COMMUNICATIONS IN SOIL SCIENCE AND PLANT ANALYSIS

https://doi.org/10.1080/00103624.2023.2282996

Barley Growth and Phosphorus Uptake in Response to Inoculation

with Arbuscular Mycorrhizal Fungi and Phosphorus Solubilizing
Bacteria
M. Mirzaei Heydaria, Robert M. Brookb, and Davey L. Jonesb
a
Department of Agronomy and Plant Breeding, Isfahan (Khorasgan) Branch, Islamic Azad University, Isfahan, Iran;
b
School of Environment and Natural Sciences, Bangor University, Bangor, Gwynedd, UK

ABSTRACT ARTICLE HISTORY

Biostimulants and bioinoculants offer the potential to enhance nutrient use Received 1 February 2023
efficiency within agricultural cropping systems and thus reduce the amount Accepted 8 November 2023
of mineral fertilizers needed to support crop growth. Considerable uncer­ KEYWORDS
tainty, however, exists about their efficacy under different management Arbuscular mycorrhizal fungi
regimes. The present field-based study investigated the effects of phos­ (AMF); nutrient dynamics;
phorus (P) solubilizing bacteria (PSB) and mycorrhizal (AMF) inoculants in phosphorus cycling; plant
the presence of a range of mineral-based P fertilizers, including triple super­ growth promoting
phosphate (TSP), struvite and rock phosphate (RP), on the growth, yield, and rhizobacteria (PGPR); rock
P uptake of barley (Hordeum vulgare L.). Two consecutive field experiments phosphate
performed in a low P soil (Eutric Cambisol) revealed that the bioinoculants
(PSB and AMF) promoted plant P uptake and grain yield. Moreover, in
comparison to P fertilizers lacking bio-inoculants, the application of external
P sources combined with bio-inoculants led to significant increases in leaf
P concentration, total P uptake, plant dry matter, grain P concentration, and
crop yield. Grain yield significantly correlated with P uptake by roots and
above-ground parts (i.e. stems, leaves, and spikes) as well as grain
P concentration in both seasons. It is concluded that bioinoculants can
help improve sustainable P use ultimately leading to greater sustainability
of cropping systems and resilience in soil P cycling.

Introduction
Phosphorus (P) underpins many biochemical reactions in plants (Lambers 2022) and contributes to
resistance against a range of abiotic stresses (Arzani and Ashraf 2016). However, its poor availability in
soil often represents a constraint to primary productivity in many agroecosystems (Lambers 2022).
Phosphorus can occur in myriad organic and inorganic forms in agricultural soils, however, plant
roots can only directly access inorganic phosphate (Rodriguez and Fraga 1999; Rose and Wissuwa
2012). Consequently, fertilizers added to soils are typically mineral based and highly soluble to
facilitate uptake. While some of this soluble P is taken up and consumed by plants, this process is
known to be highly inefficient with most of the P either becoming immobilized within the microbial
community, sorbed to the solid phase (e.g., bound to Al and Fe oxyhydroxides) or precipitated in
a poorly-available solid form (e.g., apatite; Bünemann et al. 2012).
Application of P fertilizers with a low nutrient use efficiency (ca. 10–30% of the added P taken up by
plants in the year of application) represents an economic loss to farmers but also constitutes a major
environmental concern due to its role in eutrophication (Eqbal et al. 2013). Further, P fertilizers
represent a finite resource which has led to renewed calls to reduce their use and simultaneously

CONTACT M. Mirzaei Heydari [email protected]; [email protected] Department of Agronomy and Plant

Breeding, Isfahan (Khorasgan) Branch, Islamic Azad University, Isfahan, Iran
© 2023 Taylor & Francis Group, LLC
2 M. MIRZAEI HEYDARI ET AL.

