). This method
enables the choice of specific strains of a fungus, which is why it is considered the most suitable
method, as firstly it is checked for its suitability to enhance plant growth (Marx 1980). Most of the
species of Suillus, Hebeloma, Laccaria, Amanita, Rhizopogon, and Pisolithus grow very well in
culture. Liquid substrates are more appropriate as compared to solid ones as they can be mixed very
easily, therefore they make a uniform state for crop growth. However, the risk of bacterial attack
is very high with this method and it is somewhat costly (Rossi et al. 2007; Hussain et al. 2016).
Meanwhile, the major benefit of hard solid media is limited pollution by bacteria because of less
moisture content, cheap equipment, i.e. low cost, and the simple and easy design of the bio-​reactors
(Cannel 1980). The major disadvantage of the utilization of mycelial inoculum is that various var-
ieties of ECMF are not easy to produce in the lab environment, because of a slow growth rate due
to the lack of their symbiont, and producing a huge amount of inoculum is also not very easy. Some
recent techniques have been invented using mycelium encapsulated in “beads” of calcium alginate,
but these must be kept in a refrigerator (Le Tacon et al. 1983). Inoculum beads can remain viable
for a few months if kept in refrigerator, however the outcomes vary between fungal species. It has
been tested with many plants of economic interests for several species. Therefore, these processes
have great ability and potential to be used in reforestation programmes. A bio-​reactor was designed
by Rossi et al. (2007) which have the capability of make inocula for 300,000 seedlings, which is
sufficient for the reforestation of about 200 hectares of land. On the basis of 3 billion cubic metres of
global wood demand, approximately 4.3 tons of mycelium would be required to inoculate 12 million
seedlings, i.e. 5 g of dry mycelium per plant. The benefit of alginate gel is the possibility of making
a multi-​microbial inoculant (Rossi et al. 2007).

11.6.2 Application of ECMF in Forest Management

ECMS can be injected to increase the production of edible carpophores but it is also very signifi-
cant in forest management, especially in reforestation, where it increases the quality and economic
productivity of plantations (Tommerup and Bougher 2000; Garbaye 1990). In nurseries, the growth
of plants having mycorrhized seeds depends on how quickly they obtian the nutrients and the avail-
ability of water in the soil. In mycorrhizal plantations, the diversity of mycorrhizae depends on
the introduction of other dominant species from the mixed communities. Instead, unfortunately,
the fungi which are chooses from the nursery for the best colonization have been deprived of
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202 Biofertilizers for Sustainable Soil Management

competitors in the field, particularly in the planting areas where native fungi are present (Dunabeitia
et al. 2004). There are many possible ways through which we can explain the failure of inocula-
tion to make positive impacts in the planting areas. Possibly, the fundamental cause of failure is
the persistence of inocula in the roots after transfer of plants from the nursery to the field. The soil
conditions which plants experience during planting are different from those of plants growing in a
nursery or a container and there is also a reduction in the strength of roots and their fungal associates
(McAfee and Fortin 1986). The species which show the greatest growth in the inoculation program
are Pisolithus tinctorius (15 subspecies), in conditions such as a polluted environment or a shortage
of an autochthonous mycorrhizal population. In the case of an edible ECMF of interest such as a
tuber or black truffle, with an artificially mycorrhized plant, it shows that the formation of plots has
always been the chief goal of developing fruiting bodies, leaving in the background the impact of
the ecological roles of the symbiosis (Núñez et al. 2006; Garbaye 1990). The prominent and vital
service of ECMF forest plants are restoration of the ecosystem by bioremediation, bio-​fertilization,
and control of soil pathogens. Unplanned industrial activities and unfavourable land management
ultimately lead to a degraded ecosystem. The main causes of environmental degradation all over the
world include salinity stress, soil erosion, compactness of soil, and shortage of water. A balanced
ecosystem contains composed and balanced microbiota in the soil, in such a way that there is har-
mony among potential pathogenic and mycorrhizal fungi (Geml et al. 2012). ECMF can exist in life-​
threatening conditions such as extreme temperature, drought, metal and salt concentrations, drought,
and other situations that lead to degradation of the ecosystem (Colpaert et al. 2011). ECMF have
great significance because they help to balance the ecosystem in many ways, such as by enhancing
plants’ ability to tolerate abiotic stresses, the ability to ameliorate heavy metals or to reduce organic
matter, interactions with pathogenic nematodes, soil microbes, and bacteria, and increased nutri-
tional quality, growth, and development of their associate plants. Furthermore, ECMF’s extraradical
mycelium offers a path for the translocation of photosynthesized C to microbes and a huge surface
area for the microorganisms to contact (Suz et al. 2012; Machuca 2011). It has been discovered that
ECMF help in the rhizosphere to remediate from persistent organic pollutants (POPs) (Meharg and
Cairney 2000).

11.6.3 Role of Mycorrhizal Networks in Functioning of a Forest Ecosystem

ECMFs’ extraradical mycelia join the roots of the same or a distinct ECM tree species, forming
common mycorrhizal networks (CMNs) that permit net C and nutrient exchange between host plants
(Lindahl and Tunlid 2015). Several review publications have established the role of CMNs in plant
community dynamics (Perry et al. 1992). However, problems in researching mycelial organizations
in situ in the soil without upsetting or killing them, as well as separating symbiotic from sapro-
trophic mycelia, have hampered advances in comprehending the arrangement and function of CMNs
in the field (Simard and Durall 2004). The application of molecular markers has demonstrated
that several ECMF colonize different plant hosts at the same time, implying that the creation of
CMNs in forest stands has high potential. That is confirmed by the fact that various ECM fungal
taxa may develop extremely enormous genets, up to 10 meters wide, and hence can cover a broad
region, spanning many trees of the same or different species (Ishida et al. 2007). Ectomycorrhizal
CMNs are created by Basidiomycetes and Ascomycetes fungal species (Bruns et al. 2002). Although
a few ECMF taxa are host-​specific, the majority are generalists and, at least in temperate ECM
ecosystems, are frequently more numerous than non-​generalists (Richard et al. 2005). Furthermore,
some Ascomycetes that develop ericoid endomycorrhizae with Ericaceae plants may generate
different kinds of CMN that can be incorporated into ECMF chains: some Ericaceae species form
equally ericoid endomycorrhizae and ECM networks (Dunham et al. 2003; Selosse et al. 2006). In
the same way, ECM forest trees and myco-​heterotrophic and mixotrophic orchids might have shared
Basidiomycete symbionts (Taylor et al. 2002).
203

Ectomycorrhizal Fungi 203

11.6.4 Impacts of the Interaction between CMNs and Trees

The importance of resource transmission from one tree to another via CMNs for functional ecology
and geochemical cycling has lately piqued researchers’ interest. Indeed, CMNs have the potential
to create “guilds of mutual help” between synchronized plants, or transfer means along source-​sink
gradients (Leake et al. 2014; Leake et al. 2004). The predominance of the hyphal route for C transfer
from giver to recipient green plants, and in addition to this, the destiny of the C transported and its
physiological significance, are all debatable (Fitter et al. 1998). Many orchids, on the other hand,
have been shown to transition from saprotrophic and harmful fungal associates like Rhizoctonia sp.
to ECM fungi that can deliver huge quantities of C over extended periods of time during their growth
(Simard et al. 2003). Cryptothallus mirabilis, an achlorophyllous liverwort, has been observed
forming ECM connections with Tulasnella, a genus of Basidiomycete that frequently forms orchid
mycorrhizas (Bidartondo et al. 2003). Furthermore, current investigations using steady isotope
enrichment (13C and 15N) have revealed that certain green forest orchids had 13C enrichments that
fell somewhere between entirely autotrophic plants and myco-​heterotrophic plants that obtained all
their C from their associated fungus (Tedersoo et al. 2007). In a method known as “mixotrophy”,
these green orchids get their carbon from a combination of myco-​heterotrophic and photosynthetic
sources. It has been discovered that related fungus provided 38 percent to 80 percent of the carbon to
certain pyroloid shrubs (Ericaceae) and green forest orchids (Tedersoo et al. 2007). Water transport
between conspecific and non-​conspecific neighbouring plants is also facilitated by CMNs, as has
been demonstrated employing isotopic tracers and fluorescent dyes (Querejeta et al. 2003). Water
was also transferred from adult plants to seedlings as a result of the “hydraulic lift”. Hydraulic lift,
on the other hand, is primarily a physical activity that may be carried out by rotten roots and, prob-
ably, fallen rhizomorphs (Egerton-​Warburton et al. 2007; Leffler et al. 2005).

11.6.5 Services of CMNs in the Composition and Functioning of Forest Plant

Communities
One of the most important factors governing the cohabitation of plant species, and hence the forma-
tion of plant networks, is resource competition. Resource sharing has been anticipated as a mech-
anism for reducing resource rivalry, especially in mycorrhizal plant communities (Hart et al. 2003;
Read et al. 2004). This approach is heavily reliant on CMNs as a deciding element in resource distri-
bution. The organization of tree communities is likely to be affected by their membership in a CMN,
at least at the local scale (Smith and Read 2008). Indeed, two plants can contribute uneven amounts
of C to a shared fungus or obtain unequal amounts of nutrients from a fungus that they mutually
support, meaning a net advantage to one species at the expense of the other (Selosse et al. 2006). In
other ways, negative feedback might modify dominant populations’ competitive ability, promoting
plant variety. Furthermore, CMNs can aid seedling recruitment: covered sprouts in the understory
can grow effectively alongside competing ECM tree species by joining to established mycorrhizal
systems and obtaining carbon from mature overstore trees (Cullings et al. 2000). Furthermore, seed-
ling recruitment is aided by early integration into existing mycorrhizal networks, as seedling survival
and establishment rates are determined by the pace at which they become mycorrhizal. For example,
it was found in one study that Pseudotsuga seedling survival was higher in the ECM Arctostaphylos
chaparral than in the non-​ECM Adenostoma chaparral (Horton et al. 1999). Seedlings in touch
with mature ECM trees have greater survival and mycorrhizal colonization than seedlings separated
from adult plants, according to a study in tropical forests and temperate forests (Dickie et al. 2004;
Onguene and Kuyper 2002). Adult trees can therefore serve as “nurse trees” for both conspecific and
non-​conspecific seedlings, promoting species diversity and cohabitation in forest outlooks (Grime
et al. 1987). The selection forces behind this phenomenon, however, remain unknown, as this col-
laboration may be harmful to mature plants (Newman 1988).
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204 Biofertilizers for Sustainable Soil Management

11.7 CONCLUSION
This chapter has described the ECMF’s role in ecosystems as a bio-​fertilizer and how it could be
enhanced, as they significantly contribute to a number of important ecosystem functions, especially
nutrient cycling and C fluxes. The main focus of the research should be evaluating a suitable tech-
nique for commercial manufacturing and huge-​scale inoculation of the ECMF and the use of appro-
priate arrangements for microbes and plants, carried out in a suitable well-​defined soil condition
and environment. The role of ECMF in balancing and maintaining the ecosystem is enormous as it
considerably enhances plants’ tolerance towards biotic and abiotic stresses. The ECMF are funda-
mental because of their role as a bio-​fertilizer in the restoration of the environment and reforestation.
In short, ECMF are very important for environmental sustainability and balanced ecology.

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209

12 Plant Growth-​promoting
Rhizobacteria/​Pseudomonas
as a Biofertilizer
Rafiq Ahmad,*1 Sohail Khan,2 Muhammad Hayat,3
Syed Muhammad Afzal,4 and Javed Muhammad5*
1
Department of Microbiology, The University of Haripur, KP, Pakistan
2
College of Life Science and Technology, State Key Laboratory for
Conservation and Utilization of Subtropical Agro-​bioresources,
Guangxi University, Nanning, Guangxi, China
3
State Key Laboratory of Microbial Technology, Institute of Microbial
Technology, Shandong University Qingdao, China
4
Center of Biotechnology and Microbiology, University of Peshawar, KP,
Pakistan
5
Department of Microbiology, The University of Haripur, KP, Pakistan
* Correspondence: [email protected]

CONTENTS
12.1 Introduction.......................................................................................................................... 209
12.2 Rhizobacteria that Promote Plant Growth........................................................................... 210
12.2.1 Properties of Perfect PGPR................................................................................... 211
12.3 What Are Biofertilizers and How do They Work?............................................................... 212
12.3.1 Advantages of Biofertilizers................................................................................. 212
12.3.2 Pseudomonas as a Biofertilizer............................................................................. 213
12.4 Mechanisms of Action of the Pseudomonas Biofertilizer................................................... 213
12.4.1 Solubilization of Phosphate.................................................................................. 214
12.4.2 Siderophore Biosynthesis...................................................................................... 214
12.4.3 Production of Phytohormones............................................................................... 215
12.4.3.1 Phytohormones.................................................................................... 215
12.5 Use of PGPRs as Biofertilizers: Guidelines and Precautions.............................................. 216
12.6 Challenges in Biofertilizers................................................................................................. 216
12.7 Current Challenges Due to Pseudomonas Biofertilizers..................................................... 217
References....................................................................................................................................... 217

12.1 INTRODUCTION
The rhizosphere is the layer of soil surrounding the plant roots which plays a critical role in
plant growth and development. This tiny zone surrounding the plant roots is a key hotspot for
microorganisms and is considered one of the most complicated ecosystems (Mendes et al. 2013;
Raaijmakers et al. 2009). Hiltner (1904) used the term “rhizosphere” to characterize the limited
soil zone where the root boosts microbial populations (Drogue et al. 2013; Meena et al. 2020;
Shukla et al. 2011; Zehra et al. 2021; Al-​Zahrani et al. 2022; Rajesh et al. 2022; Anam et al. 2021;
Deepranjan et al. 2021; Haider et al. 2021; Amjad et al. 2021; Sajjad et al. 2021a; Fakhre et al.

DOI: 10.1201/9781003286233-12 209

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210 Biofertilizers for Sustainable Soil Management

2021; Khatun et al. 2021; Ibrar et al. 2021; Bukhari et al. 2021; Haoliang et al. 2022; Sana et al.
2022; Abid et al. 2021; Zaman et al. 2021; Sajjad et al. 2021b; Rehana et al. 2021; Yang et al. 2022;
Ahmad et al. 2022; Shah et al. 2022). Plant roots produce diverse metabolites with a plentiful supply
of amino acids, vitamins, sugars, and organic matter. The rhizosphere microbiome is the total micro-
bial population present in the rhizosphere and significantly differs from the nearby soil microbial
population (Chaparro et al. 2013; Kumar & Dubey 2020). Microbial taxa inhabit the rhizosphere,
including prokaryotes (bacteria, archaea) and diverse eukaryotes, including (fungi, algae, protozoa,
nematode, arthropods, and viruses. However, bacteria and fungi are the most prevalent (Buee et al.
2009; Kalam et al. 2017).
According to Kloepper (1980), plant growth-​promoting rhizobacteria (PGPR) are soil bacteria
that thrive in the rhizosphere, colonize plant roots aggressively, and aid plant development (Dutta
and Podile 2010; Adnan et al. 2016). PGPR bacteria play a significant role in plant growth and help
prevent the spread of phytopathogenic microorganisms (Kloepper et al. 1980; Son et al. 2014).
Microbes may indirectly impact plant phenotypic plasticity and plant health by modulating plant
growth and defensive responses due to their lengthy co-​evolution with plants (Gao et al. 2015). They
penetrate and grow in the rhizosphere environment inside the plant microbiome, gaining traction in
agricultural operations as traditional chemical fertilizers (Compant et al. 2019; Muhammad et al.
2022; Wiqar et al. 2022; Farhat et al. 2022; Niaz et al. 2022; Ihsan et al. 2022; Chao et al. 2022, Qin
et al. 2022; Xue et al. 2022; Ali et al. 2022; Mehmood et al. 2022; El Sabagh et al. 2022; Ibad et al.
2022). PGPR plays a vital role in sustainable agriculture, enhancing crop production, soil fertility,
biodiversity, and interaction with other beneficial microbes and reducing pathogen development
and infection (Tapia-​Vázquez et al. 2020). As a result of these processes, PGPR could be used as a
biofertilizer (Vessey et al. 2003). A biofertilizer is a substance that includes live microorganisms that
colonize either the internal plant tissues or the rhizosphere and enhance plant growth when applied
to soil or plant surfaces (Verma et al. 2019). Their rise to prominence as a viable alternative stems
from the abuse of agrochemicals such as fertilizers and pesticides, which pollute the soil, fruits,
and vegetables (Adesemoye et al. 2009). Among the PGPR, Pseudomonas, Klebsiella, Alcaligenes,
Arthrobacter, Azospirillum, Enterobacter, Burkholderia, Serratia, and Bacillus are the most signifi-
cant (Bhattacharyya et al. 2012; Kumari et al. 2018).
Pseudomonas is a genus that contains the most varied group of bacteria on the planet, with over
100 distinct species (Heiman et al. 2020). It belongs to the gamma subclass of proteobacteria and
is found in diverse habitats, including marine ecosystems, freshwater, and terrestrial environments.
They have a close relationship with higher life forms and are among the most studied bacterial
species (Selvakumar et al. 2015; Saleem et al. 2015). The properties of plant growth-​promoting
(PGP) Pseudomonas strains that may improve agricultural production in both standard and stressed
settings are discussed in this chapter.

