Identification of Common Scab in Potato and its

Control Through Chemical Treatment

By
Sidra Tul Muntaha Qadeer
CIIT/SP21-RBT-006/ATD
MS Thesis
In
Biotechnology

COMSATS University Islamabad

Abbottabad Campus – Pakistan
Spring, 2021

COMSATS University Islamabad

i
Identification of Common Scab in Potato and its
Control Through Chemical Treatment
A Thesis Presented to

COMSATS University Islamabad, Abbottabad Campus

In partial fulfillment

of the requirements for the degree of

MS in Biotechnology

By

Sidra Tul Muntaha Qadeer

CIIT/SP21-RBT-006/ATD

Spring, 2021

Identification of Common Scab in Potato and its Control

Through Chemical Treatment

ii
A Post Graduate Thesis submitted to the Department of Biotechnology as partial
fulfillment of the requirements for the award of Degree of MS in Biotechnology.

Name Registration Number

Sidra Tul Muntaha Qadeer CIIT/SP21-RBT-006 /ATD

Supervisor

Name: Dr. Ayesha Baig

Assistant Professor, Department of Biotechnology
COMSATS University Islamabad,
Abbottabad Campus

Final Approval

This thesis titled

Identification of Common Scab in Potato and its Control

Through Chemical Treatment
By

Sidra Tul Muntaha Qadeer

CIIT/SP21-RBT-006 /ATD
Has been approved

iii
 For the COMSATS University Islamabad, Abbottabad Campus

External Examiner: __________________________________________

Dr.XXXXXXXXXXXXX, designation, Institution

Supervisor: __________________________________________
Dr. Ayesha Baig, Assistant Professor,
Department of Biotechnology, CUI, Abbottabad
Campus

Member Supervisory Committee: _________________________________________

Dr. XXXXXXXXXXXX, designation
Department of Biotechnology, CUI, Abbottabad
Campus
Member Supervisory Committee: __________________________________________
Dr. XXXXXXXXXXXX, designation
Department of Biotechnology, CUI, Abbottabad
Campus

Member of Supervisory Committee: _________________________________________

Dr. XXXXXXXXXXXX, designation
Department of Biotechnology, CUI, Abbottabad
Campus

Declaration

I, Sidra Tul Muntaha Qadeer, CIIT/SP21-RBT-006/ATD hereby declare that I have

produced the work presented in this thesis, during the scheduled period of study. I also
declare that I have not taken any material from any source except referred to wherever
due that amount of plagiarism is within acceptable range. If a violation of HEC rules on
research has occurred in this thesis, I shall be liable to punishable action under the
plagiarism rules of the HEC.

Date: _________________
_____________________________
Sidra Tul Muntaha Qadeer
CIIT/SP21-RBT-006/ATD

iv
Certificate

It is certified that Sidra Tul Muntaha Qadeer, CIIT/SP21-RBT-006/ATD has carried out
all the work related to this thesis under my supervision at the Department of
Biotechnology, COMSATS University Islamabad, Abbottabad Campus and the work
fulfills the requirements for award of MS degree.

Date: ___________________
Supervisor:

__________________________________
Dr. Ayesha Baig
Assistant Professor
Department of Biotechnology
COMSATS University Islamabad,
Abbottabad Campus

Head of Department:

______________________________
XXXXXXXXXXXXXXXXXX
Designation,
Department of Biotechnology,
COMSATS University Islamabad,
Abbottabad Campus

v
DEDICATION

vi
ACKNOWLEDGMENTS

Sidra Tul Muntaha Qadeer

CIIT/SP21-RBT-006/ATD

vii
 ABSTRACT
Potato is important crop in Pakistan. Minerals and vitamins are abundant in potatoes.
Potato cultivation is influenced by geography and environmental conditions. Potatoes are
best grown in temperate climates. Due to many disease and environmental conditions,
Pakistan's potato crop yield per hectare is quite low when compared to other countries.
Diseases such as Soft rot, Common scab can upset the quality and quantity of potato crop
worldwide. Thus ultimately reduces the yield of potato crop. Common scab is caused by
Streptomyces. A gram positive actinomycete it is a spore forming bacteria. Scab lesions
formed over the surface of potato and hence reduces yield of potato crop. The aim of
study was to identify specie of Streptomyces causing Common scab by using specie
specific primer. Chemical treatment was applied in order to check antibacterial activity of
Schiff bases against Streptomyces scabies. Samples were collected from different fields of
Sahiwal. Pathogen was isolated and after DNA extraction molecular identification was
done in order to identify the pathogen by using ITS primers. Antibacterial activity of
Schiff bases substitutes was checked against Streptomyces scabies through disk diffusion
method. 2, 4-disulphonamide-5-chloro substituted Schiff base, 3-carboxylic acid
substituted Schiff base, 4-carboxylic acid substituted Schiff base, 2-bromo-6-chloro-4-
nitro substituted Schiff base, 2-hydroxy substituted Schiff base were used. Among five,
2-bromo-6-chloro-4-nitro substituted Schiff base and 2-hydroxy substituted Schiff base
showed clear zone of inhibition thus showed significant antibacterial activity while 2, 4-
disulphonamide-5-chloro substituted Schiff base, 3-carboxylic acid substituted Schiff
base, 4-carboxylic acid substituted Schiff base showed no zone of inhibition thus showed
no antibacterial activity. Potatoes were affected by Streptomyces in Sahiwal and those
chemicals which showed significant activity against Streptomyces scabies can be used as
a potential drug to treat Common scab and thus productivity and marketability of potato
can be increased.

viii
TABLE OF CONTENTS
Introduction ……………………………………………………………….1
1.1 Pseudomonas aeruginosa PAO1……………………………………………...2
1.2 Biofilm………………………………………………………………………...2
1.2.1 biofilm formation……………………………………………………………3
1.2.2 Initial attachment……………………………………………………...…….3
1.2.3 Maturation ……………………………………………………………….….4
1.2.4 Detachment………………………………………………………………….5
1.3 structure of biofilm …………………………………………………………...5
1.3.1 Extracellular DNAs………………………………………………………….5
1.3.2 Extracellular proteins …….………..…………………………………..……6
1.2.3 Extracellularpolysacchrides…………………………………………………6
1.4 Quorum sensing………………….……………………………………………7
1.5 Biofilm antibiotic resistance mechanism…………………………..……….…7
1.5.1 Limited antibiotic penetration………………………………………………8
1.5.2 Horizontal gene transfer………………………………………………….…8
1.5.3 Reduced growth rate………………………………………………..………9
1.5.4 Persisted ccells…………………………………………………………...…9
1.5.5 Efflux pumps…………………………………………………………….…9
1.5.6 EPS matrix protection…………………………………………..…….……10
1.6 Inhibitors of biofilms……………………………………………………...…10
1.6.1 Inhibition of biofilms via plants……………………………………………10
1.6.2 Medicinal plants……………………………………………………...….…11
1.6.3 Argyrolobium roseum…………………………………………..………. ..12
1.6.4 Geographical distribution………………………………………………..…12
1.6.5 Taxonomy………………………………………………………………….12
1.6.6 Medicinal uses……………………………………………………….…….14
1.6.7 Phytochemicals………………………………………………………….…16

ix
1.7 Problem statment………………………………………………..…..………18
1.8 Purpose of study……………………………………………………….……18
1.9 Objectives……………………………………………………………………18
Research methodology…………………………………..………………19
2.1 Study period and location…………………………..………………………..20
2.2 Materials……………………………………………………………………..21
2.3 Sample collection…………………………………………….………………21
2.3.1 Solvent extraction…………………………………………….……………22
2.3.2 Preparation of standard solution………………………………………..….22
2.4 Inoculum preparation………………………………………………..……….22
2.5 Antibacterial activity…………………………………………………………23
2.6 Minimum inhibitory concentration…………………………………………..23
2.7 Minimum bactericidal concentration…...……………………………………23
2.8 Time kill assay…………………………………….…………………………23
2.9 Biofilm inhibition assay………………………..……………………….……23
2.10 Motility assay…………………………………………………………….…24
2.11 Virulence assay…………………………………….……………………….24
2.11.1 Pyocyanin quantification……………………………..…………………..24
2.11.2 Pyoverdin quantification…………………………………………………24
2.11.3 Protease quantification……………………………..………………….….24
2.11.4 Rhamnolipid quantification………………………………………………25
Chapter 3
Results…………………………………………………………………….23
3.1 Solvent extraction of Argyrolobium roseum…………………………………23
3.1.2 Extract percentage yield……………………………………………………25
3.2 Bacterial confirmation……………………………………….………………26
3.3 Stock solution………………………………………………………………..27
3.4 Antibacterial activity…………………………………………………………28