maximize P bioavailability in agriculture (Arrobas et al. 2022). This represents a dual challenge to the
agricultural industry and requires the design of innovative strategies to tackle the P problem (Roy et al.
2022). One potential solution is to harness the power of the rhizosphere microbial community to help
support plant P uptake (Trabelsi et al. 2017; Zhang et al. 2023). For example, it has been suggested that
the integrated use of P-solubilizing bacteria (PSBs) and arbuscular mycorrhizal fungi (AMF) may be
able to efficiently extract P from the raw material used in fertilizer production (e.g., rock phosphate)
and then transfer it to the plant (Adnan et al. 2019; Castiglione et al. 2021; Heydari et al. 2009). This
would reduce the need to process rock phosphate into more soluble P forms which are both energy
intensive and expensive (Arrobas et al. 2022; Roy et al. 2022).
Direct application of rock phosphate (RP) to P-deficient acidic soils has in recent years received
much attention as the recommended alternative to the application of soluble P fertilizers (e.g. triple
superphosphate (TSP), diammonium phosphate (DAP) (Amarasinghe et al. 2022). However, the very
low solubility of rock phosphate in nonacidic soils is low making it poorly available to plant roots.
PSBs induce chemical changes in the rhizosphere by releasing organic acid anions (e.g. citrate, malate,
2-ketogluconate) and H+ that promote the dissolution of RP, thereby enhancing both plant P uptake
and crop yield (Emami et al. 2019; Singh, Pandey, and Singh 2011). These organic acids can promote
P mineral dissolution via ligand exchange on mineral surfaces or via complexation reactions which
leads to the release of Ca, Fe, or Al bound P (Adnan et al. 2019; Gyaneshwar et al. 2002; Rodriguez and
Fraga 1999). The organic acid-mediated P solubilization process can occur in plants, however, this is
primarily a response limited to dicotyledonous crop plants (e.g. lupines, chickpea) and is a trait not
expressed in most common cereals under P deficiency (e.g. barley, wheat, maize; Khademi et al. 2010).
Biofertilizers, particularly arbuscular mycorrhizal fungi (AMF), are becoming an integral part of
sustainable agriculture and have proven to be a practical approach that improves plant production by
sustaining long-term soil fertility and health (Basiru, Mwanza, and Hijri 2021; Castiglione et al. 2021). It
is well established that symbiotic AMF can scavenge P and other nutrients from the soil and directly
transfer it to a wide range of cereals in exchange for photosynthetic carbon (Bicharanloo et al. 2020;
Khaliq and Sanders 2000; Sendek et al. 2019). The use of PSBs and AMF together may therefore increase
the bioavailability of native soil P and rock phosphate (Gyaneshwar et al. 2002). In addition, AMF
colonization may also convey tolerance to a range of other plant stresses (e.g. salinity, drought, pollution,
and pathogens; Heydari et al. 2011; Hijri and Ba 2018; Khalvati et al. 2010; Sendek et al. 2019).
In the present study, two field experiments were performed with spring barley (Hordeum vulgare
L.). The first involved an investigation of the effects of bioinoculants (AMF and PSBs) on the
availability of rock phosphate-derived P and their impact on plant growth and yield. The second
experiment evaluated whether AMF improved the sustainability of barley production through
enhanced P use efficiency in the presence of three mineral P sources with contrasting solubility.

Materials and methods

Experimental conditions
Field experiments were conducted during two growing seasons at the Bangor University
Henfaes Research Centre located at Abergwyngregyn, North Wales, UK (53° 14′ 32′′ N, 04°
01′′ 05′′ W). This site has a temperate hyperoceanic climate with a mean annual temperature
of 10.4°C and mean annual precipitation of 1060 mm. Tables 1 and 2 present the annual
precipitation, air temperatures, and soil temperatures during the two growing seasons at the
experimental site. The soil at the study site is classified as a sandy loam textured Eutric
Cambisol (US Soil Taxonomy, Typic Hapludalf) with a crumb structure. Prior to experimen­
tation, the site was a Lolium perenne L. dominated grassland under sheep grazing with no
recent history of P fertilizer application or cultivation. Soil analysis was conducted as
described by Jones et al. (2012). Soil samples taken from the topsoil (0‒30 cm, Ah horizon)
showed a very low available Olsen P content (10.6 mg P kg−1) equivalent to an Agronomic
 COMMUNICATIONS IN SOIL SCIENCE AND PLANT ANALYSIS 3

Figure 1. Mean values of root colonization affected by the P sources in the eight samples (collected at days 43, 53, 64, 73, 83, 94, 104,
and 119 after sowing) in season 1. The bars represent the standard errors of the means. The abbreviations represent the following
treatments: C) control, RP) rock phosphate, PSB) P solubilizing bacteria, M) mycorrhizae, and AMP) struvite (ammonium magnesium
phosphate hexahydrate).

P Index value of 1 (Olsen, Watenabe, and Dean 1954.), 90 mg kg−1 of available K, 42 mg kg−1
of available Mg, and a pH of 6.4.

