Organic amendment plus inoculum drivers: Who drives more P nutrition for wheat plant fitness in small duration soil experiment.

Functioning of ecosystems depends on the nutrient dynamics across trophic levels, largely mediated by microbial interactions in the soil food web. The present study investigated the use of phosphate solubilizing bacteria (PSB) and poultry manure (PM) for maintaining labile P in the soil for an extensive fertility enhancement and as a substitution of chemical fertilizers. Based on the different P solubilizing capabilities of Bacillus and Pseudomonas, a quadruple consortium of Bacillus subtilis, Bacillus cereus, Bacillus thuringiensis and Pseudomonas fluorescens, and their grazer nematodes (soil free living) supplemented with PM were studied. This study was carried out on the trophic levels of soil communities to assess the growth and availability of P to the wheat plants. Experiment was performed for 90 days. Comparing the unamended and amended predator results showed that nematode addition beyond bacterial treatment substantially increased the net available P by ≈2 times, and alkaline phosphatase (ALP) activity by 3.3 times. These results demonstrated the nematodes association with increasing nutrient availability or P mineralization. The interactive effect of PM as substrate and biological drivers was more noticeable on plant dry biomass (1.6 times) and plant P concentration (3.5times) compared to the similar unamended treatment. It is concluded that the biological drivers significantly enhanced the soil ALP and available P while the substrate and biological drivers enhanced dry biomass and plant P concentration. Bacterivore nematodes enhanced the effect of PSB for P mineralization via microbial loop and could be used for the enhancement of wheat production.

Introduction

The function of soil microflora and fauna is very important in ecosystem services such as decomposition and nutrient dynamics across trophic levels which depends on their interactions in soil food web. These important biological drivers: bacteria, fungi and nematodes act as major sink and source of key abiotic components such as carbon, phosphorus and pH. Therefore, the deficiency of either component ultimately reduces output of soil ecosystem such as plant yield [1]. It has been reported that a limited availability of the phosphorus to plants significantly affected microbial diversity, plant growth and ecosystem prolificacy [2].

Phosphorus is one of the key abiotic regulators of ecosystem functions being indispensable for development and growth of plant. Agriculture soils are strongly depleted in available P for plants, although a sufficient 50–65% amount of total P is present but in complex organic and inorganic forms [3]. Also, substantial P turnover occurs in the surface layers of soil and microbial number reduce along soil depth. Various P management strategies were executed to maintain adequate labile P in the agricultural fields [4]. For that many ecosystems globally are experiencing mineral and organic phosphate fertilizer inputs begetting accumulation of stable P minerals and organic compounds in soil through biological assimilation and chemical processes of precipitation and sorption involved in P fixation. Consequently, depending on the soil nature much (80–90%) of this remains unavailable for plant uptake leaving approximately 10–20%/(2–10 μM) for plant use in the form of mono and diorthophosphate in soil solution [4]. Soil organic phosphorus is 30–70% of the total P, largely in the form of stable inositol phosphate (soil phytate), easily hydrolysable microbial biomass P account for (2–5) % [4]. The availability of these pools for plant use relies on the soil fertility [4,5].

