Introduction
Soil-plant-microbe interactions have significant role in
plant growth, development and soil health even under a nutrient-deficient
environment (Etesami and Maheshwari 2018). Numerous bacterial genera reside in
rhizosphere and inside plant tissue are called as PGPR and endophytes,
respectively (Kumar and Sharma 2017; Singh et
al. 2017). These are diverse group of bacteria form a mutual association
with host plants and improve plant growth and development (Hussain et al. 2016; Singh et al. 2017). Endophytic bacteria live inside the plant tissue
without damaging them (Meela and Serepa-Dlamini 2019) while rhizobacteria live
in rhizosphere and make symbiotic associations with plants (Khan et al. 2017). These bacteria also
alleviate environmental stresses (Etesami and Maheshwari 2018; Danish et al. 2020). Recent studies about
plant-microbe associations revealed that plants synergize with nutrient
solubilizing PGPR and endophytic communities under nutrient-deficient
conditions (Berendsen et al. 2012;
Ahmad et al. 2019b). The symbiotic
relationships between plant and bacteria exist in different ecosystems and have
a significant role in nutrients acquisition (Luo et al. 2014).
Phosphorus
(P) is the second most important macronutrient needed by plants for their
growth and development (Minhas et al.
2020). The soil-inhabiting microbes are responsible for the P cycle
continuation within the biosphere. The mechanisms concerned in P solubilization
rely upon the nature of phosphate complexes (Nadeem et al. 2014). Although 0.05% (w/w) P is present in soil in the form
of organic and inorganic P, however, its availability for plant uptake is
restrictive and among total P, only 1% is available for immediate use by plants
(Lambers and Plaxton 2015). Phosphate solubilizing bacteria (PSB) solubilize
inorganic phosphate complexes through production of organic acids and discharge
of proton (Rfaki et al. 2014). The
extent of fixed P in soil depends on the soil pH, as in acidic pH, it forms
complexes with iron and aluminum, while in alkaline pH, it forms complexes with
calcium (Wei et al. 2018; Munir et al. 2019). Specialized microbial
enzymes called phosphatases solubilize the organic P and release it for plant
(Goswami et al. 2016). The calcareous
soil has a high buffering capacity that challenges the effectiveness of PSB in
liberating P (Stephen and Jisha 2008).
The
application of P fertilizers is increasing day by day due to extensive crop
cultivation. The dynamics of P in the soil are complicated as a large proportion of applied P
fertilizers move into the immobile pool resulting in poor fertilizer use
efficiency (FUE). However, numerous bacteria located in the rhizosphere have
the ability to solubilize immobile and precipitated P contents making it
available for plant uptake (Richardson and Simpson 2011). Therefore, the
application of PSB as biofertilizer is a promising eco-friendly strategy to
improve P uptake and reducing the use of chemical fertilizers. Mostly, PSB
belong to heterotrophic bacteria that produce organic acids as their secondary
metabolites, which solubilize the insoluble P (Glick 2014; Suleman et al. 2018). They are not only involved
in P release in soil solution but these bacteria also assimilate P for their
own use to expand their colonies thus help in improving soil microbial
population (Khan and Joergensen 2009). The PGPR and endophytes are the broad
groups of bacteria that offer huge diversity and potential to convert insoluble
P into plant-available form (Chaves et
al. 2019). Such PSB use the alkaline and acidic phosphatases which use
insoluble compounds as a substrate and free up bioavailable inorganic P (Dodor
and Tabatabai 2003). Application of multi-strain PSB biofertilizer is also
helpful in improving FUE especially for P fertilizers through solubilizing
non-labile or precipitated P (Perez et
al. 2007).
The PGPR
release organic acids in rhizosphere that solubilize unavailable source of P
while some bacteria improve P availability enzymatically by releasing
phosphatases which mineralize organic P (Nesme et al. 2018; Chen and Liu 2019). Scientists described the
mechanisms adapted by bacteria for mineralization of organic P through release
of phosphatase, phytase and C-P lyase (Sembiring et al. 2017). The P solubilization in rhizosphere is affected by
soil pH, organic matter, nature of P complexes, environmental conditions and
most prominently the interaction of P solubilizing bacteria with other
microorganisms in rhizosphere (Billah et
al. 2019). Solubilization of P by bacteria reduce the use of synthetic fertilizers
which ultimately lessen the cost of production and improve soil health (Nazli et al.
2020). In addition to increase in P availability, PGPR promote plant growth by
increasing germination, inducing resistance against environmental stresses,
improving root morphology and working as biocontrol agent against pathogens
(Ahmad et al. 2019b).
The EB live
inside plant roots and promote plant growth by
performing complex functions in regulating plant physiology (Lucava and Azevedo
2013). They are involved in biocontrol of plant-pathogens through their
antagonistic nature against pathogens especially the fungi. The EB and PGPR are
well-documented for symbiotic nitrogen fixation, solubilization of P, potassium
(K), and zinc (Zn), production of phytohormones, siderophores, allelochemicals
and interact with other useful soil-inhabiting microorganisms (Garcia-Frail et al. 2015; Naseer et al. 2019). It is evident from previous studies that soil-plant
associate bacteria improved the growth of crop plant. However, biochemical
processes involving in plant growth promotion were not clear yet. Therefore,
present study was designed to isolate and characterized endophytes and
rhizosphere bacteria for biochemical and growth promoting attributes. The
selected strains were genetically identified through 16S rRNA sequencing. The
individual and combined inoculation of selected P solubilizing EB and PGPR, was
also evaluated for improving seedlings vigor index and growth attributes of
wheat (Triticum aestivum L.) under
axenic conditions.
Materials and Methods
Isolation
of endophytic and rhizospheric bacteria
Wheat plants were randomly selected, uprooted and whole
plants including the root system were carried to the laboratory in sterile
plastic bags, and stored at 4ºC until further processing. For isolation of EB,
the root system of collected plants was separated from shoots and washed under
tap water to dispose of adhering soil debris. After that, surface sterilization
of root was done by dipping them in 1% solution of NaClO for 3 min and in 70%
ethanol for 5 min. During surface sterilization, roots were five times rinsed
with sterile distilled water before and after dipping in ethanol (Hallmann et al. 2006). Surface sterilized root
material then macerated and crushed in a sterilized mortar using
phosphate-buffered saline solution to make a homogeneous mixture. The root
extract was diluted up to 10-8 and 1 mL of aliquot was transferred
on autoclaved Reasoner's 2A (R2A) agar
media which was prepared by dissolving the following chemicals in 1L distilled
water; protease peptone (0.5 g), casamino acids (0.5 g), yeast extract
(0.5 g), dextrose (0.5 g), soluble starch (0.5 g), dipotassium phosphate (0.3
g), magnesium sulfate (0.05 g), sodium pyruvate (0.3 g), agar (15 g) and pH was
adjusted to 7.0 ± 0.2 (Reasoner and Geldreich 1985). The agar plates were incubated at 30 ± 1ºC for 72 h. For
isolation of RB, a suspension in sanitize distilled water was made with 1 g of
wheat rhizosphere soil and serially diluted up to 10-8. One mL of
aliquot from every dilution was transferred on R2A agar plates and incubated at
30 ± 1ºC for 72h. The representative different morphological colonies
resulted both from EB and RB isolates were purified through multiple streaking
method and a total of 40 pure isolates each for EB and RB were kept in 50% glycerol stock at -20ºC till more experimentations.