12.2 RHIZOBACTERIA THAT PROMOTE PLANT GROWTH

To achieve sustainable agriculture, plants should be disease-​resistant, drought-​tolerant, and metal
stress-​tolerant and have a higher nutritional value (Deepranjan et al. 2021; Haider et al. 2021; Huang
Li et al. 2021; Ikram et al. 2021; Jabborova et al. 2021; Khadim et al. 2021a,b; Manzer et al. 2021;
Muzammal et al. 2021; Abdul et al. 2021a,b; Ashfaq et al. 2021; Amjad et al. 2021; Atif et al. 2021;
Athar et al. 2021; Adnan et al. 2018a,b; Adnan et al. 2019; Akram et al. 2018a,b; Aziz et al. 2017a,b;
Chang et al. 2021; Chen et al. 2021; Emre et al. 2021; Habib et al. 2017; Hafiz et al. 2016; Hafiz
et al. 2019; Ghulam et al. 2021; Guofu et al. 2021; Hafeez et al. 2021; Khan et al. 2021; Kamaran
et al. 2017; Muhmmad et al. 2019; Safi et al. 2021; Sajjad et al. 2019; Saud et al. 2013; Saud et al.
2014; Saud et al. 2017; Saud et al. 2016; Shah et al. 2013; Saud et al. 2020; Saud et al. 2022a,b). The
use of soil microorganisms (bacteria, fungi, algae, etc.) that boost nutrient absorption capacity and
water use efficiency (Armada et al. 2014) is one option for achieving the aforementioned desirable
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Plant Growth-promoting Rhizobacteria as a Biofertilizer 211

crop attributes. The microbial populations greatly influence a plant’s health. Diverse microbiomes
populate different environments in plants, including the phyllosphere, endosphere (tissues), and
rhizosphere (Berendsen et al. 2012). The rhizosphere is described as soil adjacent to plant roots and
contains a lot of plant rhizobacteria (Bakker et al. 2013). The rhizosphere contains up to 30,000
diverse prokaryotic cells, and it includes 1011 microbial cells per gram, which positively impact
plant growth (Egamberdieva et al. 2008; Mendes et al. 2013). The procedure of nutrient efficiency
enhancement in soil to increase plant growth and production is known as rhizosphere management
(Shah et al. 2015; Zia et al. 2021; Qamar et al. 2017; Hamza et al. 2021; Irfan et al. 2021;Wajid et al.
2017; Yang et al. 2017; Zahida et al. 2017; Depeng et al. 2018; Hussain et al. 2020; Hafiz et al. 2020
a,b; Shafi et al. 2020; Wahid et al. 2020; Subhan et al. 2020; Zafar-​ul-​Hye et al. 2020a,b; Zafar et al.
2021; Adnan et al. 2020; Ilyas et al. 2020; Saleem et al 2020a,b,c; Rehman 2020; Farhat et al. 2020;
Wu et al. 2020; Mubeen et al. 2020; Farhana 2020; Jan et al. 2019; Wu et al. 2019; Ahmad et al.
2019; Baseer et al. 2019; Hafiz et al. 2018; Tariq et al. 2018). Plant roots produce and accumulate
a variety of chemicals and exudates and provide mechanical support and allow water and nutrient
intake (Walker et al. 2003). These various chemicals and exudates change the physical and chemical
properties of the soil and regulate the microbial community structure at the root surface (Dakora
and Phillips 2002). Bacteria are the most common microbes in the rhizosphere, coexisting alongside
fungi, protozoa, and algae.
Consequently, they probably substantially impact the physiology and competitiveness of root col-
onization in plants (Glick 2012). Their diversity remains powerful with repeated alterations in com-
munity structure and species abundance. PGPR include Bacillus, Serratia burkholderia, Alcaligenes,
Enterobacter, Azotobacter, Azospirillum, Arthrobacter, Klebsiella, and Pseudomonas, which either
live freely or in an association such as symbiotic, parasitic, or saprophytic (Bhattacharyya et al.
2012). However, PGPR in agriculture account for just a tiny percentage of all agricultural practices
globally. This is due to the inconsistencies in the inoculated PGPR’s characteristics, which can
impact crop output. The longevity of PGPR in soil depends on the interaction of crops with indi-
genous microflora and ecological conditions.

12.2.1 Properties of Perfect PGPR

The following characteristics should be present in an optimal PGPR strain:

(1) It should be rhizosphere-​friendly and rhizosphere-​competent.

(2) Upon injection, it should colonize the plant roots in sufficient numbers.
(3) It should be able to encourage the development of plants.
(4) It should have a wide range of actions.
(5) It needs to get along with the other bacteria in the rhizosphere.
(6) It must be resistant to physicochemical elements such as heat, desiccation, radiation, and
oxidants.
(7) It should outperform current rhizobacterial communities in terms of competitive abilities.

For decades, effective biocontrol agents and biofertilizers that have been capable of promoting
plant growth and health, respectively, have been produced. Biofertilizers offer a lot of promise for
increasing crop yields in an ecologically sustainable way. These biofertilizers are used in composting
sites or soil, which enhances the desired microbial population and plays a key role in nutrient recyc-
ling and its availability for plants (Mahmood et al. 2021; Farah et al. 2020; Sadam et al. 2020; Unsar
et al. 2020; Fazli et al. 2020; Enamul et al. 2020; Gopakumar et al. 2020; Zia-​ur-​Rehman 2020;
Ayman et al. 2020; Mohammad I. Al-​Wabel et al. 2020a,b; Senol 2020; Amjad et al. 2020; Ibrar
et al. 2020; Sajid et al. 2020; Muhammad et al. 2021; Sidra et al. 2021; Zahir et al. 2021; Sahrish
et al. 2022) (Figure 12.1).
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212 Biofertilizers for Sustainable Soil Management

FIGURE 12.1 Graphic representation of microorganisms used as biofertilizer. Reprinted with permission
from Fasusi et al. (2021).

12.3 WHAT ARE BIOFERTILIZERS AND HOW DO THEY WORK?

The usage of chemical fertilizers in agriculture has negative consequences for ecosystems (Iqra
et al. 2020; Akbar et al. 2020; Mahar et al. 2020; Noor et al. 2020; Bayram et al. 2020; Amanullah,
Fahad 2018a,b; Amanullah, Fahad 2017; Amanullah et al. 2020; Amanullah et al. 2021; Rashid et al.
2020; Arif et al. 2020; Amir et al. 2020; Saman et al. 2020; Muhammad Tahir et al. 2020; Jakirand
Allah 2020). They leache into water bodies and cause eutrophication in plants due to poor absorp-
tion by plants. They may also have various negative consequences on soils, such as loss of water
storage capacity and uneven soil fertility. They are expensive, and they also increase greenhouse gas
emissions due to the usage of fossil fuels in their manufacturing. There is a need to discover envir-
onmentally friendly, low-​cost, effective fertilizers. The biofertilizers and biocontrol agents have
shown a lot of promise in increasing crop yields in ecologically benign ways. Biofertilizer research
began in 1895 with Nobbe and Hiltner’s “Nitragin,” the first commercially produced and patented
Rhizobium culture. Biofertilizers are microbial formulations that include beneficial microbial strains
that have been immobilized or trapped on inert carrier materials and could be used to improve plant
development and soil fertility.
Biofertilizers are microorganisms found in the rhizosphere that help in plant development by
increasing nutrient accessibility to plants. Formulating microbes and their products to assist in fixing
atmospheric nitrogen, phosphate solubilization, or PGP compounds leads to nutrient solubility that
can boost agricultural plant production.

12.3.1 Advantages of Biofertilizers
1) Biological fertilizers help soils establish biological activity by mobilizing nutrients.
2) The provision of appropriate nutrients helps to maintain plant health.
3) Food is supplied, and beneficial soil worms and microbes are encouraged to develop.
4) Root development is stimulated because of the excellent structure offered to the soil.
5) They encourage the formation of mycorrhizal connections, which increases the amount of
phosphorus (P) available in the soil.
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Plant Growth-promoting Rhizobacteria as a Biofertilizer 213

TABLE 12.1
Role of Pseudomonas Species Involved in Plant and Crop Improvement, Growth, and
Production

Isolate Host Function References

1 P. fluorescens Wheat Enhance leaf and root number, root Jimtha et al. 2017
(R68) length, and fresh weight

2 P. trivialis Fs9 Sunflower Promote shoot length and weight Majeed et al. 2018
3 Pseudomonas spp. Sunflower Enhance plant height Majeed et al. 2018
AF-​54
4 P. fluorescens Sweet potato Increase weight, length of roots and density Santana-​Fernández et al.
of Santana roots 2021
5 P. flourescens Rice Increase seed germination Elekhtyar et al. 20165
6 P. flourescens Melon Effect soil nutrient distribution and Martinez et al. 2019
increase yield quality
7 P. aeruginosa Pongamia pinnata Increase biomass,dry matter uptake, sugar Radhapriya et al. 2015
RRALC3 content, amino acid content, carbon
content
8 Psudomonas spp. Improve plant biomass, relative water Sandhya et al. 2010
content, leaf water potential, and root
adherence
9 Pseudomonas Wheat Improve wheat yield 96% Selvakumar et al. 2014
putida

6) They assist in the elimination of plantar illnesses and the provision of a steady supply of
micronutrients to the soil.
7) They assist in the maintenance of nitrogen (N) and phosphorus (P) concentrations that are
stable.

12.3.2 Pseudomonas as a Biofertilizer

Pseudomonas is a Gram-​negative, non-​spore-​forming, aerobic bacteria with straight or slightly
curved rods. This bacterial genus is found in the Proteobacteria phylum, Gamma proteobacteria
class, and Pseudomonadaceae family. Pseudomonas is also a noteworthy bacterial genus since sev-
eral species are clinically relevant opportunistic human pathogens, plant pathogens, and biocontrol
agents. Pseudomonas aeruginosa is a human pathogen, Pseudomonas syringae is a plant pathogen,
and Pseudomonas putida and Pseudomonas fluorescens are non-​pathogenic biocontrol agents.
Pseudomonas species develop in a variety of ways in agro-​fields, demonstrating their capacity
to stimulate, sustain, and remediate. They produce diverse antifungal metabolites, mediate quorum
sensing, having a synergistic association with the plant root. The majority of Pseudomonas species
live as endophytes and colonize the rhizosphere, promoting plant health by controlling plant-​
pathogenic bacteria and increasing disease tolerance. Different Pseudomonas species involved in
plant and crop improvement, growth, and production are included in Table 12.1.

12.4 MECHANISMS OF ACTION OF THE PSEUDOMONAS BIOFERTILIZER

Pseudomonas are ecologically essential bacteria that plays a vital role in nitrogen and carbon cycles,
because of their capacity to secrete specific chemicals that play a critical role in phosphate solubiliza-
tion, siderophore synthesis, and nitrogen fixation for promoting plant growth. Pseudomonas species
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214 Biofertilizers for Sustainable Soil Management

may operate as both a PGR and in phytopathogen control. Pseudomonas species generate antagon-
istic substances for plant disease control, such as cell wall breakdown enzymes and antibiotics, and
maintain a mutualistic relationship with the linked plant. All of the growth-​promoting characteristics
listed above may be unusual among strains belonging to the same species. Most Pseudomonas spp.,
on the other hand, have been shown to have these basic growth-​promoting properties.

12.4.1 Solubilization of Phosphate
Phosphorus is a critical macronutrient that plants need for optimal development. It is an essential
nutrient since it is involved in metabolic activities, signal transduction, macromolecule production,
and photosynthesis. It was detected in soil at concentrations of 400–​1200 mg/​kg. It has lower solu-
bility, rendering it inaccessible to plants despite its high concentration level.
Due to the fast fixation, phosphorus becomes limited due to the formation of oxides like alu-
minium hydroxide Al(OH)3, calcium hydroxide Ca(OH)2, and ferrous hydroxide Fe(OH)2.
Agricultural areas are often fertilized with phosphate fertilizers to address soil phosphorus shortage.
Therefore, the phosphate fertilizers have weak absorption by plants, and the remaining P fertilizer
is rapidly converted into insoluble complexes in the soil. However, to address this issue, phosphate-​
solubilizing bacteria (PSB) transform insoluble phosphorus into a soluble form. Pseudomonas,
Bacillus, Rhizobium, and Azotobacter are among the most significant PSBs in the industrial world.
The phosphate-​solubilizing bacteria solubilize and mineralize phosphate, as well as produce
organic acids like citric acid and gluconic acid. Similarly, these bacteria also produce phosphatase
enzymes that convert inorganic phosphorus to mono-​or dibasic ions. In Triticum aestivum treated
with Pseudomonas spp., Ma et al. (2012) found an increase in biomass output and phosphorus con-
sumption. PSBs that generate cyanide as a secondary metabolite include Pseudomonas aeruginosa
and Pseudomonas fluorescens. This metabolite is important because it aids PSBs by controlling
phytopathogens and diseases. According to Srivastava (2020), Arabidopsis thaliana infected with
the phosphorus-​solubilizing bacterium P. putida MTCC 5279 flourished under salt stress and
P-​deficient conditions, with considerably greater acidic and alkaline phosphatases activity and
biomass.

12.4.2 Siderophore Biosynthesis
Iron (Fe) is another important nutrient for plants. In an aerobic environment, it is found in Fe2+​
and Fe3+​, oxyhydroxides, and insoluble hydroxides, making it unavailable for plant absorption.
Siderophores are low-​molecular-​weight water-​soluble organic compounds that have an affinity
for iron carriers or iron-​binding molecules. They are classified into extracellular or intracellular
siderophores. Rhizobacteria utilize the siderophores produced by other rhizobacteria of different
genera (heterologous siderophores), though some can use siderophores produced by the same
genus (known as homologous siderophores). Siderophores are mostly produced by Gram-​negative
bacteria, like Pseudomonas and Enterobacter genera, which have a significant role in controlling
rhizospheric phytopathogens. Plants use a variety of methods such as releasing or chelating iron
from siderophores, direct absorption of siderophore–​iron complexes, and a ligand exchange process.
Pseudomonas sp., such as PGPR, meets their iron needs by consuming siderophores produced by a
variety of different rhizosphere microbes. P. putida uses heterologous siderophores made by other
microorganisms to increase the amount of iron accessible in its natural environment. A luminous
siderophore (pyoverdine) plays a key role in the stimulation of plant growth (Rehman et al. 2016).
Pattnaik et al. (2019) found that a mutant strain of Pseudomonas aeruginosa with no ability for sid-
erophore production while its biocontrol activity against Pythium sp. in tomato plants showed infec-
tion rendering with inactive Pseudomonas aeruginosa siderophore complex. Sharma et al. (2003)
investigated the effect of the Pseudomonas strain GRP3 producing siderophore in Vigna radiata
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Plant Growth-promoting Rhizobacteria as a Biofertilizer 215

iron feeding. When compared to control plants, chlorotic symptoms decreased after 45 days, and
iron, chlorophyll a, and chlorophyll b levels increased in strain GRP3-​infected plants.