Experimental setup and treatments

A randomized complete block design (RCBD) with three replications and a factorial experiment in
a RCBD layout with four replications were employed for field experiments 1 and 2, respectively.
Commercial seeds pre-inoculated with AMF or PSB depending on the planned treatment (see below),
were sown in rows 20 cm apart at a planting density of 400 plants m−2 on plots 19.2 m2 (10 × 1.92 m) in
size. Rock phosphate (RP), triple super-phosphate (TSP), or struvite [ammonium magnesium phos­
phate hexahydrate (AMP)] were also applied at the time of sowing. Weather conditions were
measured at the site with a Campbell Automatic Weather Station (Campbell Scientific Ltd.,
Shepshed, UK).
In the first experiment undertaken in 2010, the effects of inoculants and a natural source of
phosphorus (RP) were investigated on the growth and yield of barley (Hordeum vulgare L.). The
experiment contained eight treatments: (i) Control with no added P or inoculants, (ii) rock phosphate
alone (RP); (iii) P solubilizing bacteria (PSB) alone; (iv) arbuscular mycorrhizal fungi (AMF) alone; (v)
RP + PSB; (vi) RP + AMF; (vii) RP + AMF + PSB; and (viii) AMF + PSB. The P2O5 content of RP was
15%. PSB consisted of a commercial mixture of two types of naturally occurring phosphate solubilizing
bacteria Pseudomonas putida strain P13 and Pantoea agglomerans strain P5 sold under the name
PhsophoBarvar-2® (GreenBiotech Inc., Tehran, Iran). These bacteria are known to mobilize P through
the production of phosphatases and organic acids, respectively (Rodriguez and Fraga 1999). The
commercial AMF inoculum [Biagro® Grass(BG)] was purchased from Glenside Co., Stirling, Scotland.
BG is in granular form, wherein one AMF taxon, Rhizophagus sp. (phylum Glomeromycota; order
Glomerales) exists, and is recommended for grass crops by the company. The inoculum contained
4 M. MIRZAEI HEYDARI ET AL.

approximately 220,000 propagules kg−1. The inocula were mixed with Arabic gum to uniformly coat the
seeds and then air dried for 2 h before sowing.
In the first experiment, the barley seeds were sown on 26th March 2010 and harvested on 22nd July.
The second experiment undertaken in 2011 was conducted to investigate the effects of AMF on
production efficiency when external P sources were used. A 2 × 4 factorial experiment was used
with AMF and P source designated as the two factors. The first factor included external P (C) in
the absence of AMF and one only included only AMF. The three external P sources plus the
control (no P fertilizer) comprised the second factor and included milled rock phosphate (RP),
triple super-phosphate (TSP), ammonium magnesium phosphate (AMP), and no P fertilizer. The
P2O5 content of TSP was 46%. Struvite or ammonium magnesium phosphate (AMP) hexahydrate
(MgNH4PO4·6H2O) was used. Struvite, being a slow-release fertilizer, contains equimolar concen­
trations (1:1:1) of phosphorous (P), ammonium (NH4+), and magnesium (Mg) with alkaline pH.
In the second experiment, barley seeds were sown on 12th May 2011 and harvested on 22nd
August.
In both experiments, N and K were applied at rates of 100 and 50 kg ha−1, as urea and K2SO4
respectively, to ensure that they were not growth limiting. Half of the N was applied at sowing and the
remainder top dressed at 40 days after sowing. All TSP, rock phosphate, and struvite (80 kg P ha−1)
were added at sowing.

Growth and yield-related traits

In the first experiment, samples at distances of 50 cm along the row length in each plot were randomly
collected on 43, 53, 64, 73, 83, 94, and 104 days after sowing (DAS) to determine leaf area index as well
as leaf, stem, and straw dry weights. At the final 8th sampling event, plants were harvested at 2 m
distances along each row in each plot to determine straw, grain, and root dry weights as well as plant
height, 1000-grain weight, number of grains per spike, and grain yield. Care was taken to select
samples at their maximum grain dry matter at the Zadoks growth stage 87 (hard dough stage) 119 DAS
(HGCA 2006).
The procedure employed for sampling in the second experiment was similar to that of the
first except for the number of sampling events that were restricted to five sampling dates at 44,
76, 91, 107, and 133 DAS. Moreover, stems and leaves in this experiment were not separated;
rather, the whole weight was reported as straw-dry matter. Finally, the crop was harvested at
grain maturity.

Root colonization and phosphorus measurement

To determine levels of mycorrhizal colonization, soil particles were washed from the roots, and five
independent samples were taken and cut into lengths of 1–2 cm. The samples were then either stored
in individual vials containing a solution of formalin, acetic acid, and alcohol (FAA) or fixed in 50%
ethanol (C2H5OH) for at least 24 h. The Trypan Blue method of Phillps and Hayman (1970) was used
for clearing and staining the root samples. Levels of infection and colonization were determined using
the grid-line intersect method (Giovanetti and Mosse 1980).
Root, leaf, stem, and grain P concentrations were determined according to the molybdate method
(Page, Miller, and Keeney 1982) after dry-ashing (450°C, 24 h) and digestion of dry ash sample with
concentrated HCl. Briefly, 180 μL of Ames Reagent was added to all the samples. Then 30 μL of
ascorbic acid 10% (w/v) was added and absorbance at 820 nm in plate reader was measured after 15
minutes. The readings were converted to mg P kg−1 dry weight from standard curves.
 COMMUNICATIONS IN SOIL SCIENCE AND PLANT ANALYSIS 5

Statistical analysis
Data were subjected to analysis of variance (ANOVA) using the GLM procedure of SAS (version 9.2;
SAS Institute Inc., Cary NC, USA). Post-hoc analysis was performed using Tukey’s test at p < 0.05
(Steel and Torrie 1980).