Livestock manures, plant residues and compost are some organic amendments and have been proposed as alternatives or supplement to mineral fertilizers. These organic amendments effect microbial biomass and their diversity with consequent long term potential benefits of improved soil nutrient turnover and other essential ecological functions such as extracellular enzyme activities e.g., phospahatase enzyme production. However, these benefits of nutrient dynamics such as P availability for plant growth remains variable depending on the different farming systems [5-7]. Therefore, Inoculation of PSBs with or without organic (such as poultry manure) [6] or inorganic fertilizers [7] has been the research concern on P dynamics often with differing responses on crop yield and P availability [7,8]. Poultry manure is a concentrated potential source of P and contains more stable mineral-associated P compared to other manures [8-10]. Soil organisms respond in different ways to phosphorus availability due to different requirements of nutrients and economic strategies [5-11]. For instance, rhizosphere bacteria incorporate large amount of P in their biomass corresponding to high available C inputs in the form of manure. Thus reduced P availability is subject to competition for plant and microbial uptake depending on the residence time of P in their microbial biomass. Microbial assimilation of soil solution orthophosphate and its timely and expedient release according to the need of the plant or future generation of microorganisms lessens the soil P fixation by chemical sorption [11]. Depending on the soil and fertilizer input the release of P from microbial biomass can be as lowest as10 days to 170 days to one year maximum reported [3], which generally constitute a prospective source of available P for plants, especially in P deficient systems. Among various phosphorus management practices, strongly positive effects of PSBs (phosphate solubilizing bacteria) have widely been recognized as key drivers for a better plant growth and soil P availability. Phosphate solubilizing bacteria (PSBs) improve availability of P for plants [10,11]. Moreover, a synergistic and complementary microbial consortium based on different functional groups of the comprising strains may effectively enhance plant growth and effect of organic fertilizers such as poultry manure [12]. The mechanism to solubilize inorganic complexes is the production of organic acids and enzymes to hydrolyze the organic compounds (PMBs) phosphorus mineralizing bacteria [11,12]. The higher effect of Bacillus and Pseudomonas inoculation amongst other PSBs have been reported on wheat (Triticum aestivum), chickpea and other crops development and P availability [13-15]. The enhanced effect of labile P status in soil is by the production of hydrolyzing enzymes and organic acids following the process of mineralization and solubilization of organic compounds and mineral complexes of P, respectively [16].

Soil bacterivores nematodes are the important indicators of soil ecosystem functioning comprising 60–80% of total nematodes [17]. The presence of higher trophic level interaction such as bacterivores nematodes in the rhizosphere soil can directly affect the net P mineralization through excretion products or indirectly by feeding upon rhizosphere bacteria [17,18]. Therefore, bacterivores nematodes beneficially affect plant by releasing nutrients such as P from bacteria after feeding upon them [19]. This mechanism of interplay between microflora and fauna is known as, the ‘microbial loop ‘triggered by high rhizosphere microbial activity and biomass through carbon pulses provided as root exudates, transmit energy to following microfaunal grazers [17-19], where approximately 30-times increase in the numbers of free-living nematodes such as bacterial feeders may occur. During the growth of nematodes large amounts of available nutrients such as P, C and N are incorporated in their biomass on the other hand biomass production through grazing utilize 50–70% of the prey carbon [20].

The P mineralization potential by co-inoculation of single bacteria (Pseudomonas, Bacillus) +bacterivores nematodes was assessed on phytate source, detritus and organic matters [17-20]. But co-inoculation of bacterial consortia and nematodes have not been investigated in the presence of organic amendment in the wheat (Triticum aestivum) rhizosphere. These works demonstrated the co-inoculation of nematodes with single PSB strain or PSB single and consortium inoculation on P dynamics. The activity of microbial exoenzymes responsible for organic P mineralization can be strengthened through microbial grazers such as nematodes [18,19]. In a research study higher effect of bacteria and their grazer nematodes on rate of organic matter utilization on the basis of organic P mineralization was observed compared to bacteria alone effect [20].

There are many integrated soil fertility management strategies available, but more efforts are always needed to improve the various functions of PSBs and interaction with microfauna besides using organic wastes as nutrient resource. Therefore, the objective of the present study was to explore the complementary effects of PMBs and their grazer nematodes within multi-trophic interactions in the presence of poultry manure on soil available P, and wheat biomass.

Material and method

Soil sampling and study area

Soil for microbial isolation was collected from forest of Nathia Gali Abbottabad, lower Himalayan region in Khyber Pakhtunkhwa (KPK) Pakistan, Asia 34° 4’ 20" North, 73° 23’ 55" East, owing to the undisturbed soil ecosystem. For each rhizosphere soil sample, the soil stuck to the pine plant roots were collected to make composite sample, sealed and placed in an iced container right after sampling [21]. This composite soil sample was used to perform bacterial isolation, soil initial pH, water available phosphorus and total phosphorus.