12.4.3 Production of Phytohormones
Plant hormones are plant growth regulators (PGRs) that works as chemical messengers through
which plants respond to their surroundings (Fahad and Bano 2012; Fahad et al. 2017; Fahad et al.
2013; Fahad et al. 2014a,b; Fahad et al. 2016a,b,c,d; Fahad et al. 2015a,b; Fahad et al. 2018a,b;
Fahad et al. 2019a,b; Fahad et al. 2020; Fahad et al. 2021a,b,c,d,e,f; Fahad et al. 2022a,b; Hesham
and Fahad 2020). These chemical compounds are effective at extremely low concentrations and
are primarily generated in certain areas of the plant before being transferred to another location.
Cross-​talk signalling is the term for this kind of communication. Phytohormones start cell devel-
opment, reproduction, and specialization in the root system. Phytohormones help with root devel-
opment by invading roots directly or indirectly through 1-​aminocyclopropane-​1-​carboxylic acid
(ACC) catalysis. The ACC deaminase hydrolyses the plant ACC and prevents the ethylene at a
phytohormone level, acting antagonistically with the plant, while plant ACC hydrolysed by ACC
deaminase prevents the phytohormone ethylene level, working antagonistically with the plant.
Bacteria, both free-​living and symbiotic, aid plant development by generating compounds that are
functionally equivalent to phytohormones produced by the plant. Auxins, cytokinins, gibberellins,
and ethylene are some of the chemicals involved in the regulation of biological processes important
for plant growth and development (Figure 12.2).

12.4.3.1 Phytohormones
12.4.3.1.1 Auxin
Auxin is a chemical that regulates most plant functions, either directly or indirectly. It plays a key
role in cell division and differentiation of plants, germination, geotropism, phototropism, metabolite
biosynthesis, and stress tolerance. Indole-​3-​acetic acid (IAA) is the most active and well-​known
auxin produced by plants (IAA). The most well-​known strains that produce auxin are Pseudomonas
putida, Pseudomonas fluorescens, and Pseudomonas syringae. These strains use L-​Trp (precursor
molecule) for PGPR activities. When comparing plants injected with an IAA-​deficient P. putida
mutant to plants treated with P. putida strain GR12-​2, Patten and Glick (2002) discovered improved
root growth in Brassica napus.

FIGURE 12.2 Plant growth regulators.

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216 Biofertilizers for Sustainable Soil Management

12.4.3.1.2 Cytokinins
Cytokinins are a class of plant hormones that stimulate cell division and vascular differentiation in
plants, as well as root hair proliferation, and inhibit lateral root growth and elongation of the root.
Several species of Pseudomonas produce cytokinins that help in the growth of roots and shoots. Salt
stress also increases the proline content in Glycine max tissues (Naz et al. 2009; Ali et al. 2016).

12.4.3.1.3 Gibberellin
Gibberellin is a plant hormone that helps in seed germination, floral stimulation, development of
flowers and fruits, and increases leaf growth (GA). Several PGPR bacteria, including Pseudomonas
sp., Bacillus spp., and Enterococcus faecium, stimulate the synthesis of gibberellins. Similarly,
P. fluorescens and P. koreensis have been reported to produce gibberellin-​like compounds in MU2
cell-​free medium.

12.4.3.1.4 Ethylene
Ethylene is a plant growth hormone that regulates fruit ripening, leaf senescence, root gravitropism,
and biotic and abiotic stress responses. High concentrations, on the other hand, may limit root exten-
sion, nodule formation, and nitrogen fixation by beneficial microbes in leguminous plants, inducing
hypertrophy and hastening maturity and leaf senescence. Bacterial genera seldom generate ethylene,
but they do have an enzyme called ACC (1-​aminocyclopropane-​1-​carboxylate) deaminase that may
reduce ethylene’s harmful effects on plants. In the presence of the P. stutzeri A1501 strain, the ACC
deaminase enzyme was revealed to be critical in boosting rice growth under salt and heavy metal
stress. Ahmad et al. (2013) showed that ACC-​deaminase-​producing Rhizobium and Pseudomonas
strains might enhance mung bean growth, physiology, and quality in salt-​affected conditions.

12.5 USE OF PGPRS AS BIOFERTILIZERS: GUIDELINES AND PRECAUTIONS

(1) It is critical that the given biofertilizer is of acceptable quality, includes 107 live cells per
gram as an inoculum, and be acquired solely from a reputable source when employing
PGPRs as biofertilizers.
(2) Due to its specificity, the biofertilizer should only be used for the crop(s) listed on the com-
mercially available product package.
(3) The name of the crop for which the culture bag is to be used should be written on the bag.
(4) Excess culture should be injected, and any remnants/​remaining culture should be promptly
placed in field grooves so that inoculum bacteria begin interacting with other microbiota in
the rhizosphere and colonize the rhizosphere.
(5) Because biofertilizers are microbial products, they should be kept in cool, shaded areas,
ideally at room temperature (25–​28oC), before being applied to fields to ensure longer
shelf life.
(6) Direct contact of biofertilizers with agrochemicals (herbicides, weedicides, insecticides)
should be avoided during storage or application.
(7) In general, 200 grams of biofertilizer may efficiently treat 10 kilograms of seeds.
(8) Soil additions such as lime or rock phosphate are frequently recommended in adverse soil
conditions, particularly if the soil is extremely acidic.

12.6 CHALLENGES IN BIOFERTILIZERS
There is a growing interest in using microbial-​based products as biofertilizers. Nonetheless, trans-
ferring from the lab to the field poses several problems. These bioinoculants have been used on
agricultural plants such as legumes and grains in the past. Furthermore, once injected into the soil,
plant growth-​promoting microorganisms (PGPM) are subjected to competitive situations, which
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Plant Growth-promoting Rhizobacteria as a Biofertilizer 217

may significantly limit their favourable effects. Therefore, the positive benefits of applying a certain
biofertilizer may vary substantially depending on the agri-​environmental circumstances, leading to
debate about the effectiveness of microbial-​based solutions. Pseudomonas aeruginosa is a powerful
strain for decomposing hydrocarbons and is often utilized in environmental research. It is also an
opportunistic pathogen that causes bloodstream, skin, and soft tissue infections, as well as otitis
externa and pneumonia.
Some of the major challenges can be summarized as (Timmusk et al. 2017):

• Lack of understanding of the environmentally friendly value of microbial biofertilizers among

farmer communities is amongst the significant obstacles.
• Farmer communities are unaware of the value of microbial biofertilizers for the environment.
• Inadequate agricultural extension worker promotion and encouragement of farmers to utilize
biofertilizer products.
• Lack of acceptable carriers for biofertilizer formulation.
• Lack of storage facilities to avoid biofertilizer product contamination and harsh weather
conditions.

12.7 CURRENT CHALLENGES DUE TO PSEUDOMONAS BIOFERTILIZERS

• It remains problematic to apply our knowledge about biofertilizer effects on microbiome
function and plant nutrition in an agroecosystem.
• Work on biofertilizers, which have limited usage because of uneven reactions across
varied soisl, crops, and environmental circumstances, as well as practical issues such as
mass production, shelf life, proper advice, and convenience of use for farmers (Debnath
et al. 2019)
• This is especially problematic in the lab-​to-​field transfer, since it is usual for a microbial strain
that performs well in vitro to function badly in a greenhouse or in field trails (Parnell et al.
2016; Hart et al. 2018; Keswani et al. 2019)
• It is difficult to anticipate the result of an immunization since we normally only evaluate and
control for a small number of factors, ignoring their complex relationships (Moutia et al.
2010; Busby et al. 2017).
• Negative interactions with the microbiota in the area (e.g., competition, predation, antagonism).
• The shelf life and survival of inoculated strains in a dramatically diverse environment are
restricted by certain biofertilizers and their long-​term effects.
• Because of the complexity and genetic variety of the soil and plant microbiome, a single for-
mulation is unlikely to be successful in all areas. However, designing customized biofertilizers
for each place is impracticable and unrealistic (Bell et al. 2019).

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233

13 Nitrogen-​fixing Biofertilizers
Muhammad Romman*1, Rainaz Parvez2,
Muhammad Adnan*3, Farhana Gul3, Muhammad Haroon3,
Rafi Ullah3, Shah Saud4, Nazeer Ahmed3, Ishfaq Hameed1,
Taufiq Nawaz5, Muhammad Hamzah Saleem6,
Sahar Mumtaz7, Amanullah8, Muhammad Arif8,
Maid Zaman9, Abdel Rahman Altawaha10, and Shah Hassan11
1
Department of Botany, University of Chitral, KP, Pakistan
2
Department of Botany, Government Girls Degree College Dargai,
Malakand, KP, Pakistan
3
Department of Agriculture, University of Swabi, Swabi, Pakistan
4
College of Life Science, Linyi University, Linyi, Shandong, China
5
Department of Food Science and Technology, The University of
Agriculture, Peshawar, Pakistan
6
MOA Key Laboratory of Crop Ecophysiology and Farming System in
the Middle Reaches of the Yangtze River, College of Plant Science and
Technology, Huazhong, China
7
Division of Science and Technology, Department of Botany University
of Education, Lahore, Pakistan
8
Department of Agronomy, the University of Agriculture Peshawar,
Khyber Pakhtunkhwa, Pakistan
9
Department of Entomology, The University of Haripur, Khyber
Pakhtunkhwa, Pakistan
10
Department of Biological Sciences Al Hussein Bin Talal University,
Ma’an, Jordan
11
Department of Agricultural Extension Education and Communication,
The University of Agriculture, Peshawar, Pakistan
* Correspondence: [email protected] (M.R): madnanses@
gmail.com (M.A)

CONTENTS
13.1 Introduction......................................................................................................................... 234
13.2 Biofertilizers: Mechanisms and Application....................................................................... 235
13.3 Quality Control and Guidelines for Biofertilizers.............................................................. 238
13.4 Major Mechanisms in Biofertilizers................................................................................... 238
13.5 Microbial Biofertilizers: Types, Applications, and Current Difficulties for
Economical Rural Production............................................................................................. 238
13.6 Production Innovation, Properties, and Quality Administration of Biofertilizers.............. 239
13.7 Multilegume Biofertilizers.................................................................................................. 239
13.8 Biofertilizers In Agribusiness.............................................................................................240
13.9 Conclusion.......................................................................................................................... 240
Acknowledgment............................................................................................................................ 240
References....................................................................................................................................... 240

DOI: 10.1201/9781003286233-13 233

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13.1 INTRODUCTION
Nitrogen (N) is a notable major supplement required by plants for their development. To accomplish
a high return, cultivating practices require synthetic fertilizers that are expensive and may also cause
environmental damage (Adnan et al. 2018a,b; Adnan et al. 2019; Amanullah and Fahd 2017; Akram
et al. 2018a,b; Aziz et al. 2017a,b; Chang et al. 2021; Chen et al. 2021; Emre et al. 2021; Habib et al.
2017; Hafiz et al. 2016; Hafiz et al. 2019; Ghulam et al. 2021; Guofu et al. 2021; Hafeez et al. 2021;
Khan et al. 2021; Kamaran et al. 2017; Muhmmad et al. 2019; Safi et al. 2021; Sajjad et al. 2019;
Saud et al. 2013; Saud et al. 2014; Saud et al. 2017; Saud et al. 2016). Due to ecological risks and
customer well-​being concerns, the utilization of compound composts in agribusiness is under-​used.
Consequently, some fertilizers, known as biofertilizers or bioinoculants, that contain microorganisms
with significant plant growth improvement impacts are used. Some of these microbial strains are
good for phosphorus solubilizing, nitrogen fixing from the air and, producing cellulytic proteins.
Biofertilizers are applied in a number of ways to soil, to enhance plant nourishment. One method is
their direct application in soil; another way is seed treatment or application with compost. However
these biofertilizers are used, they increase the amounts of favorable microorganisms in the soil to
further enhance plant requirements for different free-​living nitrogen-​fixing organisms (Kloepper and
Beauchamp 1992; Narula et al. 2000; Phillips et al. 2011; Al-​Zahrani et al. 2022; Rajesh et al. 2022;
Anam et al. 2021; Deepranjan et al. 2021; Haider et al. 2021; Amjad et al. 2021; Sajjad et al. 2021;
Fakhre et al. 2021; Khatun et al. 2021; Ibrar et al. 2021; Bukhari et al. 2021; Haoliang et al. 2022;
Sana et al. 2022; Abid et al. 2021; Zaman et al. 2021). These microorganisms greatly influence plant
growth when used as seed inoculants (Davison 1988; Reed et al. 2011; Richardson et al. 2009).
Some could impact plant growth through a mixture of advanced synthetics, fixing nitrogen, and
solubilizing rock phosphates, when used as biofertilizers (Amer and Utkhede 2000;Adnan et al.
2016; Kumawat et al. 2017; Rana et al. 2020; Sajjad et al. 2021; Rehana et al. 2021; Yang et al. 2022;
Ahmad et al. 2022; Shah et al. 2022; Muhammad et al. 2022; Wiqar et al. 2022; Farhat et al. 2022;
Niaz et al. 2022; Ihsan et al. 2022; Chao et al. 2022; Qin et al. 2022; Xue et al. 2022; Ali et al. 2022;
Mehmood et al. 2022; El Sabagh et al. 2022; Ibad et al. 2022).
Biological nitrogen-​fixing micro-​organisms enable regular uptake of nitrogen (N) in normal and
horticultural biological systems. Azotobacter species have important features of both significant
free-​living N2-​fixing microscopic organisms and potential bacterial biofertilizers with demonstrated
viability for plant nourishment and natural soil richness. Likewise, this micro-​ organism has
shown advanced attributes, e.g., supplement use effectiveness, security against phytopathogens,
phytohormone biosynthesis, and so on (National Research Council 1994; Deepranjan et al. 2021;
Haider et al. 2021; Huang Li et al. 2021; Ikram et al. 2021; Jabborova et al. 2021; Khadim et al.
2021a,b; Manzer et al. 2021; Muzammal et al. 2021; Abdul et al. 2021a,b; Ashfaq et al. 2021; Amjad
et al. 2021; Atif et al. 2021; Athar et al. 2021; Shah et al. 2013; Saud et al. 2020; Saud et al. 2022a,b;
Qamar et al. 2017; Hamza et al. 2021; Irfan et al. 2021;Wajid et al. 2017; Yang et al. 2017; Zahida
et al. 2017; Depeng et al. 2018; Hussain et al. 2020; Hafiz et al. 2020 a,b; Shafi et al. 2020; Wahid
et al. 2020; Subhan et al. 2020; Zafar-​ul-​Hye et al. 2020a,b; Zafar et al. 2021; Adnan et al. 2020;
Ilyas et al. 2020; Saleem et al. 2020a,b,c; Rehman 2020; Farhat et al. 2020; Wu et al. 2020; Mubeen
et al. 2020; Farhana 2020; Jan et al. 2020; Wu et al. 2019; Ahmad et al. 2019; Baseer et al. 2019;
Hafiz et al. 2018; Tariq et al. 2018).
Azotobacter, as a biofertilizer, has interesting properties, for example, blister development pro-
tection. Such advantageous qualities could be investigated significantly for the highest level of plan
to explore the importance of Azotobacter (Figure 13.1). Moreover, Azotobacter species are also
used address specific horticultural difficulties (e.g., supplement inadequacies, biotic and abiotic
limitations) due to their natural capacities, collaborative and multi-​trophic communication, biogeog-
raphy, and overflow appropriation (Aasfar et al. 2021; Kizilkaya 2009; Ladha et al. 2016) (Tables
13.1 and 13.2).
235

Nitrogen-fixing Biofertilizers 235

FIGURE 13.1 Classification of biological nitrogen fixation.