Results
Root colonization
In the first experiment, root colonization showed a progressive increase in all treatments over the first
2 months and then declined toward harvest (Figure 1). The amount of root colonization was sig­
nificantly affected by both the presence of rock phosphate and bioinoculant addition. The highest rates
of root colonization were observed in the AMF-only and AMF+RP treatments while the presence of
PSB-amended treatments tended to be similar to the unamended control. The presence of rock
phosphate did not affect AMF root colonization, however, when AMF was combined with PSB, it
led to a significant decline in AMF root colonization.
Analysis of the data obtained from the second experiment revealed that similar to Experiment 1, root
AMF colonization increased over the first 2 months and then started to decline (Figure 2). Relative to
the unamended control treatment, root colonization was higher in the treatments containing AMF and
either rock phosphate and struvite. In contrast, the addition of TSP tended to reduce AMF colonization

Growth parameters
At harvest, the results obtained from experiment 1 revealed a significant increase in the dry weight of
the different plant components in the RP+PSB+AMF treatment relative to the control and rock

Figure 2. Mean values of root colonization affected by the P sources in the five samples (collected at days 44, 76, 91, 107, and 118
after sowing) in season 2. The bars represent the standard errors of the means. The abbreviations represent the following treatments:
C) control; RP) rock phosphate; TSP) triple super-phosphate; M) mycorrhizae; and AMP) struvite (ammonium magnesium phosphate
hexahydrate).
6 M. MIRZAEI HEYDARI ET AL.

Figure 3. Mean values of root, stem, leaf, and spike dry weights affected by the P sources in season 1. The thin (top) bars represent
the standard errors of the means. The abbreviations represent the following treatments: C) control, RP) rock phosphate, PSB)
P solubilizing bacteria, M) mycorrhizae, and AMP) struvite (ammonium magnesium phosphate hexahydrate).

Figure 4. Mean values of root, stem, and spike dry weights as affected by the P sources in season 2. The thin (top) bars represent the
standard errors of the means. The abbreviations represent the following treatments: C) control; TSP) triple super-phosphate; PSB)
P solubilizing bacteria; M) mycorrhizae; and AMP) struvite (ammonium magnesium phosphate hexahydrate).

phosphate-only treatment (Figure 3). Overall, however, the biomass in most other treatments invol­
ving rock phosphate or bioinoculants were similar at harvest.
In the second experiment, plant dry weights were significantly enhanced by the addition of either
TSP or struvite (Figure 4), however, AMF addition had no additive effect on this response. The
addition of rock phosphate induced a small increase in dry matter yield relative to the control, which
was slightly enhanced by the addition of AMF.
 COMMUNICATIONS IN SOIL SCIENCE AND PLANT ANALYSIS 7

Figure 5. Mean values of total P uptake in the root, stem, leaf, and spike as affected by P source in season 1. The thin (top) bars
represent the standard error of the mean. The abbreviations represent the following treatments: C) control, RP) rock phosphate, PSB)
P solubilizing bacteria, M) mycorrhizae, and AMP) struvite (ammonium magnesium phosphate hexahydrate).

Figure 6. Mean values of total P uptake in the root, stem, and spike as affected by the P sources in season 2. The thin (top) bars
represent the standard errors of the means. The abbreviations represent the following treatments: C) control; RP) rock phosphate;
TSP) triple super-phosphate; M) mycorrhizae triple super-phosphate and AMP) struvite (ammonium magnesium phosphate
hexahydrate).

P uptake
As expected, at harvest in Experiment 1, most P was recovered in the ears relative to that present in the
roots, stem, and leaf (Figures 5 and 9). Across all the treatments, most P was recovered in the RP+PSB
+AMF treatment, however, all amendments had higher P recovery relative to the control. However, an
8 M. MIRZAEI HEYDARI ET AL.