Screening
for in vitro plant growth-promoting
characteristics
The bacterial isolates were screened for in vitro PGP characterization in terms
of solubilization of P and Zn, and production of exopolysaccharides (EPS) and
siderophores. Bacterial isolates were routinely grown on Dworkin and Foster
(DF) salt agar (Dworkin and Foster 1958). The P solubilization was evaluated by
spot inoculation of overnight grown isolates on Pikovskaya’s (PVK) agar media
(Pikovskaya 1948) and incubated at 30 ± 1ºC for 72 h. Zn solubilization by
bacterial isolates was evaluated through inoculating freshly grown isolates on tris-minimal salt media
(Fasim et al. 2002) and incubated at
30 ± 1ºC for 72 h. After incubation of both P and Zn solubilization bioassays,
the presence of the halo zones around the bacterial colonies was deliberated
positive both for P and Zn solubilization, respectively. The halo zone diameter
and colony diameter were measured to calculate solubilization index (SI) and
solubilization efficiency (SE) of both for P and Zn by following the formula
reported by Ahmad et al. (2019a). The
quantitative solubilization of P by isolates was assessed by using the PVK
broth medium. After incubation of inoculated PVK broth at 30 ± 1ºC for 72 h,
were centrifuged. Available P concentration in the supernatant was measured as
reported by Ryan et al. (2001). For
the determination of solubilized Zn concentration, isolates were grown in
tris-minimal salt broth modified with 0.1% ZnO and incubated for 72 h at 30 ±
1ºC. After incubation, culture supernatant was subjected to Atomic Absorption
Spectrophotometry analysis and values were compared with a calibration curve
drawn using working standards. Siderophores production was determined by
streaking isolates on chrome azurol S (CAS) agar media (Schwyn and Neilands 1987)
and plates were incubated at 30 ± 1ºC for 48 h. After incubation, orange halos
around the colonies were considered siderophores positive. The isolates were
screened for exopolysaccharides (EPS) production by propagating them on De Man, Rogosa and Sharpe (MRS)
agar media at 30 ± 1ºC for 72 h. The isolates that produce mucoid
colonies were recorded EPS positive.
The amount
of indole-3-acetic acid (IAA) production by way of bacterial isolates both in
the presence and absence of L-tryptophan (L-trp) become assessed through a
colorimetric technique using Salkowski’s method (Ehmann 1977). The 48h old
bacterial cultures were inoculated in DF salt media in the presence and absence
of L-trp and incubated for 48 h at 30 ± 1°C and 100 r.p.m. After incubation
broth cultures were filtered through sterile Whatman # 1 filter paper. The IAA
standards of various concentration viz.,
25, 50, 100, 150, 200, 250, and 300 µg mL-1 was prepared. The
1 mL of culture aliquot and 2 mL of Salkowski’s reagent was mixed and put in
dark for 60 min. The optical density (OD) was measured at 530 nm and values
were compared by plotting standard curve. The catalases, oxidase, and urease activities by selected EB and RB isolates
were performed through following standard procedures reported by Cappuccino and
Sherman (Cappuccino and Sherman 2002). Some miscellaneous plant growth promoting characterization tests in terms of chitinolytic activity, cellulose degrading ability,
protease, and esterase activities were
also carried out by following the standard protocols (Atlas 1946; Sierra 1957;
Chernin et al. 1998; Patel and Desai
2015).
Effect of PSB isolates on
growth of wheat seedlings in axenic conditions
The efficient PSB isolates were confirmed for their
prospective to escalates growth of wheat seedlings under axenic conditions.
These conditions were maintained in growth room as 70% humidity, 12 h day
length (light intensity @ 1000 flux unit area-1 unit time-1
with artificial light) and day and night temperature was adjusted as 20°C and
15°C, respectively. Seven isolates from EB and eight isolates from PGPR were
prepared through growing in R2A broth for 48 h in a shaking incubator at 30 ±
1°C. Wheat seeds were surface disinfected using ethanol (95%) and mercuric
chloride (0.2%) solution as reported by Al-Adham et al. (2012). Surface sterilized wheat seeds were inoculated with
EB and PGPR isolates through dipping in respective culture for 10 min. Plastic
jars (20 cm height) filled with sand (600 g jar-1) were moisturized
with half-strength Hoagland solution and autoclaved. Inoculated wheat seeds
were sown in each jar but control was maintained by sowing seeds soaked in a
sterilized broth. Jars were arranged in completely randomized design in
triplicate and incubated in alternative 12 h of light at 20 ± 2ºC and 12 h of
the dark period at 15 ± 2ºC. To meet nutritional and irrigational requirements,
jars were irrigated with a half strength Hoagland solution. Three plants in
each jar were maintained by thinning after establishment of seedlings. The
ability of the isolates to colonize wheat roots was estimated by taking seven
days old root samples (0.2 g) in a sterilized mortar and crushed after adding
sterilized water (5 mL). The crushed suspension was diluted up to 10-5
and 1 mL from every dilution was poured on R2A agar plates and incubated at 30
± 1°C for 72 h. After incubation, total bacterial colonies were calculated by
using digital colony counter and expressed as a colony-forming units (CFU) mL-1.
After
three-weeks of sowing, wheat seedlings were harvested and growth attributes in
terms of shoot length, root length, shoot and root dry biomass were recorded. Four most efficient isolates each from
EB and PGPR were selected on the basis of better tendency to improve wheat
growth and were evaluated for compatibility of isolates to each other. Two
isolates (one from EB and other from PGPR) were cross streaked on R2A agar
plates in perpendicular direction. Similarly, sets of compatibility tests were
conducted through cross-matching the rest of EB and RB isolates and incubated
in triplicate at 30 ± 1°C for 48 h. The inhibition zone at the point of
intersection was observed and results marked compatible for isolates having no
inhibition zone at the point of intersection. The compatible EB and RB isolates
were selected to assess for their growth promoting ability through
co-inoculation. A jar trial was conducted through sowing wheat seeds
co-inoculated with respective EB and PGPR isolates in a 1:1 ratio. All protocols
of jar experiment were followed as reported above for sole inoculation. After
three-weeks of emergence, seedling vigor index was calculated and growth
attributes were measured as mentioned above for sole inoculation.
Identification
of selected isolates
The best isolates on the basis of growth-promoting
abilities (two from EB; ZE15 and ZE32 and three from PGPR; ZR2, ZR3, and ZR19)
were selected for identification through 16S rRNA partial gene sequencing.
Crude DNA of selected isolates was extracted through treating with proteinase K
(Cheneby et al. 2004). The PCR
reactions were performed by using 2.5 µL crude DNA and PCR primers were
27F (AGAGTTTGATCMTGGCTCAG) and 1492R (TACGGYTACCTTGTTACGACTT) (Hussain et al. 2015). The length of amplified
product was confirmed through setting apart on agarose gel (1%) along with
GeneRuler. The purified PCR product was sequenced using commercial service of
MACROGEN Seoul, Korea (http://macrogen.com/eng/) by using sequenced primers
785F (GGATTAGATACCCTGGTA) and 907R (CCGTCAATTCMTTTRAGTTT). Resulted sequences
were blasted on NCBI servers and strains were identified through constructing
the phylogenetic tree with selected closely related taxa using MEGA 7.0.14
(Roohi et al. 2012; Kumar et al. 2016).