TABLE 13.1
The Important Groups of Biofertilizers

Group of Nature of
Biofertilizers Organisms Examples References

Nitrogen-​fixing Symbiotic Rhizobia, Frankia, Anabaena azollae Gaur (2010), Sharma et al.
biofertilizers (2012), Singh et al. (2015a),
and Mathivanan et al. (2015)
Free-​living Azotobacter, Beijerinkia, Anabaena, Clostridium, Sharma et al. (2012) and Singh
Klebsiella, Nostoc, Desulfovibrio, Bacillus polymyxa et al. (2020b)
Associative Azospirillum sp., Gluconoacetobacter diazotrophicus, Sharma et al. (2012) and Singh
symbiotic Enterobacter et al. (2015a); Okon et al.
(1995)

13.2 BIOFERTILIZERS: MECHANISMS AND APPLICATION

Currently, climate change is a major threat to the agriculture sector (Fahad and Bano 2012; Fahad
et al. 2017; Fahad et al. 2013; Fahad et al. 2014a,b; Fahad et al. 2016a,b,c,d; Fahad et al. 2015a,b;
Fahad et al. 2018a,b; Fahad et al. 2019a,b; Fahad et al. 2020; Fahad et al. 2021a,b,c,d,e,f; Fahad et al.
2022a,b; Hesham and Fahad 2020; Iqra et al. 2020; Akbar et al. 2020; Mahar et al. 2020; Noor et al.
236

236 Biofertilizers for Sustainable Soil Management

TABLE 13.2
Partial List of Different Biofertilizers with Their Function and Target Crops

Biofertilizer Family Function Target/​Research Crops References

Bacterial biofertilizer
Rhizobium Rhizobiaceae N-​fixation, Bengal gram (Cicer arietinum) Patil et al. (2016a,
siderophore, lentil (Lens esculenta), pea 2020b); Omer
indole acetic acid (Pisum sativum), alfalfa et al. (2016);
(IAA) (Medicago sativa), berseem Ruzzi et al.
(Trifolium alexandrinum), (2015); Yang
and soybean (Glycine max) et al. (2009)
Azotobacter Azotobacteraceae N-​fixation, produce Wheat (Triticum aestivum), Wani et al.
vitamins, plant oat (Avena sativa), barley (2016a, 2013b);
hormones viz., (Hordeum vulgare) mustard Núñez, (1999);
IAA, gibberellins, (Brassica sp.), seasum Ponmurugan
and cytokinins (Sesamum indicum), rice et al. (2012);
(Oryza sativa), sunflower Poza-​Carrión
(Helianthus annuus), castor et al. (2015);
(Ricinus communis), maize Rodriguez et al.
(Zea mays), sorghum (2017); Romero
(Sorghum bicolor), cotton et al. (2017);
(Gossypium sp.), jute Segal et al.
(Corchorus sp.), etc. (2017); Sumbul
et al. (2020)
Azospirillum Rhodospirillaceae N-​fixation, P-​ Maize (Zea mays), rice (Oryza Rodrigues et al.
solubilization, sativa), wheat (Triticum (2004), Sahoo
IAA and aestivum) et al. (2014)
siderophore
production,
increases the
number of
lateral roots and
enhances root
hairs formation
Acetobacter Acetobacteraceae Nitrogen fixation, Sugarcane (Saccharum Glick (2012);
produce auxins, officinarum), sugar beet (Beta Soumare et al.
synthesis of IAA vulgaris) and pearl millet (2020); Yadav
(Pennisetum glaucum L.) et al. (1991);
Gupta et al.
(2012)
Paenibacillus Paenibacilliaceae Biological N-​ Wheat (Triticum aestivum), Chen et al. (2006);
polymyxa fixation, produce cucumber (Cucumis Wu et al. (2006a,
plant growth sativus),watermelon 2006b); Yanni
regulators, (Citrullus lanatus), et al. (1999)
control plant soybean (Glycine
ethylene levels, max), tomato (Solanum
enhances root lycopersicum),apple (Malus
permeability and domestica)
biocontrol plant
pathogens
237

Nitrogen-fixing Biofertilizers 237

Table 13.2 (Continued)

Partial List of Different Biofertilizers with Their Function and Target Crops

Biofertilizer Family Function Target/​Research Crops References

Pseudomonas sp. Pseudomonadaceae Phosphate Wheat (Triticum aestivum), Shaharoona et al.
solubilization, cucumber (Cucumis sativus), (2006), Zahir
biocontrol, mustard (Brassica sp.), et al. (2010),
natural rice (Oryza sativa), tomato Sharma et al.
suppressor of (Solanum lycopersicum), (2012); Trivedi
specific soil borne turmeric (Curcuma longa) et al. (2008)
fungal pathogens,
produce
phytohormones
(auxins and
cytokinins)
and secondary
metabolites
Bacillus sp. Paenibacillaceae/​ Solubilize the P, Wheat (Triticum aestivum), Kang et al. (2010),
(B. subtilis, B. cereus, Bacillaceae synthesis of plant cucumber (Cucumis sativus), Swain and Ray
B. thuringiensis, growth hormones mustard (Brassica sp.), (2009); Sabier
B. pumilus, (IAA, GAs, rice (Oryza sativa), tomato et al. (2015)
B. megaterium) cytokinins and (Solanum lycopersicum)
spermidines)
Bacillus Paenibacillaceae/​ Potash Maize (Zea mays), cucumber Sangeeth et al.
mucilaginosus, Bacillaceae solubilization, (Cucumis sativus L.), (2012); Makawi
Paenibacillus nutrient uptake, Pepper (Capsicum annum (1973); Qaisrani
glucanolyticus, and biomass L.), Cotton (Gossypium et al. (2019);
Bacillus edaphicus hirsutum), rape (Brassica Eshaghi et al.
napus L), sesame (Sesamum (2019)
indicum), wheat (Triticum
aestivum), black pepper
(Piper nigrum L.)
Thiobacillus Thiobacillaceae Sulphur-​oxidizing Various crops Sharma
ferrooxidans, et al. (2012);
T. thiooxidans, Mal (2014);
T. thioparus Karuppiah et al.
(2012)
Actinobacteria, –​ Manganese-​ Various crops Sharma et al.
Firmicutes, oxidizing bacteria (2012); Malik
Proteobacteria et al.;(2002)
Thiobacillus thioxidans, –​ Zinc solubilization Rice (Oryza sativa), maize (Zea Wang et al. (2015),
T. ferrooxidans, mays), cotton (Gossypium Saravanan et al.
Microbacterium hirsutum), sorghum (2011); Zayadan
saperdae, (Sorghum bicolor) et al. (2014)
Pseudomonas
monteilli,
Enterobacter
cancerogens

2020; Bayram et al. 2020; Amanullah, Fahad 2018a,b; Amanullah, Fahad 2017; Amanullah et al. 2020;
Amanullah et al. 2021; Rashid et al. 2020; Arif et al. 2020; Amir et al. 2020; Saman et al. 2020;
Muhammad Tahir et al. 2020; Jakir and Allah 2020; Mahmood et al. 2021; Farah et al. 2020; Sadam
et al. 2020; Unsar et al. 2020; Fazli et al. 2020; Enamul et al. 2020; Gopakumar et al. 2020; Zia-​ur-​
Rehman 2020; Ayman et al. 2020; Mohammad I. Al-​Wabel et al. 2020a,b; Senol 2020; Amjad et al.
238

238 Biofertilizers for Sustainable Soil Management

2020; Ibrar et al. 2020; Sajid et al. 2020; Muhammad et al. 2021; Sidra et al. 2021; Zahir et al. 2021;
Sahrish et al. 2022).
This issue can be solved to some extent by introducing several biofertilizers in the market.
Biofertilizers are the essential contributions of supplements for practical and natural cultiva-
tion. Microbial strains which enable greater plant development ought to be segregated from
various agroecological zones and marketed. Fluid biofertilizers are superior to transporter-​based
biofertilizers. Consequently, they energize the development of fluid biofertilizers. Just 0.1% of
biofertilizer strains formed have been marketed to date, which shows that markedly less work has
been done in the field of detailing of microbial strains for biofertilizers. Be that as it may, farmers
have not benefited on the ground as the planned work has not been progressed. The interest in
biofertilizers is exceptionally high yet their manufacture is restricted. In this way, it ought to work
in the field of microbed related to foster biofertilizers as per biological zones to provide benefits to
people (Dasgupta et al. 2021; Shah et al. 2015; Nosrati et al. 2014; Rojas-​Tapias et al. 2012).

13.3 QUALITY CONTROL AND GUIDELINES FOR BIOFERTILIZERS

Biofertilizers have turned into excellent contenders avoiding damage to the ecosystem with compound-​
free techniques for horticulture. The developing interest and government support in the natural agri-
business has provided an impetus in the biofertilizer business; notwithstanding, the developing
interest and time-​restricted production of organism-​based biofertilizer has raised concerns over their
quality control. There are concerns related to the production, storage, and use of biofertilizers. In the
future, bio-​inoculants should not be restricted only to the use of microorganisms but their utilization
must include the utilization of organisms, green growth, and infections. Standardized processes have
been developed by numerous research centres for biomass age and bioactive atom creation from the
various selected organic strains. Biomass creation and dynamic particle detachments are the con-
clusive variables for quality control. The types of strains and bio-​partitions in processes are signifi-
cant observations in quality testing. The method involves collecting, detailing, and the time span of
usability of the final product, which are the properties recorded in the value control documentation.
The positive change in the acknowledgment and utilization of biofertilizers in traditional farming
requires improvements of value ideas, manuals, and guidelines. The ongoing actions are an endeavor
to comprehend the flows status of the quality control and guidelines of biofertilizer applications,
and proposes the future endeavors expected in the improvement of biofertilizer manufacture and
applications (Bharti and Suryavanshi 2021; Pandey et al. 1991; Ahmad et al. 2015; Saeed et al. 2015).

13.4 MAJOR MECHANISMS IN BIOFERTILIZERS

Biofertilizers are viewed as a natural option in contrast to synthetic compounds and substance-​based
composts, the abuse of which has caused real harm to the global climate and environment. The creation
of biofertilizers doesn’t rely just upon information on plant physiology and soil microorganisms, but
also a few innovative difficulties such as the definitions, the different species of microflora, and their
communication framework. Along these lines, the improvements in a steady bioformulation are con-
ceivable by joining the information on plant physiology with microbial and specialized perspectives.
Accordingly, biofertilizers have an exceptionally huge capacity in managing horticulture as they are
naturally harmless to the ecosystem (Ali et al. 2022; Mrkovacki et al. 2002; Tilman et al. 2011).

13.5 MICROBIAL BIOFERTILIZERS: TYPES, APPLICATIONS, AND CURRENT

DIFFICULTIES FOR ECONOMICAL RURAL PRODUCTION
Biofertilizers can be used to support farming and to help meet fulfill the needs of the expanding
global population for horticultural items while preserving and supporting the planet for future
239

Nitrogen-fixing Biofertilizers 239

generations. The significance of biofertilizers in increasing the efficiency and nature of agricultural
products has proactively been demonstrated through different research works carried out around the
world. Despite demonstrating their true capacity, biofertilizers remain underutilized to an enormous
degree. Along these lines, there is an urgent need to advance the use of biofertilizers among farmers
to ensure higher farming productivity which can be accomplished through the following:

1. Advantages of biofertilizers in ensuring soil health, supporting the efficiency of regular assets,
and achieving high efficiency and a greater cost–​benefit ratio.
2. Primary emphasis should be on the quality control during the production of biofertilizers to
safeguard their effectiveness for a long time.
3. Research into biofertilizers with multi-​strain and multi-​microorganism consortia should be
carried out for improvements in crop productivity. In contrast to single-​strain biofertilizers,
multi-​strain and multi-​microorganism consortia can achieve higher results under more hostile
conditions.
4. Biofertilizers should be made effectively accessible for all researchers in order to improve
their quality and for better utilization (Gautam et al., 2021; Marciano et al., 2012; Ortiz-​
Marquez et al., 2012).

13.6 PRODUCTION INNOVATION, PROPERTIES, AND QUALITY

ADMINISTRATION OF BIOFERTILIZERS
Biofertilizers are naturally effective microorganisms that improve plant growth by obtaining
supplements that are otherwise difficult for plants to obtain. They upgrade soil efficiency by fixing
barometrical nitrogen, solubilizing soil phosphorus, and increasing plant development. Biofertilizer
innovations have incorporated plant supplements for practical agribusiness through biological
nitrogen fixing. The plan for biofertilizer production is a pivotal multistep process that incorporates
blending of an appropriate medium with an inoculant, giving ideal circumstances for the capacity,
bundling, and dispatch, and guaranteeing endurance and foundation after placement in soil. One
of the main points of contention is the quality control of biofertilizers, which is regulated under
the Fertilizer Control Order (FCO) (Singh et al. 2021; Mirzakhani et al. 2014; Basir et al. 2015;
Rovira et al. 1991). Quality confirmation of bioproducts can go far in completing the picture for
biofertilizers. The creation of microbial plans, transportation, and commercialization are currently at
an early phase and thus production by typical means is underscored (Maan and Garcha 2021; Rashid
et al. 2004; Velmourougane et al. 2019).

13.7 MULTILEGUME BIOFERTILIZERS
Biofertilizers are indispensable in supplementing cycling in the biosphere. The biggest role is that
for natural nitrogen absorption as carried out by Rhizobium in relation to vegetables. The majority of
rhizobia are well defined for have associated plant species, however most do not act indiscriminately.
Such rhizobia with expansive hosts could be utilized for the production of multilegume biofertilizers.
Immunizing vegetables with rhizobia could accomplish significant growth in vegetable nodulation,
biomass yield, nitrogen absorption, and post-​crop soil nitrate levels. This development could help
to meet the dietary needs of the world’s constantly growing population. Multilegume biofertilizers
could be amongst the most significant advances towards economical cultivation in light of the fact
that selecting the best inoculant combination for a specific vegetable host is difficult. The prospects
of this framework are engaging in light of the fact that the whole world is trying to embrace more
natural methods of cultivation. Introduction of these biofertilizer will not only improve the environ-
ment, but also will help reducing compost data sources (Ikbal et al. 2020; Lazali and Bargaz 2017;
Mishra et al. 2016).
240

240 Biofertilizers for Sustainable Soil Management

13.8 BIOFERTILIZERS IN AGRIBUSINESS
Biofertilizers are a promising area in farming biological systems as a valuable, inexhaustible, and
ecofriendly opportunity for plant supplements. As they have the capacity to change healthful sig-
nificant components from non-​useable forms to exceptionally assimilable structures without perni-
cious consequences for the environment, they are a significant part of an integrated plant nutrient
system (IPNS) (Alley and Vanlauwe 2009; Malusá et al. 2012). The use of organic manures is
believed to be a critical component in maintaining soil health and yield efficiency at an adequate
level, which is fundamental to accomplishing on-​going cultivation. Biofertilizers may also assist
with relieving problems emerging from the growing global population for food and from adverse
effects of the far-​reaching chemicalization of agroecosystems. The changing methods to update
horticultural practices makes biofertilizers a fundamental piece of cutting-​edge crop production
which stresses of the use of organic inoculants in the futures. Various rhizosphere microorganisms
are known to have diverse plant development advancement factors, however not many have been
used as biofertilizers so far. Consequently, new procedures, taking into consideration their lengthy
application, are expected to achieve the goals of more natural cultivation (Mącik et al. 2020; Abdul
Latef et al. 2020; Ledbetter et al. 2017).