600 a 30
cd bc b c cd cd

Number grain per m 2

d ab a bc b
cd c d cd
500 25
Number spike per m 2

400 20

300 15

200 10

100 5

0 0

(a) (b)
Treatment Treatment

40 bc b ab a ab c bc 600
d
c bc a ab b bc
35 500 d
e
1000 grain weight (g)

Grain yield (g m-2)

30
25 400

20 300
15
200
10
100
5
0 0

(c) (d)
Treatment Treatment
2
Figure 7. Effects of the P sources on (a) number of spikes per plant, (b) number of grains per m , (c) 1000-grain weight, and (d) grain
yield in season 1. Different letters above the bars indicate statistically significant differences between variables using Tukey’s test at
p < 0.05. The abbreviations represent the following treatments: C) control, RP) rock phosphate, PSB) P solubilizing bacteria, M)
mycorrhizae, and AMP) struvite (ammonium magnesium phosphate hexahydrate).

increase in P content in response to the addition of bioinoculants was much greater when rock
phosphate was also present.
In experiment 2, P uptake was much greater in the TSP and struvite treatments relative to the control
(Figures 6 and 9), and this was further enhanced by the addition of AMF. In contrast, the addition of AMF
in isolation only resulted in a small increase in P uptake relative to the control. The addition of rock
phosphate caused a small increase in plant P uptake which was greatly enhanced in the presence of AMF.
Table 3 and 4 show correlation coefficients among the studied variables in the first and second seasons,
respectively. Grain yield significantly correlated with P uptake by roots and above-ground parts (i.e., stems,
leaves, and spikes) as well as grain P concentration in both seasons. Likewise, grain yield correlated
significantly with root mycorrhizal colonization, yield components, and dry weight.

Crop yield and yield components

In experiment 1 the highest grain yield was observed in the RP+PSB+AMF while, as expected, the
lowest yield was seen in the unamended (zero-P) control treatment (Figures 7 and 9). The increase in
the tri-addition treatment was due to the greater number of ears produced per plant rather than an
increase in the number of seeds per ear or seed weight which remained similar between treatments.
In experiment 2, the greatest yields occurred in the TSP and struvite treatments in the presence of AMF
(Figures 8 and 9). In contrast, the addition of AMF alone did not increase yield relative to the unamended
 COMMUNICATIONS IN SOIL SCIENCE AND PLANT ANALYSIS 9

700 a ab 30
c bc b a

Number grain per m 2

d cd c bc b a ab ab
600 c bc
25
Number spike per m 2

500
20
400
15
300
10
200
100 5

0 0

(a) (b)
Treatment Treatment

40 600 b a ab
b
b b ab ab ab ab a b bc
35 d cd c
500

Grain yield (g m-2)

1000 grain weight (g)

30
400
25
20 300
15
200
10
100
5
0 0

(c) (d)
Treatment Treatment
Figure 8. Effects of P sources on: (a) number of spikes per m2, (b): number of grains per m2, (c) 1000 grain weight (g), and (d) grain yield
(g m-2) at the final sampling event (experimental year two). Different letters above the bars indicate statistically significant differences
between variables using Tukey’s test at p < .0.05. The abbreviations represent the following treatments: C) control; RP) rock phosphate;
TSP) triple super-phosphate: M) mycorrhizae: and AMP) struvite (ammonium magnesium phosphate hexahydrate).

Table 1. Annual temperatures (soil and atmosphere) and precipitation at the experimental site in the first.
Air temperature (°C) Soil temperature (°C)
Month Min Max Min Max Precipitation (mm)
January 1.2 6.1 3.0 3.5 64
February 1.1 7.1 3.4 4.1 55
March 3.1 9.9 5.3 6.5 68
April 5.2 12.5 8.9 10.6 18
May 6.7 14.4 12.0 14.0 19
June 10.4 19.0 15.9 18.7 42
July 13.5 19.6 17.6 19.1 134
August 12.0 18.1 16.3 17.6 43
September 11.0 17.7 14.8 16.3 112
October 8.2 14.9 12.1 12.8 62
November 4.7 9.3 8.3 9.0 118
December 0.0 5.9 3.7 4.2 36
10 M. MIRZAEI HEYDARI ET AL.

Table 2. Annual temperatures (soil and atmosphere) and precipitation at the experimental site in the second year.
Air temperature (°C) Soil temperature (°C)
Month Min Max Min Max Precipitation (mm)
January 2.4 8.0 4.5 5.0 43
February 5.0 11.0 6.5 7.2 181
March 3.4 10.9 6.6 7.7 15
April 7.2 15.5 11.0 12.2 41
May 10.1 15.3 12.9 14.3 135
June 10.0 17.0 14.7 16.5 110
July 10.6 17.6 14.8 17.5 123
August 10.9 17.5 14.8 17.1 105
September 11.0 17.5 14.6 15.3 35
October 8.1 13.4 11.0 12.0 240
November 6.2 10.4 8.1 7.9 67
December 2.9 7.8 5.6 6.1 79

control treatment. The addition of rock phosphate caused a marginal increase in yield, which similar to the
TSP/struvite response, was slightly enhanced following the co-addition of AMF. As in Experiment 1, most
of the yield responses were due to the increase in the number of ears per plant.