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3.5 Minimum inhibitory concentration………………………..…………………30
3.6 Minimum bactericidal concentration………………………………………...31
3.7 Time kill curve……………………………………………………….………33
3.8 Biofilm inhibition assay……………………………………………..………34
3.9 Motility assay…………………………………………………………..……36
3.9.1 Swarming assay………………………………………………………..….36
3.9.2 Twitching assay……………………………………………………………39
3.10 Virulence assay……………………………………………………..………41
3.10.1 Pyocyanin quantification………………………………………………...41
3.10.2 Pyoverdin quantification…………………………………………………43
2.11.3 Protease quantification……………………………………………………46
2.11.4 Rhamnolipid quantification……………………………………………….
2.12. GC-MS analysis……………………………………..……………………48
Chapter 4
Discussion ……………………………………………………………..54
Conclusion……………………………………………………………...59
Chapter 5
References………………………………………………………………60

xi
 LIST OF FIGURES

Fig 1.1 Gram-negative bacteria's QS signalling …………………………………………..9

Fig 1.2 Natural plant-based anti-biofilm compounds and their probable mechanisms…. 14
Fig 2.1 Argyrolobium roseum plant ……………..………………………………………20
Fig 3.1 Argyrolobium roseum plant …..…………………………………………………27
Fig 3.2 Solvent extraction of A. roseum …………………………………………………28
Fig 3.3 Percentage yield of A. roseum crude extract …………………………………….29
Fig 3.4 Pseudomonas aeruginosa PAO1..……………………………………………….30
Fig 3.5 ZOI of A. roseum crude extract (P), Positive control (+) and negative control (-)
against P. aeruginosa PAO1…………………………………………………………….32
Fig 3.6 Minimum inhibitory concentration ……………………………………………..34
Fig 3.7 Minimum bactericidal concentration of A. roseum……………………………...35
Fig 3.8 Growth curve P. aeruginosa PAO1 treated with 1/2 MIC of A. roseum ……….36
Fig 3.9 Effect of different concentrations of A. roseum crude extract on biofilm inhibition
of P. aeruginosa PAO1………………………………………………………………….38
Fig 3.10 Effect of sub inhibitory concentrations of A. roseum crude extract against
swarming of P. aeruginosa PAO1. …………………..…………………………………40
Fig 3.11 Effect of sub inhibitory concentrations of A. roseum crude extract against
swarming of P. aeruginosa PAO1. …………………..…………………………………40
Fig 3.12 Zone of twitching of treated and un-treated P. aeruginosa PAO1…………….41
Fig 3.13 Effect of sub inhibitory concentration of crude extract of A. roseum against
twitching of P. aeruginosa PAO1……………………………….………………………42
Fig 3.14 Effect of A. roseum crude extract on pyocyanin production of Pseudomonas
aeruginosa PAO1………………………………………………………………………..43
Fig 3.15 Pyocyanin production in treated (T) with A. roseum crude extract and untreated
(C) Pseudomonas aeruginosa PAO1 ……………………………………………………44
Fig 3.16 Effect of A. roseum crude extract on pyoverdin production of Pseudomonas
aeruginosa PAO1………………………………………………………………………..45
Figure 3.17: Pyoverdin production in treated (T) with A. roseum crude extract and
untreated (C) Pseudomonas aeruginosa PAO1………………………………………….45
xii
Figure 1.18: Zone of protease activity of PAO1 treated (T) with sub inhibitory
concentration of A. roseum and untreated ( C ) PAO1…………………………………47

Figure 3.19: Effect of A. roseum extract on protease activity of P. aeruginosa PAO1..47

Figure 3.20: GC-MS chromatogram for methanolic crude extract of Argyrolobium

roseum…………………………………………………………………………………..48
Figure 3.21: Mass spectra of the identified compounds from methanolic crude extract of
A. roseum…………..……………………………………………………………………51

xiii
 LIST OF TABLES

Table 1.1 DNA Markers for Gene Mapping………………………………………………4

Table 2.1 List of Microsatellite Markers Used…………………………………………..38

xiv
 LIST OF ABBREVIATIONS

P. aeruginosa Pseudomonas aeruginosa

A. roseum Argyrolobium roseum
AWD Agar well diffusion
ZOI Zone of inhibition
QS Quorum sensing
ml Milli litr
µl Micro litr
mm Milli meter
µm Micro meter
CV Crystal violet
OD Optical density
DMSO Dimethyl sulphoxide

xv
Chapter 1

xvi
 CHAPTER 1
INTRODUCTION

17
1.1 Potato

Potatoes have high yield potential with a high nutritional value, the potato (Solanum tuberosum L.) is
an extremely alluring crop in agricultural production systems. Potato is non grain staple crop. It is a
starchy, tuberous crop from the Solanaceae family. Potato (Solanum tuberosum) with production of
approximately 376 million metric tons is the fourth major crop worldwide (Goutam et al., 2018). The
potato possesses tetrasomic inheritance characteristics and exhibits large-scale tetraploidy and
heterozygosity. From diploid to hexaploid, ploidy is seen in farmed potatoes (Andersson et al., 2018).
Potatoes have larger calorie outputs than any other grain and are one of the most widely produced
non-grain food crops (Aversano et al., 2015). Because potatoes are a storehouse of energy, vitamins,
minerals, and significant organic substances, potatoes are also regarded as a prestige crop. On
average, 100 grams of potato tubers contain roughly 75 milliliters of water, 17.47 grams of carbs, 2
grams of protein, 0.29 milligrams of vitamin B6, 12 milligrams of calcium, 421 milligrams of
potassium, 0.1 milligrams of fat, and traces of many other minerals and fibers (Beals, 2019). The
ability of potatoes to grow in a variety of habitats, from sea level to 4000 m above sea level and from
47 ˚S to 65 ˚N latitude, is well documented. In Asia, it has an average production yield of about
13t/ha that varies depending on the growing environment (Barrell et al., 2013).The potato possesses
tetrasomic inheritance characteristics and exhibits large-scale tetraploidy and heterozygosity. From
diploid to hexaploid, ploidy is seen in farmed potatoes (Andersson et al., 2018).

Whereas many other crops fail in particular environmental circumstances, it may be grown there. Its
flexible and brief vegetative cycle makes it appropriate for rotation with important crops like wheat,
rice, soy beans, or maize (Gastelo et al., 2014). Quality of the planting material and variety yield are
the two most crucial factors in utilising modern production technology in vegetatively propagated
crops like potatoes (Naik and Buckseth., 2018).

1.2 Importance

Following wheat, rice, and maize as the fourth most significant food crop in the world, the potato
(Solanum tuberosum L.) is the most widely farmed tuber crop (de Haan and Rodrihuez., 2016). A
"nutrition shift" toward more processed and energy-dense meals is being driven by rising income
levels in the majority of emerging nations, particularly in metropolitan areas. Because of this, potato
demand is increasing. In metropolitan regions of South Africa, potatoes constitute a staple diet.