13.9 CONCLUSION
Nitrogen is a fundamental supplement for plant development and improvement, however it is
inaccessible in its most pervasive form as air nitrogen. Plants rather rely on joined, or fixed, types
of nitrogen, such as ammonia and nitrate. Management of nitrogen is a challenging task and several
methods individually and in combination are in use to manage its efficiency. The use of properly
managed nitrogen manures has been prompted around the world. Organic nitrogen absorption offers
a specific method for supplying plants with nitrogen. It is a basic part of numerous aquatic, as well
as land-​based environments across our biosphere.

ACKNOWLEDGMENT
We would like to thank to the staff of the Department of Botany, University of Chitral, Khybar
Pakhtunkhwa, Pakistan.

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14 Status of Research and

Applications of Bio-​fertilizers
Global Scenario
Rafi Ullah1*, Muhammad Junaid2, Mehwish Kanwal3,
Muhammad Adnan *1, Taufiq Nawaz4, Nazeer Ahmed1,
Fazli Subhan1, Muhammad Romman5, Shah Saud6, Shakeel
Ahmad7, Anas Iqbal8, Fazli Wahid1, Muhammad Haroon1,
Muhammad Zamin1, Nazish Huma Khan9, Jamal Nasar7,
Shah Hassan10
1
Department of Agriculture, University of Swabi, Anbar, Swabi, Khyber
Pakhtunkhwa, Pakistan
2
Graduate School of Life and Environmental Sciences, University of
Tsukuba, Tsukuba, Ibaraki, Japan
3
Ministry of National Food Security & Research, Pakistan Tobacco Board,
Peshawar, Khyber Pakhtunkhwa, Pakistan
4
Department of Food Science and Technology, The University of
Agriculture, Peshawar, Pakistan
5
Department of Botany, University of Chitral, Khyber Pakhtunkhwa,
Pakistan
6
College of Life Science, Linyi University, Linyi, Shandong, China
7
Guangxi Colleges and Universities, Key Laboratory of Crop Cultivation
and Tillage, National Demonstration Center for Experimental Plant
Science Education, Agricultural College of Guangxi University,
Nanning, China
8
College of Agriculture, Guangxi University, Nanning, China
9
Department of Environmental Sciences, University of Swabi, Pakistan
10
Department of Agricultural Extension Education and Communication,
The University of Agriculture, Peshawar, Pakistan
* Correspondence: [email protected] (R.U); madnan@uoswabi.
edu.pk (M.A)

CONTENTS
14.1 Introduction.......................................................................................................................... 256
14.2 Using Bio-​Based Products.................................................................................................... 257
14.3 N-​Fixing Inoculants as Bio-​Fertilizers................................................................................. 258
14.3.1 Bio-​fertilizers in Liquid Form................................................................................ 258
14.3.2 Bioengineered Microbes........................................................................................ 258
14.3.3 Potassium-​Solubilizing Bacteria (PSB)................................................................. 258
14.3.4 Phosphate-​Solubilizing Bacteria (Ph.SB).............................................................. 258
14.3.5 Mycorrhiza............................................................................................................. 259

DOI: 10.1201/9781003286233-14 255

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256 Biofertilizers for Sustainable Soil Management

14.3.6 Blue-​Green Algae (BGA).................................................................................... 259

14.3.7 Azolla................................................................................................................... 259
14.3.8 Acetobacter.......................................................................................................... 259
14.3.9 Azospirillum......................................................................................................... 259
14.3.10 Herbspirillum....................................................................................................... 259
14.3.11 Azotobacter.......................................................................................................... 259
14.3.12 Rhizobium............................................................................................................ 260
14.4 Plant and Microorganism Interaction Mechanism.............................................................. 260
14.5 Applications and Types of Bio-​Fertilizers........................................................................... 260
14.6 Market For Bio-​Fertilizers................................................................................................... 262
14.7 Limits of Bio-​Fertilizer Promotion (Table 14.1)................................................................. 263
14.7.1 Limited Resources.................................................................................................. 263
14.7.2 Market Restrictions................................................................................................ 264
14.7.3 Production Limitations........................................................................................... 264
14.7.4 Constraints in the Field.......................................................................................... 264
14.7.5 Technical Limitations............................................................................................. 264
14.8 Conclusion............................................................................................................................. 265
Acknowledgment............................................................................................................................ 265
References....................................................................................................................................... 265

14.1 INTRODUCTION
With greater health awareness and pollution constraints, organic farming is garnering the interest
of academics and the general public. The negative impacts of pest-​resisting agrochemicals that can
build up in soil, water, and air, as well as the negative consequences of secondary pest outbreaks,
make bio-​pesticide manufacture imperative. Bio-​fertilizers are naturally dynamic additives that
can boost plant development and yield by increasing soil nutrient availability in the rhizosphere.
The most often utilized bio-​fertilizers include bacteria, blue-​green algae, and arbuscular mycor-
rhiza fungi. In the case of microbial bio-​pesticides, the most often utilized bio-​fertilizers include
bacteria, algae, and fungi. Bacillus thuringiensis (Bt) is the most often employed microbial bio-​
pesticide strain, accounting for about 90% of all bio-​pesticides. Fertilizers and insecticides made
from chemicals are currently being phased out in favour of bio-​fertilizers and bio-​pesticides, which
also are popular because they are simple to use, environmentally friendly, economically effective,
and nontoxic (Al-​Zaidi et al. 2011; Forlani et al. 2014; Adnan et al. 2016). The public’s growing
knowledge of health and ecology, as well as the dangers of uncritical use of commercial fertilizers
and fungicides, is driving demand for bio-​pesticides. Bio-​alternatives, on the other hand, account
for only 1–​2% of the whole crop protection industry, and they, too, rely on Bt. Bio-​products that are
widely used, can decrease the negative effects of phyto-​pathogens, and cover a wider range of target
crops are being developed by researchers all over the world (Mazid et al. 2011a; Arriola et al. 2015).
The world’s population is growing, which means that agriculture must improve in order to boost
food productivity and sustainability (Al-​Zahrani et al. 2022; Rajesh et al. 2022; Anam et al. 2021;
Deepranjan et al. 2021; Haider et al. 2021; Amjad et al. 2021; Sajjad et al. 2021a; Fakhre et al. 2021;
Khatun et al. 2021; Ibrar et al. 2021; Bukhari et al. 2021; Haoliang et al. 2022; Sana et al.2022; Abid
et al. 2021; Zaman et al. 2021; Sajjad et al. 2021b; Rehana et al. 2021; Yang et al. 2022; Ahmad et al.
2022; Shah et al. 2022; Muhammad et al. 2022; Wiqar et al. 2022; Farhat et al. 2022; Niaz et al.
2022; Ihsan et al. 2022; Chao et al. 2022, Qin et al. 2022; Xue et al. 2022; Ali et al. 2022; Mehmood
et al. 2022; El Sabagh et al. 2022; Ibad et al. 2022).
The most extensively utilized fertilizers are chemical fertilizers. However, to achieve this increase,
their continued and excessive usage has resulted in environmental contamination, producing signifi-
cant ecological harm as well as insect resistance and health concerns, resulting in a decrease in crop
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Status of Research and Applications of Bio-fertilizers 257

output (Youssef & Eissa 2014; Deepranjan et al. 2021; Haider et al. 2021; Huang Li et al. 2021; Ikram
et al. 2021; Jabborova et al. 2021; Khadim et al. 2021a,b; Manzer et al. 2021; Muzammal et al. 2021;
Abdul et al. 2021a,b; Ashfaq et al. 2021; Amjad et al. 2021; Atif et al. 2021; Athar et al. 2021; Adnan
et al. 2018a,b; Jan et al. 2019; Adnan et al. 2019; Akram et al. 2018a,b; Aziz et al. 2017a,b; Chang
et al. 2021; Chen et al. 2021; Emre et al. 2021; Habib et al. 2017; Hafiz et al. 2016; Hafiz et al. 2019;
Ghulam et al. 2021; Guofu et al. 2021; Hafeez et al. 2021; Khan et al. 2021; Kamaran et al. 2017;
Muhmmad et al. 2019; Safi et al. 2021). Bio-​fertilizers can be used instead of commercial fertilizers
to increase crop production while being environmentally friendly. A “bio-​fertilizer” is a commer-
cial product that contains microorganisms within the plant when applied to seed It can increase the
delivery or obtainability of essential nutrients to the host plant on plant surfaces or in the soil to boost
growth (Bhattacharjee & Dey 2014). Bio-​fertilizers use natural mechanisms to fix N2, solubilize
P, and stimulate plant development by combining growth-​promoting substances and compounds
(Malusa’ et al., 2016). Depending on their nature and purpose, they can be categorized in a variety of
ways. As an unavoidable aspect of agriculture sustainability, bio-​fertilizer technology needs to meet
the minimum necessities for its primary uses. End-​users’ social and infrastructure situations must be
accommodated by bio-​fertilizer technology. They must also be economically practical and profitable
in terms of return on investment for all farmers, regardless of their financial situation and position;
be ecologically pleasant, unchanging, and well-​organized; and be adaptive to existing circumstances
in the area and acceptable by many social sectors while meeting personal demands. Furthermore,
bio-​fertilizers must be practical to apply within a specific governmental system, conform to varied
societal cultural patterns, be simple to use and repurpose without requiring large extra inputs, and be
productive in the long term (Mushtaq et al. 2014; Suhag 2016). This chapter discusses the present
state of bio-​fertilizer research and implementation, as well as the global scenario.

14.2 USING BIO-​BASED PRODUCTS

Despite the improved food grain production brought about by the ecological imbalance caused by
the green revolution and the usage of chemical fertilizers, natural resource depletion poses a severe
threat to humanity (Sajjad et al. 2019; Saud et al. 2013; Saud et al. 2014; Saud et al. 2017; Saud et al.
2016; Shah et al. 2013; Saud et al. 2020; Saud et al. 2022a,b; Qamar et al. 2017; Hamza et al. 2021;
Irfan et al. 2021; Wajid et al. 2017; Yang et al. 2017; Zahida et al. 2017; Depeng et al. 2018; Hussain
et al. 2020; Hafiz et al. 2020 a,b; Shafi et al. 2020; Wahid et al. 2020; Subhan et al. 2020; Zafar-​ul-​
Hye et al. 2020a,b; Zafar et al. 2021). Chemical fertilizers are becoming increasingly expensive as
they are mostly made of petroleum products and feedstock that deteriorate. The energy required for
preparation of 1 kg of commercial fertilizers needs more energy and resources, such as 11.2 kWh for
nitrogen (N), 1.1 kWh for phosphorus (P), and 1 kWh for potash, respectively (Adnan et al. 2020;
Ilyas et al. 2020; Saleem et al. 2020a,b,c; Rehman 2020; Farhat et al. 2020; Wu et al. 2020; Mubeen
et al. 2020; Farhana 2020; Jan et al. 2019; Wu et al. 2019; Ahmad et al. 2019; Baseer et al. 2019;
Hafiz et al. 2018; Tariq et al. 2018). Apart from climate change it also affects the agriculture sector
significantly (Fahad and Bano 2012; Fahad et al. 2017; Fahad et al. 2013; Fahad et al. 2014a,b; Fahad
et al. 2016a,b,c,d; Fahad et al. 2015a,b; Fahad et al. 2018a,b; Fahad et al. 2019a,b; Fahad et al. 2020;
Fahad et al. 2021a,b,c,d,e,f; Fahad et al. 2022a,b; Hesham and Fahad 2020; Iqra et al. 2020; Akbar
et al. 2020; Mahar et al. 2020; Noor et al. 2020; Bayram et al. 2020; Amanullah, Fahad 2018a,b;
Amanullah, Fahad 2017; Amanullah et al. 2020; Amanullah et al. 2021; Rashid et al. 2020; Arif
et al. 2020; Amir et al. 2020; Saman et al. 2020; Muhammad Tahir et al. 2020; Md Jakirand Allah
2020; Mahmood et al. 2021; Farah et al. 2020; Sadam et al. 2020). Therefore bio-​based fertilizers are
becoming increasingly needed by growers to minimize the constraints. These bio-​based fertilizers
can also be used in conjunction with agrochemicals to help to create nutrients in the soil medium and
combat pests that cause crop damage and poor yields. As a result, research and development into bio-​
fertilizers and bio-​pesticides can only be justified if farmers are willing to use them and eventually
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258 Biofertilizers for Sustainable Soil Management

produce them. Biological N fixation is used by N-​fixing bacteria in mutual relationships with plants
to convert elemental N to NO2–​ that are conveniently accessible to them (Malusá and Vassilev 2014).

14.3 N-​FIXING INOCULANTS AS BIO-​FERTILIZERS

Using bio-​fertilizers to fix nitrogen (N) using plant growth-​promoting Rhizobacteria (PGPR) has
an advantage over bacteria that live in the soil in terms of minimizing plant pathogen impacts and
improving crop rotation by increasing N, P, and iron availability (Fe). Nitrogen is required for photo-
synthesis, as an element of protein and chlorophyll, which affects crop output, as well as vegetative and
reproductive growth (Marek-​Kozaczuk et al., 2014; Sana at al. 2015; Unsar et al. 2020; Fazli et al. 2020;
Enamul et al. 2020; Gopakumar et al. 2020; Zia-​ur-​Rehman 2020; Ayman et al. 2020; Mohammad I. Al-​
Wabel et al. 2020a,b; Senol 2020; Amjad et al. 2020; Ibrar et al. 2020; Sajid et al. 2020; Muhammad
et al. 2021; Sidra et al. 2021; Zahir et al. 2021; Sahrish et al. 2022). Azotobacter, a nitrogen-​fixing bac-
teria, is the best as a bio-​fertilizer for cereal crops, assisting in the production of PGPR, enzymes, and
phytohormones such as auxins, gibberellins, and cytokinins. Inoculating maize seeds with Azospirillum
brasilense increased root and shoot length as well as the plant’s dry weight (Forlain et al. 1998; Braccini
et al. 2012; Puente et al. 2009). Farmers generally have only marginal interest in the use of bio-​fertilizers
because of the high price compared with low-​value commodity crops.

14.3.1 Bio-​fertilizers in Liquid Form

These are unique liquid formulations containing the appropriate microorganisms and nutrients, to
increase the shelf life of resting spores/​cysts, as well as cell protectants that withstand harsh envir-
onments (Sumita et al. 2015). They can also overcome the limits of traditional (solid) bio-​fertilizers,
such as their resilience to increased temperature and UV radiation (Hegde 2008).
They have many enzyme activities that farmers prefer, and they are easy to apply as a powder
in foliar applications, hand sprayers, and as a base manure combined with poultry and farmyard
manures. The efficiency of bacteria other than Rhizobium is unknown, hence microbial inoculants
are of great interest.

14.3.2 Bioengineered Microbes
These are bacteria that have been found to have specific functional genes using rDNA technology.
The introduction of nif genes in Rhizobium meliloti protects crops from disease and degrades bio-​
based products, further improving nitrogen fixation.