Statistical
analysis
For statistical analyses, the data were compared through
one-way analysis of variance (ANOVA) technique by employing a liner model
Completely Randomized Design (CRD). The means were compared through multiple
comparisons (LSD at 5% level of probability) using Statistix v. 8.1 (Analytical
Software, Tallahassee, FL, USA) (Steel et
al. 1997).
Results
Isolation
of P solubilizing EB and RB isolates
Forty isolates each for endophytic bacteria; coded as
ZE1, ZE2… ZE40 and PGPR; coded as ZR1, ZR2, … ZR40 were isolated and screened
for the solubilizing P qualitatively and quantitatively. Results of qualitative
P solubilization showed that 11 EB and 12 PGPR isolates were able to solubilize
P (Table 1). The PGPR isolates showed more promising results in terms of
qualitative P solubilization, SE, SI and solubilized concentration than EB
isolates. Isolate ZR3 showed the maximum P solubilization halo zone diameter
(21.7 mm), SE (369%), and SI (4.7) (Table 2). The EB isolates were also better
P solubilizers. Among them, isolate ZE32 showed the maximum P solubilization
halo zone diameter (18 mm), SE (290%), and SI (3.9). Quantitative P
solubilization revealed that RB isolates ZR3 and ZR19 showed the maximum
concentration of solubilized P (32.7 mg L-1) which were
statistically similar to each other (Table 2). Among EB isolates, ZE32 showed
the maximum P solubilization (32 mg L-1) which was non-significant (P ≥
0.05) with RB isolates ZR3 and ZR19. The isolates ZE2, ZE10, ZE18, ZE28, ZR11,
ZR22, ZR36, and ZR40 were weak P solubilizers, and were not selected for
further evaluation.
Screening
for in vitro plant growth-promoting
characteristics
Phosphate solubilizing EB and PGPR isolates were screened
for solubilization of Zn, and production of EPS and siderophores. Results
(Table 1) indicated that most of the isolates were progressive for Zn
solubilization except isolates ZE2, ZE28 and ZR11. The isolate ZE19 showed the
maximum Zn halo zone diameter (24.3 mm), however the maximum SE of 244% and SI
of 3.4 was observed by the isolate ZR19. Isolate ZR19 also showed the maximum
concentration of solubilized Zn (27.7 L1) that was statistically
similar to isolates ZR2 and ZR3 (P ≤
0.05). Among EB, isolate ZE15 showed better results in terms of solubilized Zn
(26 mg L-1) which was non-significant (P ≥ 0.05) to
isolates ZR2, ZR3, and ZR19. The majority of tested PSB isolates have the
ability to produce siderophores except the isolates ZE18, ZR11, ZR22, ZR36, and
ZR40, while all isolates were able to produce exopolysaccharides. Seven EB and
eight PGPR isolates positive for all tested traits viz., P and Zn
solubilization, and EPS and siderophores production were selected for further
evaluation.
Effect
of sole inoculation of EB and PGPR isolates on growth of wheat seedlings
Effectiveness of sole inoculation of EB and PGPR
isolates to escalate growth of wheat seedlings was estimated in jar trial. The
sole inoculation of P solubilizing EB and PGPR isolates significantly (P ≤ 0.05) enhanced the shoot
length, root length, shoot and root dry biomass of wheat seedlings as related
to uninoculated control (Table 3). The mean values of growth attributes
revealed better results due to inoculation with PGPR isolates over the EB isolates. The
highest improvement in shoot length up to 22% was recorded due to application
of isolate ZR3 followed by isolate ZR19 (21% increase over control). Results of
these isolates were parallel to each other, while more than uninoculated
control (P ≤ 0.05). The isolate
ZR3 reported the absolute increase in root length (29%) that was
non-significant with the isolate ZE32. The bacterial isolates ZR2, ZR3, and
ZR19 showed the highest improvement in dry biomass which were similar to each
other. The highest improvement in root dry biomass was observed due to
inoculation with ZR19 that showed 65% increase in dry biomass of root. The EB
isolates inoculation also showed better increase in growth attributes of wheat
seedlings. The EB isolate ZE15 showed better increase in shoot length (17%), root length
(24%), shoot dry biomass (17%) and root dry biomass (39%) over un-inoculated
control. The best plant growth promoting RB isolates viz., ZR2, ZR3, ZR5, and ZR19 and EB isolates viz., ZE15, ZE19 and ZE32 were further selected for co-inoculation
compatibility assay.
Compatibility
test and effect of co-inoculation of EB and PGPR isolates on growth of wheat
The selected PGP isolates of EB and PGPR were cross
streaked on agar media for their compatibility test and results revealed
compatibility of isolate ZE15 with isolates ZR2, ZR3, and ZR19 while isolate
ZE19 was compatible with isolates ZR3 and ZR19 (Table 4). The isolate ZE32 was
compatible with isolates ZR3, ZR5, and ZR19. All other EB and RB isolates were
antagonistic to each other. The compatible isolates were co-inoculated on wheat
seeds to assess their competence to stimulate growth of wheat.
Outcomes of
the stimulus of co-inoculation with EB and PGPR isolates on wheat seedlings are
presented in Table 5. The co-inoculation with EB and PGPR isolates exhibited
considerable (P ≤ 0.05) escalation
in seedling vigor index, and growth of wheat seedlings as related to
un-inoculated control. The co-inoculation treatment ZE32 + ZR19 was the best combination to improve seedlings growth of
wheat then other co-inoculation treatments. It exhibited the maximum
improvement in seedling vigor index (69%), root length (31%), shoot length
(29%), root dry biomass (33%) and shoot dry biomass (36%) as compared to uninoculated control. The co-inoculation
combination of ZE15 + ZR2 also showed better root dry biomass of wheat
seedlings as compared to un-inoculated
control and was similar to the co-inoculation of ZE32 + ZR19.
Growth
promoting attributes and biochemical features of selected isolates
The selected two isolates of EB (ZE15 and ZE32) and three
isolates of PGPR (ZR2, ZR3, and ZR19) were tested for multiple plant growth
promoting attributes. All of these isolates showed production of IAA in
presence as well as absence of L-trp (Table 6). Accumulation of IAA by selected
isolates in presence and absence of L-trp was similar (P ≤ 0.05) to each other. Isolate ZE32 produced the maximum
IAA without L-trp (3.0 μg mL-1) and with L-trp
(16.9 μg mL-1) and was
statistically non-significant to other tested isolates. The EB isolates were
better in terms of root colonization over PGPR isolates (Table 6). The EB
isolates ZE15 and ZE32 showed root colonization up to 3.0 × 106 CFU
g-1 while all PGPR isolates ZR2, ZR3 and ZR19 showed 1.5 × 106
CFU g-1 root colonization. All the tested isolates have the ability
to produce HCN except isolates ZE15 and ZR19. The enzymatic activities in terms
of catalase, oxidase, protease, cellulase, urease, chitinase and esterase activities by EB and RB isolates were
performed and their results are depicted in Table 6. All the tested
isolates were positive for protease, cellulase, and esterase activities. Three
isolates e.g. ZR2, ZR3, and ZE15 demonstrated oxidase activity while
urease activity was only observed in isolate ZE32. Only PGPR isolates (ZR2,
ZR3, and ZR19) were catalase positive while all the tested isolates were
negative for chitinase activity.