18
Potatoes are a filling, healthy food that is low in fat and high in vital nutrients. Freshly grown
potatoes are mostly composed of water, between 75 and 80 percent of it, 16 to 20 percent carbs, 2.5
to 3.2% crude protein, 0.8 to 1.2% minerals, 0.1-0.2% crude lipids, 0.6% crude fiber, and a few
vitamins (Reddy et al., 2018). Potatoes are more nutritious than cereals even if they have a
comparatively low protein content. Leucine, isoleucine, and tryptophan are a few of the necessary
amino acids that are present in potatoes (Yadav et al., 2021). Along with dietary fiber, thiamine, iron,
and folic acid, potatoes also include dietary antioxidants that may help prevent illnesses linked to
ageing. In addition to being used as a vegetable, it is also utilized in the production of starch,
alcoholic drinks, and other processed goods like French fries and chips. Additionally, it is employed
medicinally to treat conditions including gastrointestinal and liver infections.

1.3 Potato Growth Stages

1.3.1 Growth of Sprouts

Sprouts grow upward from eyes on seed tubers and emerge from the ground. At the base of the
developing sprout, roots form.

1.3.2 Vegetative Development

Along with emergent sprouts, aboveground nodes give rise to leaf and branch stems. Nodes
underground are where roots and stolons grow. Photosynthesis begins.

1.3.3 Tuber Initiation

At the tips of the stolons, tubers develop but do not yet expand noticeably. The majority of cultivars
have early blooming at this stage's conclusion.

1.3.4 Tuber Initiation

With the buildup of water, nutrients, and carbs, tuber cells swell. Carbohydrates and mobile inorganic
nutrients are deposited mostly in tubers.

1.3.5 Maturation

Vine coloration changes, leaves fall off, photosynthesis declines, tuber growth slows, and vine death
occurs. Dry matter content of the tubers reaches a maximum, and the tuber skins harden.

(Jefferies et al., 1991; Johnson, 2008)

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 Figure 1.1: Growth stages of potato tubers (Johnson, 2008)

1.4 Major Potato Growing Season

Crop Planting Harvesting Production Share

Spring Jan-Feb April-May 07-10%
Summer March-May August-Oct 15-20%
Autumn Sept-Oct Jan-Feb 70-75%
Table 1: Seasons for potato growth.

1.5 Region and output

Around 20 million hectares of potato are planted each year, producing roughly 320 million tones
worldwide (de Haan and Rodriguez., 2016., Poczai et al., 2010). The majority of potatoes are
currently grown in China and India, which together account for over a third of global production.
While the major potato-producing nations in Africa are Egypt, South Africa, Algeria, and Morocco,
in that order, they account for more than 80% of the continent's total production. Since the early
1960s, the output of potatoes has quickly surpassed that of all other food crops in Africa and Asia (de
Haan and Rodriguez., 2016). The world's potato output is changing significantly. Up until the early
1990s, the majority of potatoes were produced and consumed in Europe, North America, and nations

20
in the former Soviet Union. According to FAO data from 2005, developing nations now produce
more potatoes than developed countries for the first time. In 2014, a total of 381,682,000 tonnes is
predicted (FAOSTAT, 2017; Quiroz et al., 2018).

Country Potato production in the % of world total

year 2013 (tonnes)
Poland 6,334,200 1.6

Netherlands 6,801,000 1.7

France 6,975,000 1.84

Bangladesh 8,603,000 2.27

Germany 9,669,700 2.56

United states 19,843,900 5.25

Ulkarine 22,258,600 5.89

Russia 30,199,100 7.99

Federation
India 45,343,600 12

China 95,987,500 25.4

Total world production = 377,863,333.33 tonnes

Table 1.2: Top 10 Potato Producing Countries in the world (FAOSTAT, 2015; Yadav et al., 2017;
Pocketbook, 2015).

1.6 Potato production in Pakistan

In India and Bangladesh, potato output and harvesting area both gradually grew throughout the
course of this five-year period. However, both growing and decreasing fluctuation were seen in

21
Pakistan. From 138 kha in 2010 to 175 kha in 2013, but then drastically fell to 159 kha in 2014, the
area under harvest for potatoes grew. Similarly, the country's potato output peaked in 2013 at 3802
kilotons but declined to 2901 kilotons in 2014 Table 1.3. Punjab has much more land used for potato
farming than other provinces. Consequently, it is Pakistan's main potato-producing province. Punjab
harvested the most potato land in 2011–12 (148 kha), followed by Khyber Pakhtunkhwa (9.9 kha),
which produced 3396 and 119 kg of potatoes, respectively. Baluchistan produced 31,000 tons of
potatoes over a harvested area of 2 kha, followed by Sindh, which produced the fewest potatoes
4,000 tons over a harvested area of 0.4 kha (Amjad et al., 2014).

Country/ Area harvested (000,ha) Production (000,tonnes)

Year 2014 2013 2012 2011 2010 2014 2013 2012 2011 2010
Afghanistan 25 22 21 20 20 340 302 230 205 246
Bhutan 5 3 5 6 3 46 41 43 52 44
Sri Lanka 5 5 4 4 3 82 78 72 59 51
Nepal 205 197 190 182 185 2817 2690 2584 2508 2517
Pakistan 159 174 185 159 138 2901 3802 3393 3491 3141
Bangladesh 461 443 430 460 435 8950 8603 8205 8326 7930
India 2024 1992 1907 1863 1835 46395 45343 41483 42339 36577
Table 1.3: Shows the area under harvest and potato output in South Asian nations from 2010 to 2014
(FAOSTAT, 2017; Quiroz et al., 2018)

1.7 Varieties of potatoes worldwide

Varieties Feature
Russet (Burbank) The traditional ruddy-skinned potato is the russet or burbank kind. Russet
potatoes are the finest for making French fries because of their
exceptionally bland flavour, which excellently complements other
flavours.
White washed White wash potatoes are the ideal all-purpose food because of their
golden exterior and creamy, fluffy white meat; they are delicious
steamed, fried, baked, boiled, or roasted. A traditional salad potato that is
devoid of fat and cholesterol and an excellent source of vitamin B6.
Red/ Pink washed The salad form of red or pink washed potatoes has a solid meat and a
thin, blushing crimson skin. Consume them whole since the skin contains

22
 a lot of the nutrients.
Chat (Baby) Chat potatoes, sometimes known as baby potatoes, are very little potatoes
with a creamy exterior and light yellow flesh. They have a bright, fresh
flavour and are delicious both warm and cold. Chat potatoes are an
excellent source of nutritional fibre, potassium, and vitamin C.
Cocktail Cocktail potatoes are a size designation rather than a kind. They have
skin that is yellowish and white meat, and are often smaller versions of
Chat or white washed potatoes. They are really simple to use because
they cook in half the time and don't need to be peeled or chopped before
use.
Desiree Desiree potatoes are perfect for smooth mash or being cooked in a sauce
since they have a solid, creamy-tasting flesh. Desirees often have an oval
form, are longer, and bigger.
Yukon Gold Yukon Gold has extremely golden meat and a very silky yellow skin. It
creates wonderful roast or chip potatoes in addition to wonderful baking
potatoes.
Purple A unique heritage potato type with purple blue flesh and indigo blue skin.
It is important to keep the skin on in order to preserve the colour.
Medley On the basis of the cooked texture and component usefulness, the 200 or
so types of potatoes that are reportedly farmed in the United States can be
divided into a number of groups.
Fingerling The solid texture and knobby look of fingerling potatoes are
complemented by a subtle nutty flavour. It pairs well with green leafy
vegetables and vegetarian cuisine.
Kipfler Small to medium in size, kipfler potatoes have an elongated, slender,
finger-like form. Due to the fact that they maintain their shape nicely
when cooked, they make great wedges and salad potatoes.
Japenese sweet potato A sweet potato cultivar from Oita prefecture is called Kanta-kun.
It resembles other Japanese sweet potatoes in appearance and is similar in
that it has a firm inside and a rich pinkish-purple skin. The best
preparations are whole, skin-on steaming or baking. Kanta-kun is
wonderfully sweet and fiber-free and needs no seasoning.
Sweet potato There are two commercial types of sweet potato, usually referred to as
23
 yams: Garnet and Jewel. An exterior with a light brown skin covering a
vivid orange, nearly copper inner.
Table 1.4: Shows different varieties of potatoes produces worldwide and their characteristics.
(Elsharif et al., 2020).