Isolation, screening and identification of PSBs

A synthetic solid medium Pikovskaya’s (PVK) was prepared with the concentration (g)/L; Mg SO4. 7H2O 0.492 g, MOP’s (pH stabilizer) 133.2 mL, Glucose 3.33 g, micro-nutrients 267 μL KNO3 0.100 g, calcium sulphate 0.543 g and calcium phosphate Ca3(PO4)2 3.956 g were added as sole inorganic P source for being the most precipitated phosphorus mineral found in neutral and alkaline soils [17]. The organic P Pikovskaya’s solid medium was added with (sodium phytate) C6H6Na12O24P6 as sole organic P source instead of Ca3(PO4)2 and MOPs for pH stabilization (pH 7–7.5) [18]. Following the dilution plate technique the isolation of PSBs was done. A sufficiently small 20μL aliquot from serially diluted (10−4–10−6) suspension of soil samples was spread on sterilized PVK solid medium and incubated for 48 hours at 30±2°C. Purification was done by repeated single colony streaking on Pikovskaya’s solid medium Then 110 strains through the observation of halo zone around colonies on Pikovskaya’s solid media qualitatively confirmed the inorganic phosphorus solubilization These isolates were further screened on the basis of organic phosphorus mineralization potential on Pikovskaya’s solid medium with organic P sourc. e and 21 strains were confirmed as P (Po & Pi) mobilizers on the basis of halo zone formation [17,18].

Biochemical assay for screening based on available inorganic P, pH and ALP activity indicators

The acquired 21 pure phosphorus mobilizing bacterial cultures were quantitatively evaluated for P (Po & Pi) mobilization on the basis of pH change, bacterial growth, orthophosphate concentration and alkaline phosphatase production “Table 1” [18,22,23]. For this all of strains were grown singly in Pikovskaya broth medium with sodium phytate as P source (pH 7.2) and sterile media without inoculation was set as blank. Bacterial growth was observed visually following incubation at 28°C for 3 days in an incubator shaker (120 rpm) [18,19]. The bacterial cultures were kept 15–20 minutes to settle down insoluble culture media. Briefly, filtered (0.45 μm size) supernatant was taken, centrifuged 10000/min for 10 minutes at room temperature and diluted accordingly to determine available P and pH determination. pH was measured with pH meter while reagent 1 and 2 [24] was added following malachite green method for determination of available phosphorus. Standards were prepared to make calibration curve and inorganic P mineralized by bacteria from organic source were analyzed by microplate reader at 620 nm [24].

Table 1

Listed is the selected efficient identified phosphorus mobilizing bacteria and their consortia with accession numbers, ALP activity and phosphorus mineralizing values.

Name Codes of bacteria	Names of bacterial strains	Accession Numbers	ALP Enzyme activity	Available P
			Moles of pNPP/ml/hr	mg/ml
Bc	Bacillus cereus	MK418810	1.39	0.92
Bs	Bacillus subtilis	MK417798	1.41	0.98
Bt	Bacillus thuringiensis	MK417800	1.40	1.57
Pf	Pseudomonas fluorescens	MK418218	1.42	1.40
Bc+Bs+Bt+Pf	Bacillus cereus+Bacillus subtilis+Bacillus thuringiensis+Pseudomonas fluorescens	----	1.44	1.76

Alkaline phosphatase (ALP) activity was determined by taking 0.25 mL supernatant from fresh bacterial cultures; followed the ALP assay described for soil in the next section [22].

Based on these defining parameters of P mobilization i.e. pH, ALP activity and P mineralization 4 efficient strains out of 21 were selected for further analyses “Table 2” and sustained on solid organic Pikovskaya’s medium.

Table 2

Showing the primers of the selected genes.