14.3.3 Potassium-​Solubilizing Bacteria (PSB)

Potassium is one of the most important macronutrients, in addition to nitrogen and phosphorus, for
a plant’s biological processes to flourish. As a result, rhizotrophic bacteria’s potassium-​solubilizing
activity is crucial. In diverse crops, the genera Aspergillus, Bacillus, and Clostridium are effective in
solubilizing K mobility in the soil (Mohammadi and Sohrabi 2012).

14.3.4 Phosphate-​Solubilizing Bacteria (Ph.SB)

The major bacteria that solubilize phosphate (Ph.SB) present in soil are Bacillus, Micrococcus,
Aspergillus, Fusarium, Pseudomonas, and others. Organic acids such as citric, oxalic, succinic,
tartaric, and malic acids are secreted in the soil rhizosphere to convert non-​available inorganic phos-
phate to soluble form, hence boosting phosphorus availability. They can increase agricultural yields
by 200–​500 kg per hectare (Ambrosini et al. 2012).
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Status of Research and Applications of Bio-fertilizers 259

14.3.5 Mycorrhiza
Mycorrhiza can create symbiotic relationships with host plants for carbohydrates in exchange for
zinc (Zn) and phosphorus (P) and is known as a disease carrier of crops. Mycorrhiza use their
mycelia to create a connection with fertile soil to absorb water and macronutrients like N, P, K, and
Ca, as well as producing growth stimulants like cytokinins and secreting antimicrobial compounds
to protect plants from invading pathogens.

14.3.6 Blue-​Green Algae (BGA)

These are free-​living photosynthetic N-​fixing organisms that promote soil porosity by contrib-
uting growth-​promoting materials such as vitamin B12. They have heterocysts, which behave as
micronodules and aid in the fixation of nitrogen. They live in symbiotic relationships with fungi,
ferns, flowering plants, and liverworts, but especially with Azolla for nitrogen fixation.

14.3.7 Azolla
This is a free-​floating symbiotic fern from the Azollaceae family that can be utilized as a dual crop or
green manuring. When Azolla inocula are soaked in super-​phosphate solution and inoculated, paddy
yield is increased, and it has been found to fix nitrogen at 45–​50 kg/​ha. The most popular species
utilized as a bio-​fertilizer in paddy crops is Azolla pinnata, which decomposes swiftly in soil and
provides the most N availability to rice and other crops (Ghosh 2004).

14.3.8 Acetobacter
This is ideal for sugarcane cultivation as it can withstand high sugar levels. It may fix up to 15 kg of
nitrogen per hectare per year.

14.3.9 Azospirillum
This is a nitrogen-​fixing bacteria known as B. polymyxa. It is a member of the Spirillaceae family, and
is good for non-​leguminous plants. This genus has a variety of species. Azospirillum amazonense,
Azospirillum halopraeferens, and Azospirillum brasilense are all Azospirillum species having a 20–​
40 kg/​ha N-​fixing capacity. These make a large difference in terms of leaf area and grain yield of
millet, sugarcane, sorghum, and maize (C4 plants), as well as other crops. They use organic acid
salts such as malic and aspartic acid to fix nitrogen (Brusamarello-​Santos et al. 2017).

14.3.10 Herbspirillum
This is an N-​fixing symbiont that lives in sugarcane roots and improves hormone production as well
as N, K, and P uptake (Khan et al. 2011a; Rasheed et al. 2015). There are bio-​protective benefits
of inoculating drought-​stricken corn with two types of bacteria of PGPR, Azospirillum brasilense
strain SP-​7 and Erbaspirillum sp. These bacteria were discovered to be useful in mitigating drought
stress that has an adverse effect on plants (Curá et al. 2017).

14.3.11 Azotobacter
The Azotobacteriaceae family includes this non-​symbiotic, aerobic, and free-​living bacterium,
although the root nodules are not evident. It sets N at 40–​200 kg/​ha and can meet the crop’s N
requirements to the tune of 80–​90%. Some of the organisms found in the rhizosphere of crops
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260 Biofertilizers for Sustainable Soil Management

FIGURE 14.1 Mechanism of plant and bio-​fertilizer interaction.

such as rice, maize, sugarcane, bajra, and vegetables include Azotobacter vinelandii, Acinetobacter
beijerinckii, Agrionoptera insignis, and Azomonas macrocytogenes, and plantations, where yields
can rise by up to 50%. These species aid in the inhibition of some root diseases through seed ger-
mination and plant growth (Mazid et al. 2011b).

14.3.12 Rhizobium
This N-​fixing symbiotic bacterium is a member of the Rhizobiaceae family and has the ability to
fix nitrogen at a rate of 50–​100 kg/​ha in pulse crops, making it a must-​have for all legumes except
mung beans. Parasponia is the only lineage outside the legume family to be able to form a nodule
symbiosis with Rhizobium. Sesbania rostrate has stem nodules as well as root nodules (Saikia and
Jain, 2007).

14.4 PLANT AND MICROORGANISM INTERACTION MECHANISM

Plants must defend themselves against infections and devise techniques to deal with them. In this
regard they are aided by beneficial microorganisms. Understanding the connection between plant and
microorganism is becoming increasingly important as the world’s population grows (Figure 14.1).
Changes in the ecology and physical environment have a significant impact on human health. Plant–​
microbe interactions can be both beneficial and harmful. Plants have a sophisticated immune system
to protect themselves from viruses and pests, as well as to avoid crop losses in agriculture. They
also increase the number of helpful microbes in their root system. Immune responses also form a
rationale for building long-​term disease resistance measures in next-​generation crops.

14.5 APPLICATIONS AND TYPES OF BIO-​FERTILIZERS

Under optimal conditions, bio-​fertilizers can be used to inoculate the seeds and roots of a variety of
agricultural crops and they can also be sprayed directly onto the soil. Nitrogenous and phosphatic
fertilizers are combined in water during seed preparation. After that, the seeds are immersed in the
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Status of Research and Applications of Bio-fertilizers 261

mixture (Abbasniayzare et al. 2012; Rai 2006). The seeds are dried after this paste has been applied
to them. They must be sowed as soon as possible after drying off to avoid being destroyed by haz-
ardous germs. On the soil where the crop is to be grown, a bed of water is placed during which the
roots of seedlings are dipped in the chemical solution for treatment. The seedlings are submerged
in bio-​fertilizer-​treated water for 8–​10 hours to allow the roots to absorb the inoculum. After that,
the seedlings are transferred. Tomatoes, rice, onions, and flowers have all been reported to benefit
from this procedure. The most common therapy is seed treatment, which accounts for 66% of the
global market. All bio-​fertilizers, as well as compost fertilizers, are combined together in the soil
treatment. They are held for one night only. The mixture is then placed on the soil where seeds must
be sowed the next day. This method is most commonly employed on fruit trees, sugarcane, and other
crops that require localized application (Bhattacharjee & Dey 2014; Qadeem et al. 2015). There are
currently a number of bio-​fertilizers available, each with its own set of functions and crop types.
To augment chemical fertilizers, bio-​fertilizers are a low-​cost, effective, and sustainable source of
plant nutrients. Bacteria, fungi, and blue-​green algae are examples of microorganisms that can be
employed as bio-​fertilizers (Singh et al. 2011; Bhattacharjee & Dey 2014).
The most popular type of bio-​fertilizer is bacterial bio-​fertilizer (Suhag 2016). These are bac-
teria that aid in the fixation of several nutrients required for plant growth in soil. They fix nitrogen,
solubilize phosphorus, and create other growth-​promoting compounds to increase plant growth.
Azotobacter, Azospirillum, and Rhizobium are examples of popular bacterial bio-​ fertilizers
(Bhardwaj et al. 2014). Nonlegume crops use Azotobacter and Azospirillum, while legume crops
need Rhizobium. Acetobacter is more sugarcane-​specific under various agro-​climatic circumstances,
and field experiments on Azotobacter have revealed that it is acceptable when treated with agricul-
tural crops under normal field circumstances (Gupta et al. 2015). Azotobacter inoculation reduces
the need for nitrogenous fertilizers by 12% to 22% (El-​Fattah et al. 2013). The inoculation of
Azospirillum improves plant vegetative growth while reducing nitrogen fertilizer use by 25–​30%.
Azospirillum lipoferum, A. brasilense, A. amazonense, and A. iraquense are the only species known
so far (Saikia et al. 2013). The production of sugarcane only be increased by the use of Acetobacter
(Raja 2013). Auxins and antibiotic-​like compounds have also been detected following its application
(Berg et al. 2013).
Bio-​fertilizers such as phosphor-​bacteria are a form of bio-​fertilizer. Phosphorus is an important
nutrient for plants, as it promotes strong development and helps plants resist disease. Phosphorus
aids in the production of roots and the growth of plants. When phosphatic fertilizers are applied
to soil, the plants absorb only 15–​20% and the remaining 80–​85 % make complexes with other
nutrients and remains in the soil for long a time. Bacillus megaterium (PSB) in the bio-​promoters
develops and releases organic acids, which dissolve the unavailable phosphate into soluble form
and make it available to plants. As a result, the soil’s remaining phosphate fertilizers can be fully
utilized, and external application can be optimized. All crops, including paddy, millets, oilseeds,
pulses, and vegetables, can benefit from PSB (Park et al. 2010).
Fungi are non-​green microbes that help in the aggregation of soil structure and availability of
phosphatic fertilizers to plants. Fungal bio-​fertilizers must create a symbiotic interaction with plant
roots in order to supply the promised nutrients. Mycorrhiza is a type of association in which the
fungal bio-​fertilizer successfully allows the nutrients to be absorbed and released, particularly phos-
phatic nutrients (Smith et al. 2011). Mycorrhiza partnerships have about eight different forms, but
arbuscular mycorrhiza is one of the most important in agriculture. Arbuscular mycorrhiza between
the roots of vascular plants and fungi is the most prevalent type of synergetic association (Akyol
et al. 2019). The accumulation of pathogens, nematodes, and heavy metals in the root zone of plants
is prevented. The soil condition is improved, making it well aerated and allowing for easy nutrient
transport. Plants develop quickly due to appropriate phosphorus availability and the generation of
phytohormones like cytokinin, because of which plant physiology has a progressive effect (Smith
et al. 2011).
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262 Biofertilizers for Sustainable Soil Management

The following are some crucial guidelines for getting the best reaction from bio-​fertilizer appli-
cation (Simarmata et al., 2016):

• Bio-​fertilizers must be devoid of contaminating microorganisms and possess a strong popula-

tion of suitable efficient strains.
• Choose the proper mix of bio-​fertilizers and utilize them before the expiration date.
• Use the recommended application methods and apply at the proper time as directed on
the label.
• A good adhesive should be used for seed treatment to obtain the best outcomes.
• Use corrective treatments for difficult soils, such as seed pelleting with lime or gypsum, or soil
pH adjustment with lime.
• Ensure that phosphorus and other nutrients are available.

14.6 MARKET FOR BIO-​FERTILIZERS

There are certain drivers and constraints in the global bio-​fertilizer industry. In these certain drivers
three are some main drivers; the first one is the growing popularity of organic agriculture. The rise
in demand for organic products is attributable to an increase in consumer awareness as well as rising
earnings, allowing individuals to consume more food. The increased demand for bio-​fertilizers has
been fueled by an increase in the eating of organic vegetables and fruits, as well as a rise in consumer
health consciousness, particularly in advanced countries such as those in Europe and North America.
Furthermore, the bio-​fertilizer market has benefited from the growing land area of organic farming,
easily availability of the product, and economic and other improvements (Malusa’ et al. 2016).
Furthermore, growing worries about synthetic agrochemicals’ adverse environmental consequences,
such as food chain contamination and pollution of soil, have prompted the use of bio-​products, such
as bio-​fertilizers. Chemical fertilizer use is limited and restricted by the common agricultural policy,
which promotes the use of bio-​fertilizers (Sheraz et al. 2010). As a result, the second driving force
is the requirement to improve soil organic matter. The final market driver is a favourable regulatory
framework that permits the bio-​fertilizer market to be regulated (Malusa’ & Vassilev 2014).
There are also some limitations to this market. The first is a lack of product efficacy in adverse
situations. The second factor is the current strong demand for synthetic fertilizers due to their lower
costs and lack of innovation compared to bio-​fertilizers, which has an impact on the product’s price.
Chemical fertilizers also produce results quickly after application. As a result, farmers prefer chem-
ical fertilizers over bio-​fertilizers in order to obtain quick results. The fourth constraint is microbes’
short shelf life, which necessitates extra caution during storing (Bodake et al. 2009).
The bio-​fertilizers market is divided into microorganisms such as Rhizobium, Azotobacter,
Azospirillum, blue-​ green algae, phosphate-​ solubilizing bacteria, mycorrhiza, and other
microorganisms, as well as technology types such as carrier-​enriched bio-​fertilizers, liquid bio-​
fertilizers, and others, application types such as seed treatment and soil treatment, and crop types
such as cereals and legume (Mordor Intelligence LLP 2019). The present worldwide bio-​fertilizer
market is depicted in Figure 14.2 based on the microorganisms utilized as bio-​fertilizers. As can
be observed, the most widely used agricultural bio-​fertilizer is Rhizobium-​based agricultural bio-​
fertilizer, which was valued at $256.8 million in 2018. Carrier-​enriched bio-​fertilizers are the most
widely used technology, with a market value of $797.5 million in 2018, followed by liquid bio-​
fertilizers. The main issue with carrier-​enriched bio-​fertilizers is that they are not UV-​resistant and
cannot withstand temperatures beyond 30–​32°C; as a result, the microbial population density of
these bio-​fertilizers falls every day as they are applied, and their marketability is likely to reduce in
the near future (Mordor Intelligence LLP 2019).
When looking at the global market for bio-​fertilizers by topography, especially by continents, it
can be observed that North America is in first place, closely followed by Europe in second place, and
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Status of Research and Applications of Bio-fertilizers 263

FIGURE 14.2 Global bio-​fertilizer market.

Asia-​Pacific in third place. South America has moved up to fourth place, and the rest of the world
has now been grouped together. For the predicted period of 2019–​2024, it is expected that the global
economy will grow steadily. Due to the rising cost of commercial fertilizers and their negative effects
on agricultural fields and the environment, as well as biotechnology advancements in the field of
fermentation technology, North America is the largest market, followed by Canada. However, in the
North American bio-​fertilizer taxonomy, Canada and Mexico remain new markets. As a result, bio-​
fertilizers’ numerous benefits contribute to their widespread application, as well as greater adoption
and use in sustainable agriculture. The country’s good agricultural outlook has boosted demand
for bio-​based products in the region. Because of the increased usage of bio-​based crop nutrition
products and rising food quality requirements, the North American region is anticipated to maintain
its market in the future (Mordor Intelligence LLP 2019) (Figure 14.2).
Bayer Crop Science AG, one of the world’s most inventive agricultural corporations, purchased
the gigantic Monsanto Company in 2018, making it the largest German acquisition to date.
Customers can choose from a wide choice of products, and the company offers them complete ser-
vices for modern and viable agriculture. Among the company’s most notable products are Optimize
(Rhizobium inoculant), Nitragin Gold (Rhizobium), Cell-​Tech (Rhizobium inoculant), JumpStart
(Penicillium bilaii), and Biodoz (Rhizobium inoculant). Similarly, the aforementioned businesses
strive to provide simple but innovative items that benefit Mother Nature, plants, humans, and
animals. Because these companies are concerned about environmental issues and the need for green
technology, they focus on natural products of microbial or plant origin for the majority of their
applications (Mordor Intelligence LLP 2019).