1.8 Varieties of potatoes in Pakistan

Red Skin White Skin

Desiree Diamant

Cardinal Ajax

Ultimus Patrones

Lal-a- Faisal Multa

Raja Symphonia Sante

Faisalabad Red Faisalabad White

PRI Red Hermes

Ruby

Sadaf

Table 1.5: Potatoes varieties in Pakistatan (Abbasi et al., 2011).

1.9 Important Potato Growing Districts of Pakistan

Punjab: Okara, Sahiwal, Kasur, Sialkot, Sheikhupura, Jhang, Lahore, Narowal, Pak pattan,
Gujranwala, T.T. Singh and Khanewal.

KPK: Nowshera, Dir, Swat, Balakot, Skardu, Abbotabad, Batakundi and Mansehra.

Baluchistan: Pishin, Killa Saifulla and Kalat.

Gilgit Bultistan: Gilgit.

(Khan and Akhtar., 2006)

24
1.10 Factors Effecting Potato

1.10.1 Abiotic Factors

The fruitful potato production faces many drastic environmental conditions, namely heat, cold,
drought and salinity and certain diseases caused by microbes effects its growth (Diallo et al., 2011).
Global climate change in drought and extreme heat is posing a great challenges to sustainable crop
production, as it is affecting crop yield and plant performance negatively. Greenhouse gas emissions
will cause increase in temperature that will result in severe drought stress, soil salinity and disease
threats. Temperature greater than 25˚C inhibit the tuber initiation and growth. High temperatures
result in certain physiological disorders such as early sprouting, unequal shape, high glycoalkaloids
concentration in tubers and bitter poisonous tubers (Levy and Veilleux., 2007). Potato plant is
extremely vulnerable to high temperature, soil salinity and drought. As potato is fourth major food
crop, enhancing the productivity of potato, adapting new cultivation practices and producing cultivars
that are stress tolerant and modified according the environmental changes is important. Drought is
considered to be the most significant abiotic factor affecting the potato yield. Acidic soils with a pH
below 5.2 can also significantly reduce the severity of common scab. Potatoes are commonly grown
in soils with a pH of 5.0 to 5.2 for control of common scab. Water stress is also known to cause delay
in flowering and tuberization, depending upon the genotype, control and extent of deficiency (Dahal
et al., 2019).

1.10.2 Biotic Factors

Potato is prone to many biotic stresses and is host to certain pathogens like virus, bacteria, fungi,
nematodes and oomycetes resulting in many diseases like Common Scab, Soft rot, Brown rot, Early
blight and Black dot (Aversano et al., 2015). Potato is generally a high yielding crop, but the
interaction between host and pathogen results in reduced yield and quality. Nearly 22% yield of
potatoes is decreased per year due to bacterial, viral, fungal and pest attack to potato crop earning an
yearly loss of over 65 million tones (Czajkowski et al., 2011).

1.11 Common Scab in Potatoes

25
Among many diseases, Common scab is a disease that is widely spread in potato-growing areas and
is characterized by the help of lesions present on the tubers (Beausejour et al., 2003). It is a serious
issue in potatoes, other root and tuber crops thereby affecting the market value and its potential yield
by causing certain types of pitted, superficial or raised lesions on the tubers making them darker or
different in color in comparison to healthy tuber skin (Wanner, 2007).

1.11.1 Disease Cycle of Common Scab

Figure 1.2: Disease Cycle of Streptomyces scabies (Wharton et al., 2007).

Common Scab of potato crop is coherent saprophyte that lives on the surface of soil, tubers and crop
residues. Filamentous bacteria, Streptomyces scabies is known to cause this soil borne disease and
the thaxtomin is a known toxin that causes common scab disease. Spores present in soil affect tubers.
As the spores are in maturation stage, they produce melanized pigmentation. When the spore interact
with suitable host, it starts germinating and the infection process begins. Temperature in which this
process continues is 25˚-30˚C. Pathogen then invades the lenticels and grow on surface of wounds.
Through lenticels, mycelia enters into the periderm layer and results in large scab lesions.

1.11.2 Causal Agent of the Common Scab

26
Streptomyces Scabies, a gram-positive, having filamentous growth pattern is known to be the main
causal agent of common scab. Streptomyces spp. Producing the CS are actually soil-borne but can be
possibly transmitted by seed tubers (Wang and Lazarovits., 2005). It is a soil inhabiting genus
majorly known for producing the antibiotics (Chater et al., 2010). Streptomyces are the soil
inhabitants and comprise of a complex genus with about 500 species (Flores-Gonzalez et al., 2008).
Along with S. scabies and S. acidiscabies, some more species such as S. griseus, S. atroolivaceus, S.
turgidiscabis, S. stelliscabiei, S. bottropensis and S. aureofaciens have been reported to cause
Common scab. In recent studies, it was identified that four actinomycete species (S.
diastatochromogenes, S. atroolivaceus, S. lydicus, and S. resistomycificus) also act as causal agents
of deep-pitted scab (Leiminger et al., 2013). Prevalence and intensity of the CS vary from year to
year and location to location in the same area. Development of the symptoms is dependent upon
vulnerability of the potato cultivar, some environmental factors, pathogen virulence and pathogen
inoculum (Lazarovits et al., 2007). All the tissues and development stages of potato are not always
prone to the Common scab. Plant pathogen Streptomyces attacks on the underground plant parts such
as developing tubers, stems and stolons but does not affects the roots. Parts of plants that are above
the ground remain healthy even after the Streptomyces infection until the scabbed part of the stem
present underground limits the nutritional and water conditions between roots and shoots (Dees and
Wanner., 2012). Streptomyces causes symptoms on the expanding tissue when the tuber is
developing. Tubers that are 0-6 weeks old after tuber initiation are more likely to be affected by the
pathogen (Khatri et al., 2010). Certain changes occur during the development of tuber such as
phellem thickness, although CS lesions might happen around lenticels. Lesions stop expanding once
the skin is mature and the disease does not develop further on tubers in storage (Loria et al., 2006).

1.11.3 Thaxtomin A; Production and virulence

Certain characteristics of these pathogenic species are controlled by thaxtomin which is a plant
phytotoxin. It is a secondary metabolite and is controlled by the cluster-situated regulator (TxtR)
which activates the expression of thaxtomin biosynthetic genes in response to cello-oligosaccharides
(Bignell et al., 2014). This phytotoxin plays role as main virulence factor for the S. scabies. The
dominating form of thaxtomin being produced by S. Scabies is thaxtomin A (Johnson et al., 2007).
Thaxtomin is famous for being the only determinant of pathogen Streptomyces causing Common
scab. The chief factor Thaxtomin A is produced by Streptomyces scabies, Streptomyces
turgidiscabies and Streptomyces acidiscabies. The morphological changes in thaxtomin A, triggers a
wide variety of cellular responses, such as; depletion of cellulose synthase complexes from the
27
plasma membrane, early and transient change in Ca2+ and H+ ion flux, reduction of crystalline
cellulose, an increase in pectins and hemicelluloses in plant cell walls, lignification, production of the
antimicrobial plant defense phytoalexin scopoletin, changes in gene expression associated with plant
cell wall synthesis and remodeling, and cell death (Bignell et al., 2014).

Figure 1.3: Structure of Thaxtomin A (King and Calhoun., 2009).