Table 1: Evaluation of endophytic and rhizospheric
bacterial strains for phosphate and zinc solubilization, and production of
exopolysaccharides and siderophores
|
Bacterial isolates |
Phosphate solubilization |
Zinc solubilization |
Production of EPS |
Production of siderophores |
|
ZE1 |
+ |
+ |
+ |
+ |
|
ZE2 * |
+ |
- |
+ |
+ |
|
ZE5 |
+ |
+ |
+ |
+ |
|
ZE7 |
+ |
+ |
+ |
+ |
|
ZE10 * |
+ |
- |
+ |
+ |
|
ZE15 |
+ |
+ |
+ |
+ |
|
ZE18 * |
+ |
+ |
+ |
- |
|
ZE19 |
+ |
+ |
+ |
+ |
|
ZE28 * |
+ |
- |
+ |
- |
|
ZE32 |
+ |
+ |
+ |
+ |
|
ZE38 |
+ |
+ |
+ |
+ |
|
ZR2 |
+ |
+ |
+ |
+ |
|
ZR3 |
+ |
+ |
+ |
+ |
|
ZR5 |
+ |
+ |
+ |
+ |
|
ZR7 |
+ |
+ |
+ |
+ |
|
ZR11 * |
+ |
- |
+ |
- |
|
ZR15 |
+ |
+ |
+ |
+ |
|
ZR18 |
+ |
+ |
+ |
+ |
|
ZR19 |
+ |
+ |
+ |
+ |
|
ZR22 * |
+ |
+ |
+ |
- |
|
ZR25 |
+ |
+ |
+ |
+ |
|
ZR36 * |
+ |
+ |
+ |
- |
|
ZR40 * |
+ |
+ |
+ |
- |
*Isolates were week phosphate and zinc solubilizers and were not
selected for further experimentations (Results for solubilization of phosphate, zinc, and production of
exopolysaccharides (EPS) and siderophores were confirmed by repeating the
bioassays in three replication)
The sign, (+) express the accuracy of the
tested attributes and the sign, (-) demonstrate the lack of the traits
Table 2: Qualitative and quantitative solubilization
of phosphate and zinc by endophytic and rhizospheric bacterial isolates
|
Bacterial isolates |
Phosphate solubilization |
Zinc solubilization |
||||||
|
HZD (mm) |
SI |
SE (%) |
SC (mg L-1) |
HZD (mm) |
SI |
SE (%) |
SC (mg L-1) |
|
|
ZE1 |
8.7 gh |
3.3 d-f |
203.3 d-f |
27.3 de |
11.0 g |
3.1 bc |
207.8 bc |
22.7 d |
|
ZE5 |
9.7 g |
3.7 b-d |
269.4 b-d |
28.3 c-e |
13.0 fg |
3.1 bc |
206.4 bc |
23.0 cd |
|
ZE7 |
7.3 h |
2.4 f |
137.8 f |
26.0 de |
18.7 bc |
2.9 c |
193.6 c |
23.0 cd |
|
ZE15 |
16.7 cd |
3.7 b-d |
265.9 b-d |
31.7 a-c |
17.7 b-d |
3.0 bc |
203.7 bc |
26.0 ab |
|
ZE19 |
13.7 ef |
3.4 c-e |
243.3 c-e |
28.7 b-e |
24.3 a |
3.2 a-c |
215.2 a-c |
23.4 b-d |
|
ZE32 |
18.3 c |
3.9 bc |
290.5 bc |
32.0 ab |
14.3 ef |
3.1 bc |
205.6 bc |
25.2 a-d |
|
ZE38 |
12.3 f |
3.7 b-d |
265.0 b-d |
29.3 a-d |
14.3 ef |
3.1 bc |
205.6 bc |
23.2 b-d |
|
ZR2 |
18.7 bc |
3.8 bc |
281.8 bc |
29.3 a-d |
19.0 b |
3.1 a-d |
211.5 bc |
27.3 a |
|
ZR3 |
21.7 a |
4.7 a |
368.9 a |
32.7 a |
16.0 c-e |
3.3 ab |
228.9 ab |
27.0 a |
|
ZR5 |
16.7 cd |
4.1 ab |
313.3 ab |
27.0 de |
14.3 ef |
3.3 ab |
233.3 ab |
25.0 a-d |
|
ZR7 |
15.0 de |
3.4 c-e |
238.9 c-e |
26.3 de |
13.3 e-g |
2.9 c |
190.58 c |
25.0 a-d |
|
ZR15 |
13.3 ef |
2.9 ef |
187.8 ef |
25.3 e |
15.3 d-f |
2.9 c |
192.1 c |
24.0 b-d |
|
ZR18 |
13.3 ef |
2.6 ff |
161.1 f |
27.3 de |
14.3 ef |
2.9 c |
186.8 c |
25.0 a-d |
|
ZR19 |
20.7 ab |
4.0 bc |
299.4 bc |
32.3 a |
18.7 bc |
3.4 a |
244.1 a |
27.7 a |
|
ZR25 |
12.7 f |
2.9 ef |
193.6 ef |
26.0 de |
13.0 fg |
3.1 bc |
205.6 bc |
25.7 a-c |
|
LSD (P ≤ 0.05) |
2.05 |
0.66 |
66.33 |
3.38 |
2.99 |
0.32 |
31.39 |
2.84 |
Means sharing same letter(s) within the
column are similar to each other according to least significant difference
(LSD) test at P 0.05
HZD= Halo zone diameter; SI= Solubilization
index; SE= Solubilization efficiency; SC= Solubilized concentration; ZE and ZR,
Z is taken from first letter of researchers’ name (Zafar Iqbal) while E and R
for endophytes and rhizospheric bacteria, respectively
Table 3: Effect of sole inoculation of endophytic and rhizospheric bacterial
isolates on growth of wheat seedlings under axenic conditions
|
Bacterial isolates |
SL (cm) |
RL (cm) |
SDB (g jar-1) |
RDB (g jar-1) |
|
Control |
7.1 i |
6.8 i |
0.05 e |
0.04 i |
|
ZE1 |
7.4 gh |
6.9 hi |
0.05 e |
0.04 f |
|
ZE5 |
7.6 fg |
7.1 gh |
0.05 e |
0.04 fg |
|
ZE7 |
7.7 ef |
7.3 f |
0.05 e |
0.04 h |
|
ZE15 |
8.3 bc |
8.4 b |
0.06 bc |
0.05 de |
|
ZE19 |
7.6 fg |
7.9 c |
0.05 de |
0.04 f |
|
ZE32 |
8.2 cd |
8.7 a |
0.06 cd |
0.05 e |
|
ZE38 |
7.4 gh |
8.0 c |
0.05 de |
0.04 gh |
|
ZR2 |
8.0 de |
8.3 b |
0.07 a |
0.06 b |
|
ZR3 |
8.6 a |
8.8 a |
0.07 a |
0.06 a |
|
ZR5 |
7.3 hi |
7.4 ef |
0.06 bc |
0.05 cd |
|
ZR7 |
7.4 h |
7.3 fg |
0.06 bc |
0.05 de |
|
ZR15 |
7.5 f-h |
7.7 d |
0.06 b |
0.05 c |
|
ZR18 |
7.4 gh |
7.9 c |
0.06 b |
0.05 c-e |
|
ZR19 |
8.5 ab |
8.4 b |
0.07 a |
0.06 a |
|
ZR25 |
7.3 hi |
7.6 de |
0.06 b |
0.05 c-e |
|
LSD (P ≤ 0.05) |
0.26 |
0.21 |
0.004 |
0.002 |
Means sharing same letter(s), within the
column are statistically similar to each other according to least significant
difference (LSD) test at P ≤
0.05; Three plants were maintained in each jar
SL=Shoot length; RL = Root length; SDB = Shoot
dry biomass; RDB = Root dry biomass
Table 4: Compatibility
test of endophytic and rhizospheric bacterial isolates
|
Combination |
Compatibility |
Combination |
Compatibility |
|
ZE15 + ZR2 |
Compatible |
ZE19 + ZR5 |
Not compatible |
|
ZE15 + ZR3 |
Compatible |
ZE19 + ZR19 |
Compatible |
|
ZE15 + ZR5 |
Not compatible |
ZE32 + ZR2 |
Not compatible |
|
ZE15 + ZR19 |
Compatible |
ZE32 + ZR3 |
Compatible |
|
ZE19 + ZR2 |
Not compatible |
ZE32 + ZR5 |
Compatible |
|
ZE19 + ZR3 |
Compatible |
ZE32 + ZR19 |
Compatible |
Incompatible pairs of isolates were not
selected for further evaluation (Compatibility of endophytic and rhizospheric
bacterial isolates were confirmed by repeating the bioassays in three
replications)
Isolates were cross streaked on a petri plate
and incubated at 30°C ± 1 and observed the growth after 24, 48 and 72 hours.