Discussion
The results obtained from the two field experiments showed that the bioinoculants (AMF and PSB) in
combination with P sources were able to increase foliar P concentration, grain P uptake, and yield.
Moreover, their co-application improved P use efficiency and the apparent soil P balance, both of which
are beneficial to plant growth and crop production. The current study suggested that without an additional
P source, the advantage of bio-inoculants was significantly diminished. This strongly suggests that the
bioinoculants are not accessing native soil P to a much greater extent than uninoculated plant roots. The
findings of the current study, therefore, support previous experiments conducted on contrasting cropping
systems and soil types, which have shown the benefits of bio-inoculants
The application of bio-inoculants alone or combined with P sources plays significant roles in
optimizing P solubilization, enhancing plant growth and dry mass, increasing nutrient levels, and
enhancing organic phosphate mineralization. It also helps decrease P fertilizer application in barley
cropping systems with higher economic returns but without any considerable yield loss (Fernández
Bidondo et al. 2012; Groppa, Benavides, and Zawoznik 2012; Munda et al. 2018; Singh, Pandey, and
Singh 2011; Trabelsi et al. 2017). Soil application is the most dominant practice of AMF inoculant
followed by seed coating (Basiru, Mwanza, and Hijri 2021). Nonetheless, Basiru, Mwanza, and Hijri
(2021) assumed that seed coating is more resilient to large-scale crop production due to minimum
inoculum use. The close symbiotic relationships between the AMP and the host can promote the root
surface extension and in turn, enhance the absorption of nutrients (Hijri 2016).
Based on the results of the first field experiment, the combination of AMF, PSB, and RP led to
significantly increased dry matter (grain yield in particular), P concentration, and P uptake in plant parts
but decreased root mycorrhizal colonization. Plants suffering from nutrient deficiency during their
reproductive development might rely on reserves within the roots, stems, and leaves for their grain
nutrient requirements (Lopez et al. 2023). The higher grain P concentrations observed in the AMF+PSB
+RP treatment as compared to those in the other fertilizer treatments indicated the efficiency of the bio-
fertilizers (namely, AMF and PSB) in releasing insoluble soil P to make it available for uptake by the
roots. Application of bio-inoculant in the absence of P seems to have had a positive effect on both
P uptake and plant yield (Figure 9). The poor results obtained from the first experiment might have been
due not only to the low soil fertility but also to the weather conditions, in particular, the drought stress at
the anthesis stage, which possibly reduced yield and yield components such that grain yield failed to rise
even with increasing P concentration in the grains. Rehman and Nautiyal (2002) reported that, under
Table 3. Correlation coefficients and significance levels for the studied traits in season 1.
Total Total Total Total No. No. 1000-
Root Dry P uptake P uptake P uptake P uptake P uptake P uptake P uptake P uptake spikes grains grain Grain Grain
colonization weight in root in stem in leaf in spike in root in stem in leaf in spike per plant per m2 weight yield P concentration
Dry weight 0.88 **
P uptake in root 0.76 ** 0.34
ns
P uptake in 0.39 ns 0.78 ** 0.62 **
stem
P uptake in leaf 0.30 ns 0.86 ** 0.50 * 0.89 **
P uptake in 0.38 ns 0.83 ** 0.61 ** 0.65 ** 0.93 **
spike
Total P uptake 0.81 ** 0.57 ** 0.70 ** 0.58 ** 0.58 ** 0.59 **
in root
Total P uptake 0.43 * 0.72 ** 0.68 ** 0.78 ** 0.69 ** 0.77 ** 0.66 **
in stem
Total P uptake 0.55 ** 0.71 ** 0.60 ** 0.86 ** 0.88 ** 0.88 ** 0.49 * 0.78 **
in leaf
Total P uptake 0.67 ** 0.74 ** 0.65 ** 0.83 ** 0.69 ** 0.81 ** 0.56 ** 0.93 ** 0.80 **
in spike
No. spikes per 0.61 ** 0.20 0.81 ** 0.57 ** 0.38 ns 0.54 * 0.33 ns 0.67 ** 0.56 ** 0.76 **
plant ns
No. grains 0.50 * 0.42 * 0.68 ** 0.72 ** 0.63 ** 0.71 ** 0.38 ns 0.80 ** 0.83 ** 0.87 ** 0.62 **
per m2
1000-grain 0.64 ** 0.43 * 0.80 ** 0.71 ** 0.55 ** 0.69 ** 0.48 * 0.85 ** 0.75 ** 0.93 ** 0.83 ** 0.87 **
weight
Grain yield 0.81 ** 0.42 * 0.83 ** 0.74 ** 0.62 ** 0.73 ** 0.54 * 0.78 ** 0.83 ** 0.87 ** 0.87 ** 0.80 ** 0.87 **
Grain
P concentration 0.76 ** 0.34 0.48 * 0.41 * 0.26 ns 0.39 ns 0.40 * 0.66 ** 0.38 ns 0.69 ** 0.48 ns 0.68 ** 0.79 ** 0.89 **
ns
Total P uptake 0.49 * 0.40 * 0.71 ** 0.66 ** 0.54 * 0.65 ** 0.36 ns 0.77 ** 0.61 ** 0.65 ** 0.70 ** 0.43 * 0.81 ** 0.86 ** 0.93 *
in grains
**, * and ns: indicate significance at p < 0.01, p < 0.05 and no significance, respectively.
COMMUNICATIONS IN SOIL SCIENCE AND PLANT ANALYSIS
11
 12