1.11.4 Detection methods of Streptomyces species

The taxonomic identification and distribution of Streptomyces species mostly based on
morphological and physiological characteristics related with the production of thaxtomin A,
pathogenicity tests in-vitro and in-vivo (Wanner, 2004). However, recently, the molecular
characterization techniques have become more important and authentic source in which DNA-based
methods enabled the fast and accurate detection of thaxtomin A, which helped in identification and to
distinguish among the various Streptomyces species. Although Streptomyces taxonomy is very much
complicated, however; phylogenetic analysis using 16S ribosomal (rRNA) gene sequencing and
DNA-DNA hybridization have been found as successful tool for the detection of Streptomyces
species by using specie specific primers through PCR amplification method, which is considered a
more rapid detection technique with in short time duration and is also cost effective (Barrera et al.,
2013). The virulence factor of Streptomyces, Thaxtomin A. can also be easily detected with bioassays
(Loria et al., 1995). Mostly it can be measured by using liquid chromatography (LC) or mass
spectrometry (MS) or high-performance liquid chromatography (HPLC) analysis (Flores-Gonzalez et
al., 2008).

1.12 Host Range

Streptomyces species that are responsible in producing Common scab disease are basically not host
or tissue-specific. Bacteria are capable of infecting seedlings of a range of monocots and dicots under
desired conditions (Loria et al., 2006). The infection of the seedlings differentiates from other
underground stems or stools because in this roots are affected. Under field conditions, host range of

28
CS for certain Streptomyces species carries a number of tap root crops such as radish, carrot, beet and
parsnip in addition to potato (Bignell et al., 2010).

1.13 Symptoms of Common Scab

Common Scab produces raised or deep-pitted lesions on tubers surface and can be present in tubers
from the same plant. Lesions produced by CS are brown in color, can be discrete, or may coalesce to
cover large patches of the tuber surface. Netted scab symptoms have been described as superficial,
brown corky lesions on the tuber periderm (Dees and Wanner., 2012). Common Scab causes lesions
on the outer layer of potato tuber resulting in significant economic losses following reduced
marketability. Tuber symptoms of common scab vary in extent and appearance. Common scab
lesions are usually circular and 0.25 to 0.33 inch (6 to 8 mm) in diameter, but they can be smaller in
early stages of development and larger if they coalesce. Lesions typically possess a raised margin and
slightly depressed center. Some characteristic symptoms have descriptive names: russet scab appears
on tubers as superficial tan to brown corky lesions; pitted scab is characterized by lesions with
depressions beneath the tuber surface; and raised scab appears as cushion like, warty lesions.
Common scab lesions can be confused with tuber lesions of powdery scab caused by Spongospora
Subterranea and patchy russeting caused by Rhizoctonia solani. In addition to tuber symptoms,
Streptomyces spp. can cause brown stem and stolon lesions. These lesions can affect just a small
portion of the tuber surface, or may completely cover it. Sometimes the ridged portions are in broken
concentric rings.

1.14 Control and Management of Potato Common Scab

Environmental factors as pH, soil moisture and the microbial flora of soil in combination with potato
cultivars and virulence of pathogen makes management of Common Scab a difficult task.
Physiological differences such as thaxtomin sensitivity and skin properties can partly describe
differential cultivar susceptibility. Streptomyces as saprophyte on the debris of plant in soil further
reduces the effectiveness of disease control (Wiggins and Kinkel., 2005). Existence of the CS-
suppressive soils suggests that microbial flora is important too (Neeno-Eckwall et al., 2001). The
diseases in plants can be controlled by the use of few pesticides, fungicides and agrochemicals. In
agricultural practices around the globe, environmentally safe methods are now being used for the
control of diseases in plants. For this purpose, plants are considered to be the best choice as they are
human friendly and naturally important sources of agricultural agent for the control of diseases
(Kagale et al., 2004). Plants are famous in producing natural phytochemicals in a wide variety.
29
Medicinal plants play a pivotal role in this aspect and have been tested for their antimicrobial
activities because of the secondary metabolites in all plant cells (Hassawi and Kharma., 2006).

1.15.1 Cultural Practices

Ideal conditions for common scab infection are; low soil moisture i.e. less than 65-70% soil moisture
during tuber initiation, soil pH between 5.2 and 8.0 and temperatures during daytime above 20 oC, so
increasing soil moisture to 80-85% during tuber initiation until tubers reach 1 to 1.5 inches in size
results in reduced common scab incidence (Agrios, 2005). Use of healthy seed tubers and cultivation
in un-infested fields may help also. Destruction of diseased plant debris is the most important factor
for eliminating any kind of existing disease in the fields. Crop rotation and destruction of host plants
is fruitful. Using the manure of different plants to control the Common scab is an emerging technique
that helps in reduction of the disease.

1.15.2 Biological Control

Controlling the Common scab disease in potato biologically offers and interesting alternative in
comparison to classical control strategies. Being the production house of antibiotics and certain
extracellular enzymes, non-pathogenic Streptomyces species are also capable of working as
biological control agents against CS (Doumbou et al., 2001). Streptomyces naturally present in potato
fields can suppress CS. In sustainable agricultural practices, biological control has been recognized as
a viable approach for the treatment of plant diseases. In this context, several studies have been
conducted for the biological control of common scab, including non-pathogenic Streptomyces spp.
(Wanner et al., 2014; Hiltunen et al., 2009) Pseudomonas spp. (Arseneault et al., 2016), and Bacillus
spp. (Meng et al., 2012). These microorganisms produce secondary metabolites which are used as
antibiotics, considered a best disease control technology. Some of other biological agents such as
soymeal, meat and bone meal (Lazarovits et al., 1999) fish emulsion (Abbasi et al., 2006) lopsided
oat green manure (Sakuma et al., 2011) and mustard green manure (Larkin and Griffin, 2007) have
been also reported as a beneficial role in controlling common scab.

1.15.3 Chemical Control

Chemical management for common scab includes a good seed treatment to maintain plant health for
the production of healthy tubers. A number of studies have been reported for the chemical treatment
of potato seed tuber such as use of copper sulphate, mercuric chloride, formaldehyde, tetracycline,
borax, boric acid, plantomycin, NaCl, Dithane M-45, and quintozene for the control of Common scab

30
in soil (Razdan and Sabitha., 2009). Certain fertilizers such as urea, gypsum, TSP and MOP can also
help in treating the tubers chemically (Iqbal et al., 2019). These chemical treatments reduced the risk
of common scab infection spreading from the mother infected plant to the progeny and virgin soil.

1.15.4 Schiff bases

Schiff bases are molecules that resemble aldehydes or ketones but instead of having a carbonyl
group, they have an imine or azomethine group. Under particular circumstances, any primary amine
combines with an aldehyde or a ketone to produce Schiff bases, which bear Hugo Schiff's
name(Shiff, 1864). They demonstrate a wide spectrum of biological activity in addition to being
widely employed for industrial reasons. Building-wise, a Schiff base (also known as imine or
azomethine). It is an aldehyde or ketone analogue that has an imine or azomethine group in lieu of
the carbonyl group (C double bond O).

Figure 1.4: General structure of a Schiff base (Silva et al., 2011).

Some of the most popular organic compounds are schiff bases. They serve as dyes and pigments,
catalysts, steps in the synthesis of chemical compounds, and stabilisers for polymers (Dhar and
Taploo., 1982). A wide spectrum of biological activities, such as antifungal, antibacterial,
antimalarial, antiproliferative, anti-inflammatory, antiviral, and antipyretic characteristics, have also
been demonstrated for schiff bases (Dhar and Taploo., 1982; (Przybylski et al., 2009).

31
Figure 1.5: An illustration of a bioactive Schiff base. Each molecular structure's imine or azomethine
group is indicated by shading (Silva et al., 2011).

32
 Figure 1.6: Chemical composition of a few artificial antibacterial Schiff bases, among which
compound 5 is an antimalarial (Silva et al., 2011).