Growth inhibition at the point of intersection means isolates were incompatible
and vice versa.
Table 5: Effect of co-inoculation of endophytic and
rhizospheric bacterial isolates on growth of wheat seedlings under axenic
conditions
|
Co-inoculation |
SVI |
SL (cm) |
RL (cm) |
SDB (g jar-1) |
RDB (g jar-1) |
|
Control |
6.2 e |
6.5 g |
6.4 h |
0.041 f |
0.037 d |
|
ZE15 + ZR2 |
9.4 b |
7.0 de |
7.5 de |
0.053 b |
0.048 a |
|
ZE15 + ZR3 |
7.4 d |
7.9 b |
8.2 b |
0.046 de |
0.040 c |
|
ZE15 + ZR19 |
8.3 cd |
7.2 d |
7.7 c |
0.049 cd |
0.044 b |
|
ZE19 + ZR3 |
7.4 d |
6.8 f |
7.4 ef |
0.045 e |
0.044 b |
|
ZE19 + ZR19 |
8.1 cd |
7.5 c |
7.7 cd |
0.044 e |
0.039 cd |
|
ZE32 + ZR3 |
8.7 bc |
6.8 f |
6.8 g |
0.049 cd |
0.045 b |
|
ZE32 + ZR5 |
8.4 b-d |
6.9 ef |
7.3 f |
0.050 c |
0.040 c |
|
ZE32 + ZR19 |
10.5 a |
8.4 a |
8.4 a |
0.056 a |
0.049 a |
|
LSD (P ≤ 0.05) |
1.06 |
0.24 |
0.18 |
0.003 |
0.003 |
Means sharing same letter(s) within the column
are statistically same according to least significant difference (LSD) test at P ≤ 0.05; Three plants were
maintained in each jar
SVI= Seedling vigor index; SL= Shoot length;
RL= Root length; SDB= Shoot dry biomass; RDB= Root dry biomass
Identification
of selected isolates
The selected plant growth promoting EB and PGPR isolates viz., ZE15, ZE32, ZR2, ZR3, ZR19 were
identified through 16S rRNA partial gene sequencing. The isolate ZE15, ZR2, and
ZR3 showed resemblance with Bacillus
subtilis up to 99.85, 99.55 and 99.71%, respectively. These isolates were
identified as a B. subtilis ZE15 (Fig. 1), B. subtilis ZR2 (Fig. 2), and B. subtilis ZR3 (Fig. 3) and submitted to NCBI under accession
number MN003400, MN007184 and MN007185, respectively. The isolates ZE32 and
ZR19 showed similarity of 99.55 and 97.78%, respectively, with B. megaterium and
identified as B. megaterium ZE32 (Fig. 4) and B. megaterium ZR19 (Fig. 5),
respectively. These isolates were submitted to NCBI under accession numbers
MN003401 and MN007186.
Discussion
The sole as well as co-inoculation of compatible EB and
PGPR isolates significantly promoted the growth of wheat seedlings and the
selected EB and PGPR isolates were identified as Bacillus spp. through bioinformatics analysis (Table 1–6).
In the
present study, the EB and PGPR were isolated and selected on the basis of solubilization
of insoluble source of P. Qualitative P solubilization revealed the
solubilization index was in the range of 2.3 to 3.9 by EB and 2.6 to 4.6 by
PGPR isolates while solubilization efficiency ranged from 138 to 290% for EB
and 161 to 369% for PGPR isolates. Inoculation of EB isolates solubilized P up
to 26 µg mL-1 and PGPR isolates solubilized P up to 27 µg
mL-1. Solubilization of P by EB and PGPR isolates in the present
study could be due to production of organic acids. One of the most common mechanism
to increase P solubility from insoluble P source is the accumulation of organic
acids (Mumtaz et al. 2019). For
example, Saeid et al. (2018) informed
the solubilization of P by B. megatarium through
production of different organic acid like gluconic, lactic, acetic, succinic
and propionic acids that solubilized insoluble source of P. Different Bacillus spp. (B. cerus, B. subtillis, B.
thurengenses, B. megatarium, etc.)
present in soil and improve the availability of P (Meena et al. 2017). Bacterial strains
showed acid phosphatases activity which improved mineralization of P (Cheng et al. 2017). Acid phosphatase activity
in rhizosphere stimulate plant roots to produce organic acids which boost
solubilization of P (Eisenhaur et al. 2017). The
mechanism adopted by bacterial strain for solubilization of phosphate might be
helpful for the solubilization of insoluble zinc. It can be correlated with the
release of organic acids by bacteria which react with insoluble zinc compounds
and release plant available form of zinc (Mumtaz et al. 2019).
%201439-1448_files/image002.png)
Fig. 1: Neighbor-joining phylogenetic evaluation consequent from the various
associations of 16S rRNA gene sequence of Bacillus
subtilis ZE15 with existences of different bacterial strains from Gene Bank
database
%201439-1448_files/image004.png)
Fig. 2: Neighbor-joining phylogenetic evaluation consequent from the various
associations of 16S rRNA gene sequence of B.
subtilis ZR2 with existences of different bacterial strains from Gene Bank
database
%201439-1448_files/image006.png)
Fig. 3: Neighbor-joining phylogenetic evaluation consequent from the various
associations of 16S rRNA gene sequence of Bacillus
subtilis ZR3 with existences of different bacterial strains from Gene Bank
database
%201439-1448_files/image008.png)
Fig. 4: Neighbor-joining phylogenetic evaluation consequent from the various
associations of 16S rRNA gene sequence of B.
megateriam ZE32 with existences of different bacterial strains from Gene
Bank database
%201439-1448_files/image010.png)
Fig. 5: Neighbor-joining phylogenetic evaluation consequent from the various
associations of 16S rRNA gene sequence of B.
megateriam ZR19 with existences of different bacterial strains from Gene
Bank database
The EB isolates in the present study,
solubilized insoluble Zn compound up to 26 µg mL-1 while PGPR
isolates exhibited solubilization of Zn up to 27 µg mL-1.