Table 4. Correlation coefficients and significance levels for the studied traits in season 2.
Total Total Total Total No. No. 1000-
Root Dry P uptake P uptake P uptake P uptake P uptake P uptake P uptake P uptake spikes grains grain Grain Grain
colonization weight in root in stem in leaf in spike in root in stem in leaf in spike per plant per m2 weight yield P concentration
Dry weight 0.92 **
P uptake in root 0.83 ** 0.67 **
M. MIRZAEI HEYDARI ET AL.

P uptake in 0.87 ** 0.81 ** 0.65 **

stem
P uptake in leaf 0.39 ns 0.90 ** 0.53 * 0.65 **
P uptake in 0.45 * 0.87 ** 0.64 ** 0.65 ** 0.81 **
spike
Total P uptake 0.80 ** 0.60 ** 0.70 * 0.61 ** 0.61 ** 0.62 **
in root
Total P uptake 0.45 * 0.75 ** 0.62 ** 0.81 ** 0.72 ** 0.81 ** 0.69 **
in stem
Total P uptake 0.58 ** 0.74 ** 0.63 ** 0.80 ** 0.92 ** 0.78 ** 0.51 ** 0.82 **
in leaf
Total P uptake 0.70 ** 0.78 ** 0.65 ** 0.72 ** 0.73 ** 0.86 ** 0.59 ** 0.81 ** 0.84 **
in spike
No. spikes per 0.64 ** 0.40 * 0.50 * 0.60 ** 0.40 * 0.57 ** 0.35 ns 0.70 ** 0.58 ** 0.80 **
plant
No. grains per 0.53 * 0.45 * 0.71 ** 0.75 ** 0.67 ** 0.75 ** 0.40 * 0.84 ** 0.87 ** 0.92 ** 0.66 **
m2
1000-grain 0.67 ** 0.42 * 0.84 ** 0.74 ** 0.57 ** 0.72 ** 0.50 * 0.89 ** 0.78 ** 0.97 ** 0.87 ** 0.91 **
weight
Grain yield 0.92 ** 0.51 * 0.87 ** 0.78 ** 0.65 ** 0.77 ** 0.76 ** 0.81 ** 0.87 ** 0.91 ** 0.86 ** 0.85 ** 0.88 **
Grain
P concentration 0.80 ** 0.57 ** 0.50 * 0.43 * 0.39 ns 0.41 * 0.55 ** 0.70 ** 0.65 ** 0.72 ** 0.67 ** 0.72 ** 0.83 ** 0.93 **
Total P uptake 0.60 ** 0.32 0.75 ** 0.69 ** 0.57 ** 0.68 ** 0.51 * 0.81 ** 0.85 ** 0.91 ** 0.74 ** 0.45 ** 0.88 ** 0.91 ** 0.89 **
in grains ns
**, * and ns: indicate significance at p < 0.01, p < 0.05 and no significance, respectively.
 COMMUNICATIONS IN SOIL SCIENCE AND PLANT ANALYSIS 13

600 600 n a a
b a ab b a a b

Grain yield (g m-2)

Grain yield (g m-2)
500 bc 500 bc
c d c cd
400 400

300 300

200 200

100 100

0 0

(a) (d)
Treatment Treatment

3.5 ab a 3.5 a
P concentration (mg -1. grain)

ab ab ab

P concentration (mg -1. grain)

b b c b c bc
c
3 d 3 d cd

2.5 2.5
2 2
1.5 1.5
1 1
0.5 0.5
0 0

(b) (e)
Treatment Treatment

a ab
P uptake in grain (mg m-2)