1.15.5 Antibacterial activity of Schiff bases

Bacteria that display multiple antibiotic resistance are closely linked to the rise in the mortality rate
of infectious illnesses. The primary source of this issue is the absence of efficient therapies (Baquero,
1997; Alekshun and Levy., 2007). There is unquestionably an urgent medical need for the

33
development of new antibacterial drugs with inventive and more effective modes of action (Rice,
2006).

It has been suggested that schiff bases are effective antibacterial substances. For instance, N-
(salicylidene)-2-hydroxyaniline has a MIC value of 8 g/mL and is effective against Mycobacterium
TB H37Rv (Souza et al., 2007). By conducting tests on J774 macrophages, the selectivity of N-
(Salicylidene)-2-hydroxyaniline was verified. Despite being tested at concentrations as high as 1000
g/mL, N-(Salicylidene)-2-hydroxyaniline had no cytotoxic effects on J774 macrophages. At these
experimental circumstances, more than 80% of macrophage cells were still alive, demonstrating the
great selectivity of N-(Salicylidene)-2-hydroxyaniline. A number of Schiff bases produced via the
condensation of 5-chloro-salicylaldehyde with primary amines have recently been shown to have
antibacterial action (Shi et al., 2007). The most effective antibacterial agents against at least one of
the tested bacterial species were the 5-chloro-salicylaldehyde-Shiff base derivatives 6–15. The strain
with the highest sensitivity to chemicals 6-11 and 13-15 was Pseudomonas fluorescens, with MIC
values ranging from 2.5 to 5.2 g/mL. The reference medication kanamycin's MIC value for the
identical bacterial strain was 3.9 g/mL. The MIC values for the Schiff bases 6, 7, 9–11, 14, and 15
against Escherichia coli ranged from 1.6–5.7 g/mL, while the MIC value for kanamycin was 3.9
g/mL. Only the Schiff base 14 proved toxic to Bacillus subtilis (MIC = 1.8 g/mL). Compounds 6 and
7 have MICs of 3.1 and 1.6 g/mL against Staphylococcus aureus, respectively (Shi et al., 2007).
Schiff bases generated from isatin have also been found to have antibacterial properties (Pandeya et
al., 1999).

1.15.6 Derivatives of Schiff bases

Sr No. Names Structure Molecular Weight

1 2,4- 419
disulphonamide-
5-chloro
substituted Schiff
base

2 3- carboxyalic 271
acid substituted
Schiff base

34
 3 4- carboxylic acid 271
substituted Schiff
base

4 2-bromo-6- 385
chloro-4-nitro
substituted Schiff
base
5 2-hydroxy 243
substituted Schiff
base

Table 1.6: Showing Schiff bases synthesized by Chemistry Department Comsats Islamabad
Abbotabad Campus. Samples with names and molecular mass.

1.16 Problem Statement

One of the most common potato disease in Pakistan is Common scab, which affects potato
marketability. The significant prevalence of bacterial illness reduces the efficiency of the crop.
Common scab limits yield and marketability of potato crop.

1.17 Purpose of study

Common scab of effect potato crop limiting its yields and marketability. Therefore, the aim of
study is the identification and characterization of Common scab pathogen and its possible control
through chemical treatment.
1.18 Objectives

1. To identify potato Common scab pathogen

2. To molecular characterize Common scab pathogen using species and specific

primers
3. To check the antibacterial activity against Streptomyces scabies

35
 CHAPTER 2

MATERIAL AND METHODS

36
2.1 Research Methodology

The study for isolation and characterization of pathogenicity determinant of Common scab and its
control through chemical treatment was conducted in Biotechnology Laboratory, COMSATS
University Islamabad. Abbottabad Campus by the following methods and procedures.

2.2 Sample Collection

For determination of Common scab on potato and management of scab with the help of synthetic
chemicals, fields in Sahiwal were visited and scabbed samples were collected from different regions.
Scabbed samples were kept in air tight bags, and labelled. Those bags were placed in 4˚C until the
process of isolation and characterization of disease were done. Similarly different plants were also
collected and anti-bacterial activity was done. Plant extract were made and kept at 4 0C for use against
bacterial pathogen.

2.3 Media Preparation

2.3.1 Yeast Malt Extract media (YME)

YME media was prepared in 1000 ml of autoclaved distilled water. 4g of glucose was added, 10g of
malt extract, 4g of Yeast extract and 2g of CaCOȝ were added. After that pH was maintained between
7.2-7.5 and 12g of Agar was added. Ingredients were mixed in reagent bottle by shaking gently and
then the media was kept for autoclaving at 121˚C with 15 Psi of pressure. After this, autoclaved
media was poured in petri plates inside the laminar flow hood. Let the plates dry. Once the media
gets solidified, plates will be covered with lid and will be sealed will the help of Para film. These
plates will be kept at 37˚C overnight to observe if any contamination is there.

2.3.2 YME Broth

37
YME broth were prepared in 500 ml of autoclaved distilled water in which 4g of glucose was added,
10g of malt extract, 4g of yeast extract was added and was autoclaved.

2.4 Molecular Analysis

Serial Stock solutions Volume (ml) Activity

No.

1 Cloroform– 24:1, 24ml chloroform and Helps in removing

Isoamylalcohol 1ml of Isoamylacohol proteins and lipids
during extraction
2 Ice-chilled Isopropanol 50ml Helps in precipitating
the DNA

3 70% Ethanol 70ml of ethanol dissolved in Required for washing

30ml of distilled water the DNA pellets

4 TE Buffer 100µl of 0.1M Tris and 200µl Helps in the DNA pellets
of 0.5M EDTA is dissolved in
80ml of distilled water

5 5X TBE 13.75g boric acid , 27g Tris Helps in maintaining pH

and 10ml of 0.1M EDTA is during the gel
added in 500 ml of distilled electrophoresis
water
Table 2.1: Overall view of the stock solutions made.

2.5 Preparation of DNA Extraction Stock Solutions

38
2.5.1 5M NaCl
NaCl helps in neutralizing the negative charge of the DNA in a solution. To make its 5M stock
solution, 141.6 g NaCl was added in 500ml of distilled water.

2.5.2 2M Tris HCl (pH 9.5)

2M Stock solution of the Tris-HCl was prepared by adding 60.5gm of Tris-HCl in 450 ml dH 20 and
its pH was maintained up to 9.5.

2.5.3 0.5M EDTA

EDTA works as a chelating agent that is they help in formation of stable water-soluble complex.
93.06g of EDTA was dissolved in 450ml of distilled water to prepare 0.5M of its stock solution.
NaOH pellets were then added to dissolve the EDTA, and the pH was maintained up-to 8.0 by adding
1M HCL solution and then volume was raised up to mark.

2.5.4 Chloroform – Isoamylalcohol

Chloroform and Isoamylalcohol are used as in combination with (24:1) respectively, the purpose of
these solution is to remove proteins and lipid impurities during extraction of DNA. 24ml of absolute
Chloroform and 1 ml of Isoamylalcohol was added in a falcon tube, after labeling placed at 4 0C to
chill.

2.5.5 Isopropanol
Isopropanol is used to precipitate the DNA pellet after extraction. Chilled isopropanol was used and
the desired amount was taken and stored at 4oC.

2.5.6 70% Ethanol

This helps in washing the DNA pellets and to remove extra impurities. 70ml absolute ethanol was
taken in the graduated cylinder and the volume was raised up to 100ml by adding 30ml of distilled
water and placed in refrigerator at 4 oC.

2.5.7 TE Buffer
After extracting the DNA, TE buffer was used to dissolve the pellet. For preparing 50ml TE buffer,
100µl of 0.5M EDTA and 500µl of 0.1M Tris was mixed and final volume was made up to 50ml by
adding distilled H2O.