Similarly, solubilization of Zn in agar and broth culture were reported by
Ramesh et al. (2014). In another
study, Mumtaz et al. (2019) described
the possible mechanism of Zn solubilization by B. cereus and Bacillus spp. ZM20 through accomulation
of lactic acid and acetic acid as major Zn solubilizing metabolites. They
stated that lactic acid released by B.
cereus up to 22 mM with an increase up to 691% under ZnO amended
media as compared to lactic acid production in media without ZnO that was 0.035
mM. The solubilization of Zn could also be due to production of formic,
isobutyric, isovaleric, citric and succinic acids identified as minor
metabolites for release of Zn. In current study, inspected EB and PGPR isolates
were also progressive for production of EPS and siderophores. Parallel
findings were stated by Mumtaz et al.
(2017) who described that P and Zn solubilizing rhizobacteria strains could
produce EPS and siderophores, and could promote plant growth through
facilitation of iron availability for plant uptake. Siderophores production
improve plant growth through supply of Fe or indirectly by controlling the
uptake of Fe by other microbes (Ahmad et
al. 2008). The ability of microbes to solubilize P and Zn, and production
of exopolysacchrides and siderophore has been well documented to increase the
nutrient use efficiency even under harsh environments (Ahmad et al.
2019b) that can be attributed to improvement in germination, root development
and growth.
Results of
the current study showed that co-inoculation of PSB isolates was
better in improving the seedling vigor index, root and shoot length, fresh and
dry biomass over uninoculated control as compared to sole inoculation.
Improvement in growth of wheat seedlings would be due to direct and indirect
plant growth promoting traits of the isolates. These mechanisms could be in
terms of increase in nutrient availability viz.
fixation of atmospheric nitrogen, solubilization of P and Zn (found positive in
current study) and suppression of pathogenic microbes (Nazli et al. 2020), accumulation of plant
hormones (Ahmad et al. 2019b) and
siderophores (Wilson et al. 2006).
Improvement in plant growth due to individual and combined application of PGPR,
and endophytes was also described in former studies (Ahmad et al. 2019b; Hussain et al.
2020).
Table 6: Characterization for biochemical and plant
growth promoting attributes of selected strains
|
Characteristics |
ZE15 |
ZE32 |
ZR2 |
ZR3 |
ZR19 |
|
IAA without L-tryp (μg mL-1) |
2.0 a |
3.0 a |
2.9 a |
2.8 a |
2.2 a |
|
IAA with L-tryp (μg mL-1) |
15.0 ab |
16.9 a |
15.3 ab |
16.1 ab |
14.0 b |
|
Root colonization (CFU g-1) |
3.00 × 106 |
3.03 × 106 |
1.53 × 106 |
1.47 × 106 |
1.50 × 106 |
|
Catalase activity |
- |
- |
+ |
+ |
+ |
|
Oxidase activity |
+ |
- |
+ |
+ |
- |
|
HCN production |
- |
+ |
+ |
+ |
- |
|
Protease production |
+ |
+ |
+ |
+ |
+ |
|
Cellulose degradation |
+ |
+ |
+ |
+ |
+ |
|
Urease activity |
- |
+ |
- |
- |
- |
|
Chitinase activity |
- |
- |
- |
- |
- |
|
Esterase activity |
+ |
+ |
+ |
+ |
+ |
Indole-3-acetic acid (IAA) production values
both in presences and absences of L-tryptophan (L-trp) are mean of three
replicates and means followed by the same letter(s) are not significantly
different according to the least significant difference test at 5% probability
The symbol, (+) represent the presence of
traits and symbol, (-) represent the absence of traits; HCN= Hydrogen cyanide
The two EB isolates (ZE15 and ZE32) and three PGPR isolates (ZR2, ZR3 and ZR19) showing
better efficiency in improving wheat growth were evaluated for plant growth
promoting traits. These isolates possess multiple plant growth promoting
characteristics which support the increase in plant growth due to their
inoculation in wheat. In the current study, selected isolates showed the
production of IAA in presence as well as in absence of L-trp which is a
valuable in PGP attributes (Patten and Glick 2002). IAA produced by bacteria
act as a signaling compound for the expression of rubisco that promote the
production of amino acids and organic acids (Defez et al. 2019). These biochemical characteristics of bacterial
strains promote plant growth through creation of resistance in plants against
different stresses and suppressing pathogens activity in rhizosphere as well
(Ahmad et al. 2019b).
In the
current study, the selected isolates possess more than three plant growth
promoting attributes in terms of HCN production, catalase, oxidase, protease,
cellulase, and esterase activities which might synergistically helped to
increase plant growth. The selected isolates ZE15,
ZE32, ZR2, ZR3 and ZR19 were identified as Bacillus spp. through 16S rRNA partial gene sequencing. In the
present study, we identified P solubilizing EB and PGPR strains possessing PGP
traits, however, these inoculants need to be evaluated for their effectiveness
under natural conditions before recommendation to be applied as biofertilizer.
Furthermore, before launching them as phosphate solubilizing biofertilizer, it
is highly recommended that they should be evaluated for production of organic
acids, and phytase gene expression and quantification is also required to find
out as possible P solubilization mechanism.
Conclusion
Phosphorous solubilizing strains viz., B. megaterium ZE32,
B. subtilis ZR3 and B. megaterium ZR19 have several plant growths
promoting features in terms of solubilization of zinc, production of
exopolysaccharides, hydrogen cyanide, and siderophores and enzymatic
activities. The sole as well as co-inoculation of endophytes and rhizosphere
bacteria improved the vigor index and growth attributes of wheat seedlings.
These strains could be potential bio-inoculants to overcome the problem of
phosphorous deficiency; however, their extensive evaluation under natural
conditions is required before their recommendation to be used as biofertilizer
to improve nutrient use efficiency.
Acknowledgement
The current study is a part of Ph.D. dissertation of Mr.
Zafar Iqbal (the first author) and he would like to express his gratitude to
the Soil Microbiology and Biotechnology Laboratory, Department of Soil Science,
the Islamia University of Bahawalpur, Pakistan, for providing the working space
and other facilities. Authors highly acknowledged the help from Dr. Muhammad
Zahid Mumtaz (Assistant Professor) Institute of Molecular Biology and
Biotechnology, The University of Lahore, Main Campus, Lahore, Pakistan, during
the preparation of this manuscript.
Author Contributions
All authors contributed to the study commencement and
design. The experiment plan, material preparation, data collection, analyses
and preparation of figures and tables were performed by Zafar Iqbal, Maqshoof
Ahmad, Moazzam Jamil and Muhammad Fakhar-U-Zaman Akhtar. The initial draft of
manuscript was prepared by Zafar Iqbal and edited by Maqshoof Ahmad. All the
Authors, reviewed drafts of the paper, commented and approved the final draft.