1800 a 2000
P uptake in grain (mg m-2)

ab b bc
1600 1800 bc c
c b b bc
c 1600 d
1400 d e
1200 1400
1200
1000
1000
800
800
600 600
400 400
200 200
0 0

(c) (f)
Treatment Treatment
Figure 9. Mean values of the effects of P sources on grain yield, P concentration, and total P uptake in grains in seasons 1 and 2.
Experimental year 1: grain yield (a), P concentration (b), total P uptake in grains (c). Experimental year 2: grain yield (d),
P concentration (e), total P uptake in grain (f). Different letters above the bars indicate statistically significant differences between
variables using Tukey’s test at p < 0.05. The abbreviations represent the following treatments: C) control; RP) rock phosphate; TSP)
triple super-phosphate; M) mycorrhizae; and AMP) struvite (ammonium magnesium phosphate hexahydrate).

drought conditions, the P-solubilizing and N-fixing bacteria fail to exploit their full potential for soil
P and N utilization. It may be claimed that the concentrations of minerals, especially that of P, in plant
tissues, increase under drought stress or abiotic conditions unfavorable to vegetative growth, as also
reported by Horst et al. (2001). It has also been reported that water deficit during seed development leads
to enhanced grain protein, P, and N concentrations in barley (Rogers, Hu, and King 2023) and wheat
14 M. MIRZAEI HEYDARI ET AL.

(Houshmand, Arzani, and Maibody 2014; Javed et al. 2022; Lonbani and Arzani 2011). It may, therefore,
be hypothesized that water deficit under drought stress conditions enhances grain nutritional value.
The results obtained from the second field experiment indicated that the conjunctive application of
P sources (namely, RP, TSP, and AMP) and AMF led to significantly increased yield and yield components,
P concentration, total P uptake, and plant dry matter. The observed increases might be attributed to the
ability of mycorrhizae to increase nutrient uptake, especially P uptake, via mycorrhizal hyphae and root
extension. This is in agreement with the results reported in barley (Khaliq and Sanders 2000; Khalvati et al.
2010; Sendek et al. 2019), and in other crop plants (Bicharanloo et al. 2020; Maji et al. 2017). The current
results support their findings that mycorrhizae are capable of taking up, translocating, and transferring
water and nutrients from the soil to plant roots. Moreover, mycorrhizae have been suggested to play an
important role in the absorption of poorly available forms of nutrients, thereby increasing nutrient
bioavailability. The mechanism involved is that of mobilizing key nutrients (especially P) as a result of
depletion zones developed by mycorrhizal hyphae which extend from the root surface to the area
surrounding the root system (Bicharanloo et al. 2020; Munda et al. 2018; Rashid et al. 2016).
Further work should focus on evaluating the legacy effects of bioinoculants in successive cropping
cycles and their potential to work within wider rhizosphere microbial consortia designed to improve
other aspects of crop nutrition (e.g. N use efficiency) and resilience (e.g. drought and pathogen). It
would also be beneficial to look at the synergies between bio-inoculants and biostimulants (e.g. humic
substances, hormones, microalgae polysaccharides).

Conclusion
Overall, the current experiments showed that commercial bioinoculants appeared to improve
P availability and led to improvements in the growth and yield of barley. In addition, the synergistic
effects of the bioinoculants appeared to be independent of the form of P supplied offering the potential
for this type of technology to be widely adopted within different management regimes. Further, clear
evidence was found that bioinoculants can enhance P-use efficiency and thus may be able to maintain
satisfactory crop growth and yield at lower P fertilizer rates. It is concluded that the co-application of
AMF and PSB offers one potential strategy to promote resource use efficiency in barley cropping systems.

Acknowledgements
The authors would like to thank Mark Hughes and Llinos Hughes at the Henfaes Experimental Station for their
assistance in setting up the field trials and the collection of samples.

Disclosure statement
No potential conflict of interest was reported by the author(s).

Funding
M.M.H. received a PhD scholarship from Azad University, Iran.

Author contributions
All the authors contributed to the conception and design of the study. Material preparation as well as data collection and
analysis were performed by M.M.H. The first draft of the manuscript was written by M.M.H. and the second draft by D.
L.J. All authors read and approved the final submitted version. M.M.H. received a PhD scholarship from Azad
University, Iran.
 COMMUNICATIONS IN SOIL SCIENCE AND PLANT ANALYSIS 15

Availability of data and materials

All data related to this article will be available.

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