2.5.8 5X TBE Buffer

Tris-borate-EDTA (TBE) buffer is utilized for gel electrophoresis. 5 X TBE Stock solutions were
prepared by dissolving 27gms Tris, 13.75gms Boric acid and 10ml 0.5M EDTA in 500ml distilled
water.
39
2.5.9 CTAB Extraction Buffer Recipe
CTAB buffer was made by mixing 25ml of Tris, 10ml of EDTA, 70ml of NaCl, 2% of CTAB i.e.
10g in 500ml and volume was made up to mark by distilled water. Then 1% β-marcaptoethanol was
added in a 250 ml of CTAB buffer.
2.6 Isolation of Pathogen

Scabbed potato samples were taken as a whole and washed with 2% Sodium hypochlorite. After
washing with sodium hypochlorite for one minute the whole potato was again rinsed with autoclaved
distilled water two to three times. Diseased part or lesion of almost 1 cm was cut with the help of
sterilized blade and transferred to mortar. This portion of potato was grinded finely by adding 500µl
of distilled water. The suspension was transferred to 1.5 ml eppendorf tubes and the tubes were
labelled. These eppendorf tubes were placed in water bath for incubation at 65˚C for 1 hours.

2.6.1 Serial Dilution

Serial dilutions were performed on all the samples. Eppendorf tubes were taken out of the water bath
and were serially diluted in 10 tubes approximately inside the laminar flow hood to avoid any
contamination. 50 µl of 1x, 5x and 10x suspension was taken each and spreaded on petri plates
having water agar respectively. These plates were kept at 28˚C for 3-5 days in order to obtain the
bacterial colony. Bacterial colonies were then observed and with careful handling single colony was
picked up with the red hot sterilized wire loop and was streaked on YME media prepared plates to
get the single bacterial colony. These plates were again kept in 28˚C for the bacterial colony. Once
the bacterial colony appeared, these plates were taken out from incubator and kept in refrigerator at
4˚C.

40
 Figure 3: YME agar plates for spreading of Streptomyces.

2.6.2 DNA Extraction

Single colony obtained was then picked up from the YME plate with the help of wire loop that was
made red hot before starting the procedure to avoid any contamination. The colony will be then
inoculated in the falcon containing 20ml of YME broth. Those falcons will be then kept on shaking at
37˚C for 48 hours until the bacterial suspension became turbid. These falcons containing bacterial
suspension were then centrifuged at 14000 rpm for 2-5 minutes. Supernatant was discarded while the
pellet was left behind. The protocol then followed for DNA extraction is given below

1. Pellet was resuspended in 567µl of TE buffer and with the help of re-pipetting the pellet was
transferred to fresh 1.5ml eppendorf tube.
2. In the next step, 100ml of 10% SDS and 10ml of Proteinase K was added. After mixing gently
the tubes were kept in water bath at 65˚C for 1 hour until the solution becomes visible.
3. 100µl of 5M NaCl was added and mixed thoroughly.
4. After that, 80µl of CTAB was added and mixed gently to and fro and eppendorf tubes were
again kept at 65˚C for 10 minutes.
5. In the next step, approximately equal volume i-e 700µl of chloroform/isoamyl alcohol was
added and the tubes were kept in micro-centrifuge for 4-5 minutes at 4000rpm.

41
 6. Aqueous, viscous supernatant was then transferred to fresh eppendorf tube leaving the
interface behind and 700µl of phenol/chloroform/isoamyl alcohol was added and again
centrifuged for 5 minutes in micro-centrifuge.
7. Supernatant was then transferred to a fresh tube and 600µl of isopropanol was added in
order to precipitate out the nucleic acids. By shaking the tubes back and forth, stringy white
DNA precipitate became visible.
8. Pellet was then transferred to fresh eppendorf tube and was washed with 70% ethanol and
was again spin for 5 minutes.
9. Supernatant was carefully discarded and pellet was dried and dissolved in 100µl of TE buffer
and kept in -20˚C to avoid any degradation of the DNA.

2.6.3 DNA verification by Gel Electrophoresis

For preparing 1% of agarose gel, 0.6g of agarose was added in 60ml of 1X TBE buffer and was
heated until the clear solution appeared. After that, the solution was left for 30 seconds to cool down
and 3µl of Ethidium bromide was added. This mixture was then poured in gel tray having comb and
left to solidify for 30-40 minutes. After the gel was solidified, combs were taken out carefully and the
gel tray was placed in gel tank. This tank was filled with 1X TBE running buffer. After that 2µl of
loading dye and 5µl of DNA samples were loaded in the wells. DNA samples were run on the gel for
40 minutes at 400mA and 100V. Gel was visualized on trans illuminator.

2.7 PCR Analysis

After the bands of DNA were visualized, PCR was carried out for the confirmation of the presence of
pathogen. Specific primers for Streptomyces were used for the characterization and identification of
the Common scab.

2.7.1 Primers for PCR

Primers Size Sequence

42
ITS-F 650 bp 5 - GTC AAG TCA TCA TGC
CCC TT 3

ITS-R 5 – AAA CTT GGC CAC AGA

TGC TC3

Table 2.2: Primers and their sequence.

2.7.2 PCR reaction

Serial No. Reagents Volume (µl/reaction)

1 2X master mix 7.5
2 Forward primer 0.5
3 Reverse primer 0.5
4 DNA 2
5 PCR Water 4.5
Total Volume 15
Table 2.3: Reagents and their concentrations.

2.7.3 Conditions for PCR

Serial No. Steps Temperature (˚C) Timing

1 Initial denaturation/1st 94 5 minutes
hold
2 Denaturation 94 30 seconds
3 Annealing 54 30 seconds

43
4 Extension 72 90 seconds
5 Final extension/ 2nd 72 7 minutes
hold
35 number of cycles

Table 2.4: Conditions and temperature for PCR.

2.8 Gel Electrophoresis

The PCR product was analyzed by performing gel electrophoresis. For which 2% agarose gel was
prepared in by adding 1.2g of agarose in 60ml of TBE buffer (1X). After that it was heated in oven
for at least for 50-60 seconds until the solution becomes clear. 3µl of Ethidium bromide was then
added into the TBE buffer solution and was poured into the gel tray having a comb for solidification.
Comb was carefully removed and the gel tray was kept in gel tank having 1X TBE running buffer. In
the first well, 1kb ladder was loaded in order to get the band size and samples were sequentially
loaded in the next wells. The samples were run on the gel for 25-30 minutes at 110V and 400mA for
PCR. Visualization of gel was done on trans illuminator.

2.9 Antimicrobial Activity Assay

2.9.1 Chemicals

Five chemicals synthesized by Chemistry Department Comsats Islamabad Abbotabad Campus. Schiff
bases having antibacterial activity were selected. 2, 4-disulphonamide-5-chloro substituted Schiff
base, 3- carboxyalic acid substituted Schiff base, 4- carboxylic acid substituted Schiff base, 2-bromo-
6-chloro-4-nitro substituted Schiff base, 2-hydroxy substituted Schiff base were used.

2.9.2 Preparation of chemicals concentration

5 mg/mL concentration of chemicals were prepared. 5 mg of each chemical was added in 1 mL of

DMSO.

2.9.3 Preparation of YME Agar plates

YME agar media was made and autoclaved and about 25ml was poured in each petri plate. 9 petri
plates were made ready and left for solidification inside laminar flow hood. After the media was
solidified, plates were covered with lid and sealed with the help of parafilm and were placed in
incubator at 37˚C for overnight to check if any contamination was present or not.