References
Ahmad F, I Ahmad, MS
Khan (2008). Screening of free-living rhizospheric bacteria for their multiple
plant growth promoting activities. Microbiol Res 163:173‒181
Ahmad
M, Z Adil, A Hussain, MZ Mumtaz, M Nafees, I Ahmad, M Jamil (2019a). Potential
of phosphate solubilizing Bacillus strains for improving growth and
nutrient uptake in mungbean and maize crops. Pak J Agric Sci 56:283‒289
Ahmad
M, SM Nadeem, ZA Zahir
(2019b). Plant-Microbiome interactions in agroecosystem: An application. In:
Microbiome in Plant Health and Disease,
pp: 251‒291. Kumar V, R Parasad, M Kumar, D Choudhary (Eds.). Springer,
Singapore
Al-Adham I, R
Haddadin, P Collier (2012). Types of Microbicidal and Microbistatic Agents. In:
Russell, Hugo and Ayliffe’s: Principles
and Practice of Disinfection, Preservation and Sterilization, pp: 5‒70.
Fraise AP, JY Maillard, S Sattar (Eds.). Wiley-Blackwell, Oxford, UK
Atlas RM (1946). Handbook
of Microbiological Media, 4th edn. CRC Press Taylor &
Francis Group 6000 Broken Sound Parkway NW, Suite 300 Boca Raton, Florida, USA
Berendsen RL, CMJ
Pieterse, PAHM Bakker (2012). The rhizosphere microbiome and plant health. Trend Plant Sci 17:478‒486
Billah M, M Khan, A
Bano, T Hassan, A Munir, AR Gurmani (2019). Phosphorous and phosphate
solubilizing bacteria; keys for sustainable agriculture. Geomicrobiol J 36:904‒916
Cappuccino JG, N
Sherman (2002). Microbiology; A Laboratory Manual, 6th edn.
Pearson education Inc., San Francisco, California, USA
Chaves EIDO, VF
Guimarães, ECG Vendruscolo, MFD Santos, FF Oliveira, JAC Abreu, MP Camargo, VS
Schneider, EM Souza, LM Cruz, ES Vasconcelos (2019). Interactions between E.B
and their effects on poaceae growth performance in different inoculation and
fertilization conditions. Aust J Crop Sci
13:69‒79
Chen Q, S Liu (2019).
Identification and characterization phosphate solubilizing bacterium Pantoaea spp. S32 in reclamation soil in Shanxi, China. Front Microbiol 10; Article 2171
Cheneby D, S Perrez,
C Devroe, S Hallet, Y Couton, F Bizouard, G Iuretig, JC Germon, L Philippot
(2004). Denitrifying bacteria in bulk and maize rhizospheric soil: diversity
and N2O-reducing abilities. Can
J Microbiol 50:469‒474
Cheng J, W Zhuang, NN Li, CL Tang, HJ Ying (2017). Efficient
biosynthesis of d-ribose using a novel co-feeding strategy in Bacillus subtilis without acid
formation. Lett Appl Microbiol 64:73‒78
Chernin LS, MK
Winson, JM Thompson, S Haran, BW Bycroft, I Chet, P Williams, GSAB Stewart
(1998). Chitinolytic activity in Chromobacterium
violaceum: substrate analysis and regulation by Quorum sensing. J Bacteriol 180:4435‒4441
Danish S, M
Zafar-ul-Hye, F Mohsin, M Hussain (2020). ACC-deaminase producing plant growth
promoting rhizobacteria and biochar mitigate adverse effects of drought stress
on maize growth. PLoS One 15; Article
e0230615
Defez R, A Andreozzi,
S Romano, G Pocsfalvi, I Fiume, R Esposito, C Angelini, C Bianco (2019).
Bacterial IAA delivery into Medicago root
nodules triggers a balanced stimulation of C and N metabolism leading to a
biomass increase. Microorganisms 7;
Article 403
Dodor DE, AM
Tabatabai (2003). Effect of cropping systems on phosphatases in soils. J Plant Nutr Soil Sci 166:7‒13
Dworkin M, J Foster
(1958). Experiments with some microorganisms which utilize ethane and hydrogen.
J Bacteriol 5:592‒601
Ehmann
A (1977). The Van Urk-Salkowski reagent-a sensitive and specific chromogenic reagent for silica gel thin-layer
chromatographic detection and identification of indole derivatives. J Chromatogr 132:267‒276
Eisenhaur N, A Lanoue, T Strecker, S Scheu, K Steinauer,
MP Thakur, L Mommer (2017). Root biomass and exudates link plant diversity with
soil bacterial and fungal biomass. Sci
Rep 7; Article 44641
Etesami H, DK
Maheshwari (2018). Use of plant growth promoting rhizobacteria (PGPRs) with multiple
plant growth promoting traits in stress agriculture: action mechanisms and future
prospects. Ecotoxicol Environ Saf 156:225‒246
Fasim F, N Ahmed, R
Parsons, GM Gadd (2002). Solubilization of zinc salts by a bacterium isolated
from the air environment of a tannery. FEMS
Microbiol Lett 213:1‒6
Garcia-Frail P, E
Menendez, R Rivas (2015). Role of bacterial biofertilizers in agriculture and
forestry. AIMS Bioeng 2:183‒205
Glick BR (2014).
Bacteria with ACC deaminase can promote plant growth and help to feed the
world. Microbiol Res 169:30‒39
Goswami D, JN
Thakker, PC Dhandhukia (2016). Portraying mechanics of plant growth promoting
rhizobacteria (PGPR); a review. Cogent
Food Agric 2:1‒19
Hallmann J, G Berg, B
Schulz (2006). Isolation procedures for endophytic microorganisms. In: Microbial Root Endophytes, pp:
2099-2320. Schulz BJE, CJC Boyle, TN Sieber (Eds.). Springer-Verlag Berlin,
Heidelberg, Germany
Hussain A, ZA Zahir,
HN Asghar, M Imran, M Ahmad, S Hussain (2020). Integrating the potential of Bacillus spp. Az6 and organic waste for
zinc oxide bio-activation to improve growth, yield and zinc content of maize
grains. Pak J Agric Sci
57:123‒130
Hussain A, M Arshad, ZA Zahir, M Asghar (2015).
Prospects of zinc solubilizing bacteria for enhancing growth of maize. Pak J Agric Sci 52:915‒922
Hussain M, Z Asgher, M
Tahir, M Ijaz, M Shahid, H Ali, A Sattar (2016). Bacteria in combination with fertilizers improve growth, productivity and net returns of wheat (Triticum aestivum L.). Pak J Agric Sci 53:633‒645
Khan KS, RG
Joergensen (2009). Changes in microbial biomass and P fractions in biogenic
house hold waste compost amended with inorganic P fertilizers. Bioresour Technol 100:303‒309
Khan MS, A Rizvi, S
Saif, A Zaidi (2017). Phosphate solubilizing microorganisms in sustainable production
of wheat; current prospective. In: Probiotic
in Agroecosystem, pp: 51‒81. Kumar V, M Kumar, S Sharam, R Prasad (Eds.).