List of target specific primers, their amplicon length and nucleotide sequences used for gene presence identification. Enzyme	Amplicon length (bp)	Primer sequence 5’-3’	Primer name	Reference
Acid phosphatase (class A)	159	GGAAGAACGGCTCCTA CCCIWSNGGNCA	phoN-FW
		CACGTCGGACTGCCAG TGIDMIYYRCA	phoN-RW
Phosphonoacetaldehyde hydrolase	147	FWCGTGATCTTCGACtGGGCNGGNAC	phnX-FW	Berkgemper et al., 2016
		GTGGTCCCACTTCCCC ADICCCATNGG	phnX-RW
Quinoprotein glucose dehydrogenase	330	CGGCGTCATCCGGGSITIYRAYRT	gcd-FW
		GGGCATGTCCATGTCC CAIADRTCRTG	gcd-RW

Molecular characterization of selected bacterial strains

The selected bacterial strains were identified using 16sRNA gene sequencing and submitted in gene bank with accession number “Table 2”. The bacterial strains were stored in glycerol stocks at −80°C and revived as and when required.

Presence of phosphorus mineralizing gene was done using q-PCR: In bacteria phoN, phnX gene encoding enzymes are responsible for organic P mineralization and gcd for inorganic P solubilization i.e., Genes encoding phoN included in bacteria responsible for enzymatic hydrolysis of organic P to orthophosphate during phosphate starvation [25]. The presence of responsible genes was analyzed in selected bacterial strains.

DNA extraction

DNA of each selected bacterial strain was extracted by adapted method from Irshad et al [18]. Supernatant from fresh cultures of selected bacterial strains was centrifuged and pellette was suspended in 500μL of 1X PBS repeatedly to remove all the media. after repeated thawing, tubes were centrifuged and the supernatant containing DNA fragments was treated with isopropanol and washed with 70% ethanol and pellet was dried and dissolved in 1x phosphate buffer saline and kept at -80°C [18].

PCR was performed to amplify the genes involved in P mineralization from each DNA extracts of selected PMB (phosphorus mobilizing bacteria); Bc, Bs, Bt and Pf, using primers of selected genes “Table 2”. A ready to use master mix 10μl (containing Taq DNA polymerase, dNT P, MgCl2, PCR buffer and PCR stabilizer), 1.5μL DNA extract of each strain, 1μL (R and F) primers, diluted to 20 μL final volume with molecular grade water H2O. The reaction mixture was placed in thermocycler at condition according to Bergkemper et al. [25] Initial temperature (95°C; 5F min) following denaturation; 30 cycles (95°C; 1 min) then (57.6°C; 1 min) annealing and elongation (72°C; 30s) followed by a final step of elongation (72°C; 4 min). This optimum annealing temperature of the primers was investigated in a preliminary test performing a gradient PCR (annealing temperature: phnX 57.6°C, phoN 59°C, gcd 55°C [25]. Finally, PCR amplified product was evaluated by electrophoresis on 1.5% agarose gel in 1xTAE running buffer with 50bp to 1kb DNA ladder [25].

Isolation of nematodes

Extraction of nematodes was carried out according to the Cobbs’ sieving and decanting method as described by Irshad et al [26]. Following sequential sieving (sieves mesh size ranging from 20–325) and decanting to isolate soil free-living nematodes, the final backwashed material was spread on filter, supported on wire mesh and placed on water filled pertiplates at 22–25°C for one week. Then nematodes move through filter in petriplate. Finally, the recovered extracted nematodes were counted and identified to bacterivores in 20ml of subsamples from pertiplate under microscope at magnification of 100· and 400· respectively. While a small aliquot was poured on sterilized TSA, incubated at 30°C and maintained for further use.

Microbial compatibility experiment

Bacteria-bacteria compatibility

The synergistic activity between the selected efficient isolates was performed following Cross Streak method [18-26]. According to this technique, bacterial strains were cross streaked one following the other, then incubated at 30±2°C 48 h. The cultured strains merged together with no reduced growth were considered compatible, while non-compatibility between them was assessed by growth suppression. A group of 4; Bacillus cereus S Bacillus subtillis, Bacillus thuringiensi and Pseudomonas fluorescens strains showed the higher compatibility for no growth suppression together. This shows their potential to be mixed as consortium to make bio inoculants.