14.7 LIMITS OF BIO-​FERTILIZER PROMOTION (TABLE 14.1)

14.7.1 Limited Resources
• Inadequate resource constraints/​unavailability of appropriate strains
• Manufacturers’ obliviousness to product quality
• Lack of quality resources and quality control procedures
• Infrastructural obstacles, such as the lack of room for a laboratory, production, and storage
• •A shortage of inoculum cold storage equipment
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264 Biofertilizers for Sustainable Soil Management

14.7.2 Market Restrictions
• Farmers’ lack of awareness/​quality assurance, as well as marketing constraints
• Bank-​related financial limitations
• Smaller production units suffer as a result of lower sales returns compared to loan
payments

14.7.3 Production Limitations
• A lack of quality assurance research and development, resulting in limited output
• Inability to meet seasonal demand/​requirements due to a skilled workforce shortage

14.7.4 Constraints in the Field

• Unfavourable environmental conditions such as soil and climate factors, microbiological
factors, and population density
• There are no quick visible consequences, unlike with inorganic fertilizers
• There is a lack of knowledge in agricultural activities, as well as a short period of sowing/​
planting in a given area

14.7.5 Technical Limitations
• Mutations, mishandled methods, and unauthenticated inoculum/​microbes
• A lack of understanding of the advantages of technology
• Issues with technology uptake due to differing inoculation methods

TABLE 14.1
Bio-​Fertilizer Advantages and Disadvantages

Benefits Limitations

Bio-​fertilizers can help soils establish biological Nutrient contents in compost products are quite varied
activities by mobilizing nutrients
The addition of appropriate nutrients to the soil Extensive and long-​term use could lead to an addition of
improves plant health salts, minerals, and toxic elements, which could harm plant
growth, soil organism development, quality of water, and
human health
Nutrient availability, and beneficial soil worms and In comparison to chemical fertilizers, large amounts are
microbes are encouraged to develop required for application to the desire land to overcome
nutritional content deficits
Root growth is improved as it increases the porosity and Plant growth and development may be hampered by a lack of
fertility of the soil essential macronutrients
The organic matter level of the soil increases to more Low translocation of micro-​and macronutrients could result
than the normal level in nutritional deficits
Promotes the formation of mycorrhizal connections, The expense of implementation is higher than those of some
which enhances phosphorus (P) availability in the soil chemical fertilizers
Assists in the elimination of plantar illnesses and the For the manufacturing of bio-​fertilizers, there is a lack of
provision of a steady supply of essential nutrients to quality assurance and restricted resource generation
the soil
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Status of Research and Applications of Bio-fertilizers 265

14.8 CONCLUSION
Bio-​fertilizers are naturally dynamic foodstuffs that can boost plant development and yield by
increasing soil nutrient availability in the rhizosphere. With the intensive use of chemical fertilizers by
farmers, nutrient accumulation in soil increases, and ultimately destroys the soil fertility and quality.
As a result, developing efficient and long-​lasting bio-​fertilizers for crop plants, where inorganic
fertilizer use can be decreased greatly to avoid further pollution issues, is a major research priority.
Bio-​fertilizers are live, high-​cell-​density microbial preparations, and the necessary microorganisms
must be carefully monitored throughout the manufacturing process. This makes sense, given that the
quality of inoculants in a bio-​fertilizer is one of the most crucial aspects determining its success or
failure, as well as farmer acceptance or rejection. The presence of the proper type of microbe in an
active form and in the desired quantities is a measure of its quality. Given these challenges, it is clear
that thorough and extensive education of professionals, dealers, and farmers regarding the import-
ance of bio-​fertilizer technology and the economic feasibility of application is required. As a result,
in order to better understand and apply bio-​fertilizer technology, substantial information, practical
training, adoption, and perception are required.

ACKNOWLEDGMENT
We would like to thank to staff of the Department of Agriculture, University of Swabi, KP, Pakistan.

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15 Bio-​fertilizer Effects on
Plant-​parasitic Nematodes
Taufiq Nawaz*1, Muhammad Junaid2, Mehwish Kanwal3,
Saeed Ahmed4, Nazeer Ahmed*5, Rafi Ullah5,
Muhammad Adnan5, Muhammad Saeed5,
Muhammad Romman6, Shah Fahad7, Maid Zaman8,
Muhammad Haroon5, Muhammad Shahab5, Shah Saud9,
and Shah Hassan10
1
Department of Food Science and Technology, The University of
Agriculture, Peshawar, Pakistan
2
Graduate School of Life and Environmental Sciences, University of
Tsukuba, Tsukuba, Ibaraki, Japan
3
Ministry of National Food Security & Research, Pakistan Tobacco Board,
Peshawar, Khyber Pakhtunkhwa, Pakistan
4
Agricultural Research Center, Londrina State University, Londrina, Brazil
5
Department of Agriculture, University of Swabi, Swabi, Khyber
Pakhtunkhwa, Pakistan
6
Department of Botany, University of Chitral, Khyber Pakhtunkhwa, Pakistan
7
Department of Agronomy, Abdul Wali Khan University Mardan, Khyber
Pakhtunkhwa, Pakistan 8Department of Entomology, The University of
Haripur, Khyber Pakhtunkhwa, Pakistan
9
College of Life Science, Linyi University, Linyi, Shandong, China
10
Department of Agricultural Extension Education and Communication,
The University of Agriculture, Peshawar, Pakistan
** Correspondence: [email protected] (T.N.)
[email protected] (N.A.)

CONTENTS
15.1 Introduction......................................................................................................................... 280
15.2 Artificial Fertilizers vs. Bio-​fertilizers vs. Organic Fertilizers............................................ 280
15.3 Importance of Bio-​fertilizers............................................................................................... 280
15.4 Plant-​Parasitic Nematodes (PPNs)...................................................................................... 281
15.5 Classification of PPNs......................................................................................................... 281
15.6 Attack Mechanism of PPNs................................................................................................ 281
15.7 Control Strategies for PPNs................................................................................................ 281
15.8 Microorganisms as Bio-​fertilizers....................................................................................... 282
15.9 Fungi as Bio-​fertilizers against PPNs.................................................................................. 282
15.10 Bacteria as Bio-​fertilizers against PPNs.............................................................................. 282
15.10.1 Effects of Some Bacterial Bio-​fertilizers on the Root-​Knot Nematode,
Meloidogyne incognita Infecting Some Vegetable Crops��������������������������������� 283
15.10.2 Effects of Some Bacterial Bio-​fertilizers on the Citrus Nematode,
Tylenchulus semipenetrans Infesting Citrus Trees�������������������������������������������� 285

DOI: 10.1201/9781003286233-15 279

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280 Biofertilizers for Sustainable Soil Management

15.10.3 Effects of Some Commercial Bio-​fertilizers and Nutrients on the Root-​Knot

Nematode, M. incognita Infesting Some Field and Vegetable Crops��������������� 285
15.11 Conclusion........................................................................................................................ 285
Acknowledgment............................................................................................................................ 285
References....................................................................................................................................... 285

15.1 INTRODUCTION
In agriculture, increased yields can be obtained by the increased use of fertilizers. However, they
are also expensive and depreciate the environment by causing adverse effects on living things and
soils’ physio-​chemical properties. Due to fractional uptake of fertilizers by plants, the leftover
fertilizers leach down with rainwater to the waterbodies, causing eutrophication and affecting living
organisms, including microorganisms responsible for growth inhibition. In addition, these fertilizers
adversely affect soil fertility causing a disparity of soil nutrients and reducing the water-​holding
capacity of soils (Youssef and Eissa 2014). Eco-​friendly and cost-​efficient fertilizers that do not dis-
turb natural resources are therefore required to be produced (Deepali and Gangwar 2010). Hence,
several fertilizers have been developed recently that work as natural stimulators for the development
and growth of plants (Khan et al. 2009). The knowledge of microbial inocula or natural stimulators
has an extensive history, with the use of small-​scale compost production passed down through
generations of farmers (Halim 2009). This particular type of fertilizer includes products based on
microorganisms promoting plant growth called “microbial nutrients” or “bio-​fertilizers” that con-
tain live efficient strains of phosphate-​solubilizing, nitrogen-​fixing, or cellulolytic microorganisms.
These microorganisms are applied in seed, soil, or composting areas to increase their numbers and
hasten microbial activities to aid the availability of nutrients to plants in a readily available form
(Khosro and Yousef, 2012). These types of bio-​fertilizer are essential factors in the integrated nutrient
management of soils. At the same time, they have an important role in soil sustainability and prod-
uctivity and also play a vital part in resisting different parasites, pathogens, and pests in susceptible
crops (Ashry et al. 2018; Abd-​Elgawad 2020). Bio-​fertilizers are replacing chemical fertilizers in
modern agriculture because they are ecological, economical, and renewable. This chapter highlights
the need for bio-​fertilizers and their roles in managing plant-​parasitic nematodes.

15.2 ARTIFICIAL FERTILIZERS VS. BIO-​FERTILIZERS VS. ORGANIC FERTILIZERS

NPK is the most significant and indispensable of plant nutrients. Nitrogen (N) is used in solid form
and the conversion of gaseous N to the solid state is through nitrogen fixation. However, N fixation
in chemical fertilizers is known as artificial nitrogen fixation (Gaur 2010).
Microorganisms in bio-​fertilizers colonize the rhizosphere of soil or plant surfaces or within plant
surfaces after application, and improve plant growth by providing essential primary nutrients for host
plants. The source of organic manure is plant and animal wastes after the complete breakdown of raw
material. It comprises all essential nutrients in small amounts required in the maximum amount for
crop growth and could provide a suitable medium for bio-​fertilizers (Gaur 2010; Zaki et al. 2015).

15.3 IMPORTANCE OF BIO-​FERTILIZERS
Bio-​fertilizers are considered the safest substitute for chemical fertilizers to decrease environmental
hazards. They are eco-​friendly, cost-​effective, and can be made at any time. They are the most eco-
nomical source of microelements and micronutrients, stabilize chemical fertilizers’ negative effects,
and enhance the release of growth hormones. They can improve crop production by up to 40% and
fix nitrogen by 50%. One of the advantages of these fertilizers is that after 34 years of continuous
application, there is no further need for bio-​fertilizer applications, as the parental inocula will be
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Bio-fertilizer Effects on Plant-parasitic Nematodes 281

sufficient for the growth, multiplication, and improvement of soil texture, pH, and other soil prop-
erties (Gaur 2010).

15.4 PLANT-​PARASITIC NEMATODES (PPNS)

Plant roots contain various beneficial and harmful organisms that interact with each other to affect
plant growth. Crop production and plant growth are adversely affected by harmful organisms (Oerke
2006; Khan et al. 2015) and among these harmful organisms, plant-​parasitic nematodes (PPNs)
cause significant damage to crop plants worldwide (Kenney and Eleftherianos 2016). PPNs affect
agricultural and horticultural crops, including major crops, adversely. Losses are predicted to be up
to 15% in agriculture production, amounting to USD 125–​157 billion (Watkins et al. 2012; Singh
et al. 2015).

15.5 CLASSIFICATION OF PPNS
More than 4100 species of PPNs have been discovered to date, most of which are major plant
pathogens. However, some are specific to a limited range of crops, which cause huge effects on
economically important crops. PPN species classification was established based on the effects of
nematodes on crops. A researcher survey enlisted the “top 10” PPN based on scientific and economic
importance, and these are Aphelenchoides besseyi (foliar nematode), Xiphinema index (dagger nema-
tode; a virus vector nematode), Nacobbus aberrans (false root-​knot nematode), Bursaphelenchus
xylophilus (pine wilt nematode), Rotylenchulus reniformis (reniform nematode), Radopholus similis
(burrowing nematodes), Ditylenchus dipsaci (stem and bulb nematode), Heterodera and Globodera
spp. (cyst nematodes), Pratylenchus spp. (root lesion nematodes), and Meloidogyne spp. (root-​knot
nematodes) (Jones et al. 2013).

15.6 ATTACK MECHANISM OF PPNS

The PPN infestation symptoms are sometimes mistaken for water stress, soil-​related conditions,
nutrient deficiencies, or bacterial or fungal infections (Mesa-​Valle et al. 2020). PPNs inject enzymes
while attacking their host with their saliva, and a feeding site starts to be developed in the plant
tissues; consequently, physiological changes start. Cell walls and plasma membrane areas may
increase, and these changes begin to transport plant nutrients to the parasites. Cysts, galls, or lesions
are formed. These morphological and biochemical modifications result in water and nutrient uptake
and efficacy changes. The development of the plant reduces, its productivity decreases, and the plant
starts to wilt and becomes yellow. Moreover, potential alarming characteristics of PPN infections
are that the chances of secondary infections by soil-​borne pathogens also increase because of mech-
anical deformations. Resistance to infections also is reduced. Therefore, the damage by these soil-​
borne pathogens is also increased (Rodiuc et al. 2014).

15.7 CONTROL STRATEGIES FOR PPNS

The economic impact of parasitic nematodes has resulted in many nematode control strategies
being developed in agriculture, including the use of chemical nematicides. The need for nema-
tode resistance increased with the loss of pesticides due to EU regulations (EC No. 1107/​2009),
as they are unsafe for human health and contaminate the environment (Zhang et al. 2014, 2017).
Strategies linked with biological control are believed to be a safe alternative and highly realistic for
plant-​parasitic nematodes management. Bio-​control of nematodes is defined as the regulation of
populations of nematodes and a reduction in nematode damage by the action of organisms antag-
onistic to them which occurs naturally or by the environmental manipulation or the introduction of
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282 Biofertilizers for Sustainable Soil Management

antagonists (D’Addabbo et al. 2019; Hadi et al. 2014; Khan and Kim 2007). Among the different
biological control strategies, the application of bio-​fertilizers to combat and control various species
of PPNs has been found to be very efficient, effective, and economical.

15.8 MICROORGANISMS AS BIO-​FERTILIZERS
Fungi, actinomycetes, bacteria, and other microorganisms are host specific and can kill PPNs. Among
these microorganisms, soil-​borne bacteria and fungi can more effectively control nematodes. They
are efficient in promoting plant growth, and they secrete plant growth promoters such as auxin, gib-
berellic acid, cytokinins, abscisic acid, and ethylene, and improve the germination of seeds and the
growth of roots. They decay organic matter and enhance compost formation in soil. These microbes
efficiently control infestations of nematodes in different crops. Fatty acids, volatile compounds,
hormones, enzymes, hydrogen sulphide, phenolic compounds, and alcohol are among the bacterial
products used to control PPNs (Blyuss et al. 2019; El-​Eslamboly et al. 2019).

15.9 FUNGI AS BIO-​FERTILIZERS AGAINST PPNS

Nematophagous fungi have a specific feature to compete against PPNs and particularly against the
root-​knot nematode, Meloidogyne spp. (Collange et al. 2011). Some nematophagous fungi can trap
nematodes, and their various trapping systems like Arthrobrotrys oligospora and A. superb have
network trapping systems. Fungi feed on nematodes such as A. dactyloides, Dactylaria brochopaga,
and A. anchonia, forming constrictive rings. These fungi have a saprophytic nature but can trap
nematodes in their first larval or adult stage in the soil (de Freitas Soares et al. 2018). Most fungi
are saprophytic and can attack eggs. Some of these fungi belong to the genera Paecilomyces,
Paecilomyces lilacinus, Pochonia, and Verticillium. Pochonia chlamydosporia is thought to be the
most active egg parasite. P. lilacinus has been already reported to manage root-​knot nematodes
effectively, including M. incognita and M. javanica on brinjal, tomato, and several other vegetable
crops (Van Damme et al. 2005; Goswami et al. 2006; Haseeb and Kumar 2006; Khan et al. 2013).
Root nematodes inoculate tomato roots, where the mycorrhizal fungus Glomus fasciculatus
increases infestation and spore production. The size and number of the root-​knots created by the
root-​knot nematode decreases with the inoculation of the fungus Meloidogyne incognita and the
improvement of crop growth criteria (Bagyaraj et al. 1979). Suresh et al. (1885) reported that
M. incognita causes a remarkably low number of giant cells, and also the formation of giant cells
in mycorrhizal produced in minimal amounts. In addition, mycorrhizal plant root extract produced
nearly 50% mortality in nematode larvae in 96 hours. Hajra et al. (2013) evaluated an increase in
vegetative growth of crops while infested plants showed retardation in growth. Many carbohydrates
are present in the roots of mycorrhizal plants, which indicates the transfer of photosynthesis to
fungal partners. Less carbohydrates are recorded in nematode-​infested roots, displaying a great
carbon sink to the rhizosphere. Classification of nematophagous fungi was done based on their
mode of inhibitory action against PPNs (Cayrol et al. 1992). Important fungal strains that act as bio-​
fertilizers for PPN control and management are summarized in Table 15.1.