44
2.9.4 Disk Diffusion Method

Agar well diffusion method or disk diffusion method was done to check the antimicrobial activity of
the chemicals selected for this study. For this method, single bacterial colony was again picked with
the help of sterilized wire loop and dipped in 15 ml of YME broth and left for shaking at 37˚C
overnight. Next day O.D for the bacterial suspension was checked that showed to be 0.5. 50µl of this
bacterial suspension was taken and spreaded on the nutrient agar plates with the help of glass
spreader inside the laminar flow hood. 4 wells were then made in the YME agar plates with the help
of borer and were labelled as one for the negative control that was DMSO and rest of the three wells
for 40µl, 70µl and 100 µl of 2, 4-disulphonamide-5-chloro substituted Schiff base at the concentration
of 5mg/mL was loaded in each well. In the middle of the plate 10 µg streptomycin disk was placed.
Similarly more plates were made for the other chemicals. 3 replicates were made preparing 9 plates.
These plates were then kept in incubator for to check the zone of inhibition.

45
 CHAPTER 3

RESULTS

3.1 Isolated common scab pathogen

46
Figure 3.1: Single bacterial colonies isolated on YME media from Common scab diseased tuber
with 5X dilution.

47
Figure 3.2: Single bacterial colonies isolated on YME media from Common scab diseased tuber with
10X dilution.

Figure 3.3: Plate showed single bacterial colony streaked from 5X spreaded plate.

48
Figure 3.4: Plate showed single bacterial colony streaked from 10X spreaded plate.

49
Figure 3.5: Plate showed single bacterial colony streaked from 5X spreaded plate.

50
3.2 DNA Extraction

S1 S2 S3 S4 S5 S6 S7

Figure 3.6: Genomic DNA of potato sample having scab. The symbols in the lane represented S1,

S2, S3, S4, S5, S6 and S6.

S8 S9 S10 S11 S12 S13

Figure 3.7: Genomic DNA of potato sample having scab. The symbols in the lane represented S8,
S9, S10, S11, S12, and S13.

51
3.3 Molecular Analysis of Results

-ve C +ve S1 S2 S3 S4 S11 Ladder

C

Figure 3.8: Lane 1 represented 1,000 bp Ladder; Negative control, positive control and sample S1 to
S1. Samples showed bands at 200bp with ITS primers at 60˚C from DNA extracted from Common
scab tuber samples from Sahiwal Region.

3.4 Chemical treatment

Chemical 40 µL 70 µL 100 µL Positive Negative

Name control control
Streptomycin DMSO
2,4- 0 0 0 24±2 mm 0
disulphonamide-
5-chloro
substituted
Schiff base
3- carboxyalic 0 0 0 25±1 mm 0
acid substituted
Schiff base
4- carboxylic 0 0 0 24±1 mm 0
acid substituted

52
Schiff base
2-bromo-6- 16.75±2.78mm 17.66±3.7 21±5.1 mm 29±1 mm 0
chloro-4-nitro mm
substituted
Schiff base
2-hydroxy 26.66±4.1 mm 32±5.2 mm 36.6±5.7 mm 23±3.5 mm 0
substituted
Schiff base
Table 3.1: Showed Antibacterial activity of chemicals and their zones of inhibition at different
concentration against Streptomyces.

Figure 3.9: 2, 4-disulphonamide-5-chloro substituted Schiff base showed no zone of inhibition.

53
Figure 3.10: 3- carboxyalic acid substituted Schiff base showed no zone of inhibition.

54
Figure 3.11: 4- carboxylic acid substituted Schiff base showed no zone of inhibition.

55
Figure 3.12: 2-bromo-6-chloro-4-nitro substituted Schiff base showed clear zones of inhibition.

56
 Figure 3.13: 2-hydroxy substituted Schiff base showed clear zones of inhibition.

40
Antibacterial Activity
Zone of inhibition in mm

35

30
2-bromo-6-chloro-4-
25 nitro substituted
20 schiff base
15
2-hydroxy substituted
10
schiff base
5

0
40μL 70μL 100μL Positive Negative
Control Control
Different concentrations of chemicals

57
 Figure3.14: Antibacterial activity of 2-bromo-6-chloro-4-nitro and 2-hydroxy substituted Schiff
bases against S. scabies.

2, 4-disulphonamide-5-chloro substituted Schiff base, 3-carboxylic acid substituted Schiff base, 4-

carboxylic acid substituted Schiff base, 2-bromo-6-chloro-4-nitro substituted Schiff base, 2-hydroxy
substituted Schiff base were used in my study to check their activity against Streptomyces scabies. 2-
bromo-6-chloro-4-nitro substituted Schiff base and 2-hydroxy substituted Schiff base showed clear
zone of inhibition thus were effective against Streptomyces scabies as shown in fig 3.11, 3.12 while
2, 4-disulphonamide-5-chloro substituted Schiff base, 3-carboxylic acid substituted Schiff base and
4-carboxylic acid substituted Schiff base showed no zone of inhibition so were ineffective as shown
in fig 3.8, 3.9, 3.10.

58
CHAPTER 4

DISCUSSION
Discussion

Due to its extraordinarily high nutritional value and wide commercial appeal, the potato
(Solanum tuberosum L.) is a very significant crop in agriculture. It is allegedly grown in
180 nations throughout the world (Koleva et al., 2012). The potato, a member of the
Solanacae family, is a rich source of vitamins, minerals, dietary fiber, and mostly carbs.
Although it may be cultivated in a variety of settings, it is essentially a cool-weather crop.
Although it may be cultivated in a variety of settings, it is essentially a cool-weather crop
(Haverkort et al., 1990). However, there is a high likelihood that the crop is afflicted by a
number of nematode, bacterial, viral, and fungal diseases that reduce the crop's yield or
utility. It has been shown that the common scab disease is the most prevalent disease
affecting potatoes globally (Zhao et al., 2008). Common scab disease is thought to be
mostly caused by the bacterium Streptomyces scabies, which produces rough, corky sores
and deeply pitted holes (Braun et al., 2017). These symptoms were found in all the
samples collected from Sahiwal. The colonies obtained resembled Streptomyces rather
closely in terms of morphology, being generally convex and white to creamy in colour as
shown in fig 3.1, 3.2 as that these qualities were previously reported by (Leimingera et
al., 2013).

To identify the particular specie known to be responsible for spreading the Common scab
disease in the Sahiwal region, molecular characterization was used. To accomplish this,
the species of Streptomyces isolated from scabbed symptomatic tubers were detected and
distinguished using standard polymerase chain reaction (PCR) tests based on ITS genes
(Lehtonen et al., 2004; Wanner, 2009). Within the genus Streptomyces, a species
complex rather than a single species is responsible for Common scab (Loria et al. 1997;
Wanner, 2009). But with the samples taken from Sahiwal, the ITS primers for
Streptomyces spp. described by (Wanner, 2009) were amplified satisfactorily fig 3.7

It has been suggested that Schiff bases are effective antibacterial substances. For instance,
N-(salicylidene)-2-hydroxyaniline has a MIC value of 8 g/mL and is effective against
Mycobacterium TB H37Rv (Souza et al., 2007). A number of Schiff bases produced via
the condensation of 5-chloro-salicylaldehyde with primary amines have recently been
shown to have antibacterial action (Shi et al., 2007). Schiff bases generated from isatin

2
have also been found to have antibacterial properties (Pandeya et al., 1999). 2, 4-
disulphonamide-5-chloro substituted Schiff base, 3-carboxylic acid substituted Schiff
base, 4-carboxylic acid substituted Schiff base, 2-bromo-6-chloro-4-nitro substituted
Schiff base, 2-hydroxy substituted Schiff base were used in my study to check their
activity against Streptomyces scabies. 2-bromo-6-chloro-4-nitro substituted Schiff base
and 2-hydroxy substituted Schiff base showed clear zone of inhibition thus were effective
against Streptomyces scabies as shown in fig 3.11, 3.12 while 2, 4-disulphonamide-5-
chloro substituted Schiff base, 3-carboxylic acid substituted Schiff base and 4-carboxylic
acid substituted Schiff base showed no zone of inhibition so were ineffective as shown in
fig 3.8, 3.9, 3.10.

3
CHAPTER 5
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