Springer nature, Singapore
Kumar S, G Stecher, K
Tamura (2016). MEGA7: Molecular evolutionary genetics analysis version 7.0 for
bigger datasets. Mol Biol Evol
33:1870‒1874
Kumar V, N Sharma
(2017). Plant growth promoting rhizobacteria as growth promoter for wheat; a
review. Agric Res Technol 12:110-116
Lambers H, WCP
Plaxton (2015). Phosphorous: Back to the roots. Annu Plant Rev 48:3‒22
Lucava
PT, JL Azevedo (2013). Endophytic Bacteria: A Biotechnological Potential in
Agrobiology System. In: Bacteria
in Agrobiology: Crop Productivity, pp: 1‒44. Maheshwari
D, M Saraf, A Aeron (Eds.). Springer, Berlin, Heidelberg, Germany
Luo Q, L Sun, X Hu, R
Zhou (2014). The variation of root exudates from the hyper accumulator Sedum alfredii under cadmium stress:
metabonomics analysis. PLoS One 9;
Article e115581
Meela PM, MH
Serepa-Dlamini (2019). Current understanding of bacterial endophytes, their
diversity, colonization and their role in promoting plant growth. Appl Microbiol 5; Article 1000157
Meena VS, BR Maurya, SK Meena, RK Meena, A Kumar, JP
Verma, NP Singh (2017). Can Bacillus
species enhance nutrient availability in agricultural soil. In: Bacilli and Agrobiotechnology, pp:367‒397.
Islam M, M Rahman, P Pandey, C Jha, A Aeron (Eds.). Springer, Cham, Switzerland
Minhas
WA, M Hussain, N Mehboob, A Nawaz, S Ul-Allah, MS Rizwan, Z Hassan (2020).
Synergetic use of biochar and synthetic nitrogen and phosphorus fertilizers to
improves maize productivity and nutrient retention in loamy soil. J Plant Nutr 43:1356‒1368
Mumtaz
MZ, KM Barry, AL Baker, DS Nichols, M Ahmad, ZA Zahir, ML Britz (2019).
Production of lactic and acetic acids by Bacillus spp. ZM20 and Bacillus cereus following exposure
to zinc oxide: A possible mechanism for Zn solubilization. Rhizosphere 12; Article 100170
Mumtaz MZ, M Ahmad, M
Jamil, T Hussain (2017). Zinc solubilizing Bacillus spp. potential
candidates for biofortification in maize. Microbiol
Res 202:51‒60
Munir I, A Bano, M
Faisal (2019). Impact of phosphate solubilizing bacteria on wheat (Triticum aestivum) in the presence of
pesticides. Braz J Biol 79:29‒37
Nadeem SM, M Ahmad,
ZA Zahir, A Javaid, M Ashraf (2014). The role of mycorrhizae and plant growth
promoting rhizobacteria (PGPR) in improving crop productivity under stressful
environments. Biotechnol Adv 32:429‒448
Naseer
I, M Ahmad, SM Nadeem, I Ahmad, Najm-ul-Seher, ZA Zahir (2019). Rhizobial
inoculants for sustainable agriculture: prospects and applications. In: Biofertilizers for Sustainable Agriculture and
Environment; Soil Biology, Vol. 55, pp:245‒283. Giri B, R
Prasad, QS Wu, A Verma (Eds.). Springer, Cham, Switzerland
Nazli
F, Najm-ul-Seher, MY Khan, M Jamil, S Nadeem, M Ahmad (2020). Soil microbes and plant health. In: Plant Disease Management Strategies
for Sustainable Agriculture through Traditional and Modern Approaches, Vol.
13, pp:111‒135. Ul-Haq I, S Ijaz (Eds.). Springer, Cham, Swirzerland
Nesme T, GS Metson,
EM Bennett (2018). Global P flows through agricultural trade. Glob Environ Change 50:133‒141
Patel PV, PB Desai
(2015). Isolation of rhizobacteria from paddy field and their traits for plant
growth promotion. Res J Rec Sci 4:34‒41
Patten CL, BR Glick
(2002). Role of Pseudomonas putida indole acetic acid in development of
the host plant root system. Appl Environ Microbiol 8:3795‒3801
Perez E, M Sulbarán,
MM Ball, LA Yarzabál (2007). Isolation and characterization of mineral
phosphate-solubilizing bacteria naturally colonizing a limonitic crust in the
southeastern Venezuelan region. Soil Biol
Biochem 39:2905‒2914
Pikovskaya RI (1948). Mobilization of phosphorous in
soil in connection with vital activity of some microbial species. Mikrobiologiya 17:363‒370
Ramesh A, SK Sharma,
MP Sharma, N Yadav, OP Joshi (2014). Inoculation of zinc solubilizing Bacillus
aryabhattai strains for improved growth, mobilization and biofortification
of zinc in soybean and wheat cultivated in vertisols of central India. Appl
Soil Ecol 73:87‒96
Reasoner DJ, EE Geldreich (1985). A new
medium for the enumeration and subculture of bacteria from potable water. Annu
Meet Amer Soc Microbiol 49:1‒7
Rfaki
A, L Nassiri, J Ibijbijen (2014). Genetic diversity and phosphate solubilizing
ability of Triticum aestivum rhizobacteria
isolated from Meknes region, Morocco. Afr
J Microbiol Res 8:1931‒1938
Richardson AE, RJ
Simpson (2011). Soil microorganisms mediating phosphorus availability update on
microbial phosphorus. Plant Physiol
156:989‒996
Roohi A, I Ahmed, M
Iqbal, M Jamil (2012). Preliminary isolation and characterization of
halotolerant and halophilic bacteria from salt mines of Karak, Pakistan. Pak J Bot 44:365‒370
Ryan J, G Estefan, A Rashid (2001). Soil and Plant Analysis
Laboratory Manual, 2nd edition, p: 172. International
Center for Agriculture in Dry Areas (ICARDA), Aleppo, Syria
Saeid A, E Prochownik, J Dobrowolska-Iwanek (2018).
Phosphorous solubilization by Bacillus
species. Molecule 23:2897-2914
Schwyn B, JB Neilands
(1987). Universal chemical assay for the detection and determination of
siderophores. Anal Biochem 160:47‒56
Sembiring M, D
Elfiati, ES Sutarta, T Sabrina (2017). Phosphate solubilizing agents in
increasing potatoes production on andisol sinabung area. Asian J Plant Sci 16:141‒148
Sierra GA (1957). A
simple method for the detection of lipolytic activity of microorganisms and
some observations on the influence of the contact between cells and fatty
substrates. Anton Leeuw 28:15‒22
Singh M, A Kumar, R
Sing, KD Pandey (2017). Endophytic bacteria; a new source of bioactive
compounds. Biotechnology 7:315-328
Steel RGD, JH Torrie, DA Dicky (1997). Principles
and Procedures of Statistics- A Biometrical Approach, 3rd edn. McGraw-Hill
Book International Co., Singapore
Stephen J, MS Jisha
(2008). Buffering reduces phosphate solubilizing ability of selected strains of
bacteria. Amer-Euras J Agric Environ Sci 4:110‒112
Suleman M, S Yasmin,
M Rasul, M Yahya, BM Atta, MS Mirza (2018). Phosphate solubilizing bacteria
with glucose dehydrogenase gene for phosphorous uptake and beneficial effect on
wheat. PLoS One 13; Article e0204408
Wei Y, Z Wei, Y Zhao,
M Shi, Z Cao (2018). Effect of organic acids production and bacterial community
on the possible mechanism of phosphorus solubilization during composting with
enriched phosphate solubilizing bacteria inoculation. Bioresour Technol 247:190‒199
Wilson KA, MF Mcbride, M Bode (2006). Possingham, H.P.
Prioritizing global conservation efforts. Nature
440:337‒340