15.10 BACTERIA AS BIO-​FERTILIZERS AGAINST PPNS

Species of the genus Pasteuria are cosmopolitan and are used as biological control agents against
PPNs (Table 15.2). Pasteuria is a mycelial, endospore-​forming bacterial parasite that destroys
and reduces the nematode population (Bekal et al. 2001) and causes considerable damage to crop
plants globally (Bird et al. 2003). P. penetrans has a symbiotic relationship with PPNs, especially
with the root-​knot nematodes, significantly damaging crop plants (Charles et al. 2005). Pasteuria
species effectively infect and kill nematodes where root lesion nematodes (Pratylenchus spp.) are
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Bio-fertilizer Effects on Plant-parasitic Nematodes 283

TABLE 15.1
List of Some Important Fungal Species used as Bio-​fertilizers against Plant Parasitic
Nematodes (PPNs) and Their Modes of Action

Fungal Strains PPNs Mode of Action References

Paecilomyces Heterodera zeae, Reduced cyst population in soil; Ashraf and Khan, 2010;
lilacinus Meloidogyne javanica improves growth and protection Baheti et al. 2017
against nematodes
Trichoderma M. incognita Decreased root galls, egg masses, and Feyisa et al. 2015;
harzianum nematode population, including root-​ Devi et al. 2016
knot nematode
T. viride Meloidogyne spp., Increased plant growth and yield; Narasimhamurthy et al.
M. graminicola, decreased root galls and egg masses 2017
M. incognita
Pochonia Globodera spp., H. zeae, Lowered cyst nematode population; Baheti et al. 2017
chlamydosporia M. incognita reduced eggs and juveniles in soils

infected and killed by P. thornei, P. penetrans successfully controls the population of root-​knot
nematodes (Meloidogyne spp.) and the cyst nematodes are killed by P. nishizawae (Atibalentja
et al. 2000).
Three blue-​green algae, Nostoc calcicola, Anabena oryzae, and Spirulina sp., were reported by
Youssef and Ali (1998) to decrease the number of galls and egg masses created by the root-​knot
nematode, Meloidogyne incognita infesting cowpea and enhanced plant growth criteria. The pene-
tration of Pratylenchus penetrans and M. chitwoodi was reduced by B. megaterium in potato roots
by 50% (Al-​Rehiayani et al. 1999). Padgham and Sikora (2007) mentioned that B. megatherium
treatment resulted in a 40% decrease in rice nematode penetration and gall formation. Compared
to chemical fertilizers, plant growth microbe-​based bio-​fertilizers, such as phosphate-​solubilizing
microbes, can effectively manage nematode-​caused diseases (Khan et al. 2007).

15.10.1 Effects of Some Bacterial Bio-​fertilizers on the Root-​Knot Nematode,

Meloidogyne incognita Infecting Some Vegetable Crops
Significant increases in growth, quality, and yield parameters were observed by Khan et al. (2012)
when PPN-​infested chilli (Capsicum annum L.) was inoculated with the nitrogen fixation biological
agents Azosperillum and Azotobacter. Azosperillum performance was found to be better in com-
parison to Azotobacter. Repetitive inoculations with bio-​fertilizers increased plant growth, quality,
and yield. The nematocidal effects of various bio-​fertilizers bacteria, including Paenibacillus
polymyxa, and phosphate-​and potassium-​solubilizing bacteria Bacillus megatherium, were tested
separately on tomatoes in sandy soils for infestation by the root-​knot nematode M. incognita (El-​
Haddad et al. 2011). Relating with the un-​inoculated nematode-​infested control, the B. mega‑
therium PSB2, P. polymyxa NFB7, and B. circulans KSB2 inoculations augmented the number of
total bacterial spores in plants potted in soil from 1.2 to 2.6 times in about 60 days after inoculation.
Likewise, P. polymyxa NFB7 inoculation resulted in enhanced plant growth, quality, and yield
parameters, and within 60 days of inoculation a significant reduction in the nematode population was
observed. The strains B. megatherium PSB2, P. polymyxa NFB7, and B. circulans KSB2 produced
a better result in nematode population control. Hence, bacterial-​based bio-​fertilizers enhance the
soil nutrient (NPK) mobility and provide biological control against parasitic nematodes, including
M. incognita.
284

284 Biofertilizers for Sustainable Soil Management

TABLE 15.2
List of Some Important Bacterial Species Used against Plant Parasitic Nematodes (PPNs)
and Their Modes of Action

Bacterial Strains PPNs Mode of Action References

Bacillus firmus Meloidogyne incognita, Secondary metabolites and Xiong et al. 2015;
Ditylenchus dipasi, Sep 1 protease Geng et al. 2016
Radopholus similis,
Heterodera, and
Pratylenchus spp.
B. licheniformis Bursaphelenchulus xylophilus, Protease and chitinase Jeong et al. 2015;
M. incognita El-​Nagdi et al. 2019
B. cereus Heterodera avenae, Secondary metabolites, Gao et al. 2016;
M. incognita, M. javanica sphingosine, protease, Ahmed 2019;
chitinase, antibiotic Jiang et al.2 020
production, and ISR
B. thuringiensis H. glycines, M. incognita Bt crystal protein Wei et al. 2003;
(toxin protein) Mohammed et al. 2008
and Thuringiensin
(β-​exotoxin)
B. megaterium H. glycines, M. incognita, Secondary metabolites and Padgham et al. 2007;
M. graminicola protease Mostafa et al. 2018
B. subtilis Rotylenchulus reniformis, Secondary metabolites, Mazzuchelli et al. 2020
Helicotylenchus lipopeptide antibiotics,
multicinctus, and hydrolytic enzymes
M. graminicola,
M. incognita, M. javanica,

B. coagulans M. incognita Hydrolytic enzymes Xiang et al. 2018

B. pumilus & B. pumilus L1 H. glycines, M. arenaria Protease, chitinase, ISR Lastochkina et al. 2017;
and SAR Forghani and Hajihassani
et al. 2020
Rhizobium etli Meloidogyne spp ISR Reitz et al. 2000
Serratia marcescens M. incognita, M. javanica, Volatile metabolites, Rahul et al. 2014
R. similis prodigiosin
Pseudomonas aeruginosa Caenorhabditis elegans, Hydrogen cyanide (HCN), Fatima and Anjum, 2017;
M. incognita, M. javanica ISR, and SAR Singh and Siddiqui, 2010

P. fluorescens CHA0 M. incognita, M. javanica Pyoluteorin, Extracellular Siddiqui et al. 2005

protease, HCN, 2,4-​
diacetylphloroglucinol
(DAPG)

P. stutzeri M. incognita HCN Khan et al. 2016

P. fluorescens Wood1R M. incognita DAPG Timper et al.2 009
Corynebacterium M. incognita Hydrogen sulphide, Mena and Pimentel 2002
paurometabolum chitinase
285

Bio-fertilizer Effects on Plant-parasitic Nematodes 285

15.10.2 Effects of Some Bacterial Bio-​fertilizers on the Citrus Nematode,

Tylenchulus semipenetrans Infesting Citrus Trees
Bacterial strains Pseudomonas fluorescens strain 843 and Azospirillum brasilense strain W24
effectively improved soil quality and orange production (Shamseldin et al. 2010). The inoculation
of bio-​fertilizers with P. fluorescens strain 843 in Washington navel orange as a growth-​inducing
rhizobacterium significantly enhanced plant growth parameters, including fruit length, weight,
and yield, total soluble solids, and juice volume. In contrast, inoculations with A. brasilense strain
W24 did not significantly improve these parameters. Three types of nematodes mainly attack the
roots of Washington navel orange: saprophytic nematodes, Tylenchulus spp., and Pratylenchus spp.,
Meanwhile, Tylenchulus semipenetrans is the most abundant species. The use of bio-​fertilizers
improves and maintains soil fertility and soil health. According to researchers, bio-​fertilizers effect-
ively control plant-​parasitic nematodes and contribute to achieving sustainable agriculture globally.

15.10.3 Effects of Some Commercial Bio-​fertilizers and Nutrients on the Root-​Knot

Nematode, M. incognita Infesting Some Field and Vegetable Crops
Ismail and Hasabo (2000) tested six commercial Egyptian bio-​fertilizers and five commercial
Egyptian plant nutrients to control M. incognita in sunflower cv. Giza 101 under greenhouse
conditions. These products considerably decreased (P ≤ 0.05 and 0.01) the numbers of egg masses,
juveniles, females, nematode build-​up rate, gall formation on roots, and therefore gall and egg
mass indices. Nematode populations and galls were significantly suppressed by the seed coating
of rizobacterin, followed by phosphorine and nitrobein as bio-​fertilizers. Menawhile, nameless
biocides and blue-​green algae resulted in a minimal nematode population. Moreover, plant nutrients,
kapronite, Kotangein, potassein F, and citrein controlled the nematode population earlier. In other
research into the control of M. incognita in cowpea, with the use of phosphorine and nirobien as
bio-​fertilizers, no significant results were found to control nematode infestation or improve plant
growth, however, nitrobien performed better than the phosphorine.

15.11 CONCLUSION
Plant-​parasitic nematode biocontrol techniques are an effective alternative to hazardous chem-
ical nematodes. The use of bio-​fertilizers to control plant-​parasitic nematodes is one of the most
important biological control strategies that leads not only to soil enrichment but also is compatible
with long-​term sustainability. Furthermore, they are environmentally friendly and do not threaten
the environment. Thus, they can be used in place of chemical fertilizers. In conclusion, bio-​fertilizers
are a promising long-​term biocontrol method for plant-​parasitic nematodes in agriculture, owing to
the vast variety of modes of action that, in most cases, function in concert. However, further research
is needed to answer certain fundamental problems.

ACKNOWLEDGMENT
The authors would like to thank to the staff of the Department of Agriculture, University of Swabi,
KP, Pakistan.

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290

Index
A chemolithotrophs 164
chitin 175
abiotic stresses 1
chitosan derivatives 73
Acc-​deaminase 100
chlorides 80
Achromobactin 94
chlorophyll content 2
actinomycetes 174
clay minerals 125
Aerobacter 94
climatic condition 166
agricultural waste 186
cobalt (Co) 7
agriculture 2
commercial fertilizers 28
agriculture sustainability 257
compost biofertilizer 165
agrochemicals 258
contamination 192
agroecological zones 238
copper (Cu) 186
Alcaligenes 7
cost-​efficient fertilizers 280
Amanita 121
crop production 280
amino acids 127
crop yield 89
ammonia 128
cyanobacteria 32
ammonium nitrate 120
cysts 95
ammonium sulphate 120
cytokinins 215
anabolic steroids 186
antibiotics 214
D
anticancer 200
antioxidant 200 Dactylaria brochopaga 282
Aphelenchoides besseyi 281 Dactyloides 282
arbuscular mycorrhizal fungi 6 detoxification 5
arbuscule 78 di-​ammonium phosphate 120
arsenic 7 Ditylenchus dipsaci 281
Arthrobacter 210 drip irrigation 187
Aspergillus 258 drought stress 2
auxin 258 dryland region 92
Azolla 259
Azospirillum 259 E
Azotobacter 259
economy 99
ectomycorrhizae fungi 198
B
effective microorganisms 239
Bacillus 261; B. firmus 284; B. pumilus 5 effluents 7
bacteriodetes 190 endomycorrhizae 127
beneficial microbes 210 endophytic microbes 1
best management practices 35 Enterobacter 5
biochar 30 environment 6; conditions 6; stress 48;
biofertilizer efficiency 168 threat 187
biofertilizer production 168 enzymatic oxidation 7
bio fertilizers 43 Erwinia 94
biostimulants 67 ethylene 100
biotic factors 143 exopolysaccharides 178
blue green algae 176 extrametrical hyphae 199
Burkholderia 210
Bursaphelenchus xylophilus 281 F
farmer awareness 1
C
fertilizer control order 239
cadmium 7 fertilizers 239
calcium ammonium nitrate 120 flavonoids 70
calcium phosphate 124 fluorapatite 179
carbonates 80 francolite 179
chemical fertilizers 89 Fusarium 190

290
291

Index 291

G non-​specific acid phosphatases

176
geological factors 26 nucleic acids 179
gibberellin 69
global climate change 186 O
global population 239
Globodera spp. 281 organic acid 258
organic compounds 2
H organic manure 240
organic nitrogen 240
heavy metals 8 organic phosphates 27
Hebeloma 201 osmolytes 71
Heterodera 281
high-​cell-​density 265 P
homeostasis 4
hormones 5 Pasteuria 282
human food supplements 186 phosphates 32
humic acid 1 phosphate solubilizing bacteria 8
humic substances 66 phosphate solubilizing microbes 32
phosphites 32
I phosphorus cycle 174
photosynthetic apparatus 5
immobilization 174 photosynthetic organisms 198
indol-​3-​acetic acid 5 phytases 31
inorganic compounds 79 phytohormone 32
integrated nutrients management 164 phytopathogens 190
integrated plant nutrient system 240 Pisolithus 201
internal transcribed spacer 198 plant growth promoting rhizobacteria
isotope enrichment 203 209
plant-​microbe interaction 167
K plant-​parasitic nematodes 279
Klebsiella 210 plant smoke 72
polysaccharides 1
L potassium 34
powdered rock phosphates 120
Laccaria 121 protein hydrolysates 70
lead (Pb) 122 proteins 69
legacy phosphorus 25 proteobacteria 190
legume 46 Pseudomonas 209
psychotolerance 145
M
macromycetes 198 Q
management techniques 92 quality of inoculants 1
Meloidogyne spp. 283 quality standards 1
metabolic exchange 198
microalgae 71 R
microbial strain 94
microbiome 143 Radopholus similis 281
microorganisms 143 reactive oxygen species ROS) 5
mineralization 179 reforestation 198
multilegume biofertilizer 239 Rhizobium 212
mushrooms 198 Rhizopogon 201
mycorrhizal inoculation 200 rhizoremediators 138
rhizosphere 148
N Rotylenchulus reniformis 281

nematicides 281
S
nematophagous 282
N-​fixing inoculants 141 salinity 4
nitrogen-​fixing cyanobacteria 189 scavenging enzymes 5
292

292 Index

seaweed extracts 44 sustainable agriculture 123

sedimentary rock 26 synthetic fertilizer 164
seed treatment 48
Serratia sp. 1 T
sesquioxide surface 175
siderphore production 178 taxonomic identification 198
silicates 34 tricalcium phosphate 33
soil 34; contamination 199; fertility 210;
microbial activity 175; organic carbon 192; V
organic matter 101 Variovorax spp. 7
soil pH 121 vitamins 47
sphingomonas 7
Streptomyces 32
Z
sugar phosphates 34
sugars 68 zeolites 34
Suillus 201 zinc (Zn) 75
superphosphates 120 zinc solubilizes 47