Bacterial nematode interaction was assessed by the adapted method Hayat et al [27]. Briefly TSA sterilized large glass pertri plates were spot inoculated in center with the PSB isolates at 1 cm distance from each other, incubated overnight at 28±2°C. Thereafter, 50 nematodes were emplaced towards the periphery at 2cm distance (approx.) from center. Two controls were also set with only bacteria at center and only nematodes with sterile water inoculation. The observation was made under microscope with nematodes movement path and bacterial colonies disturbance indicative of positive interaction between 4 selected isolates and nematodes.

Preparation of inoculum

Bacterial inoculum

Individual liquid cultures of each of four isolates; Bacillus subtillis, Bacillus cereus, Bacillus thuringiensis and Pseudomonas fluorescens were prepared in TSB media according to the adapted method [16], incubated overnight at 30±2°C. These liquid cultures were repeatedly centrifuged (3000 rpm for 1 minute at room temperature) and suspended in sterile distilled water until thoroughly washed [18]. By taking small aliquots from these suspensions’ cells were counted under microscope using cell counter and diluted accordingly. (Bc; 4.15x106 Bs; 3.9x107 Bt; 3.7x106 Pf; 4.45x105)/ml. Finally adjusted 1m of each of these suspensions (approximately equal no. of cells of each strain) were mixed to form inoculum pH (7.2) 4ml diluted to final volume 250ml.

Inoculation

Bacterial consortia inoculation was done after 12 days of germination (15th) day of experiment with drop and pour method. A pierce was done in soil near the vicinity of roots and inoculated with 5 mL of bacterial inoculum with concentration (5.25x107 cells/mL). Afterwards all the pots including uninoculated ones were watered with 10 mL of distilled water.

Nematode inoculation

Nematodes were inoculated on 30th day of experiment. TSA Plate cultures of nematodes were washed with sterile distilled water and collected in falcon tube. Centrifuged and washed 3 times and resuspended in 5ml distilled water. Diluted and enumerated under microscope and 2ml of inoculant (1200 Ind/ml) were added in pots with drop and pour method after which all pots including uninoculated ones were watered with 10ml of distilled water [17].

Experimental design

Sampling and processing of soil and poultry manure

Microcosms were set up by first sampling, homogenizing, and processing the soil and poultry manure. The soil for the pot experiment was recovered from depth of 0 to 25 cm from a small crop field of COMSATS university Islamabad, Abbottabad campus (N 34° 13’22” and E 73° 16’ 0”). Field was previously sown with maize and wheat on rotational basis. The soil contains loamy clay texture with deficiency in available plant P possessing slight alkaline properties.

The soil and fresh poultry manure samples were dried and sieved at 2 mm/10 mesh size [9]. Soil and processed poultry manure after initial analyses of total P, water available P and pH in triplicates “Table 3” were homogenized. According to the literature different rates of same amendments are applied from 1% to 10% on different basis. So the lower rate was considered for being economical [8-28]. Poultry manure (PM) as amendments was mixed with soil at 5% by weight i.e. 50g/kg.

Table 3

Soil and poultry manure initial pH and available P analysis.

Sample	pH	Water available P (mg/g)	Total P (mg/g)	Unavailable P (mg/g)
Soil	7.79	0.314±0.2	98.81±5.61	98.496
Poultry manure	8.1	17.8±13.3	166.68±2.25	148.88
Soil+Poultry manure	7.86	60.41±1.2	269.29±4.21	208.88

Overall, 24 experimental units/pots were arranged with 2 biological treatments, 2 biological controls (four treatments), with and without poultry manure (PM) addition and triplicated each. To exploit trophic level, it was either added with bacteria/nematodes with and without wheat plant or added bacteria alone and with nematodes in wheat rhizosphere. We amended half of the microcosms with poultry manure while other half of the microcosms were not amended with poultry manure. Each small plastic pot was labelled and filled with 200 grams of soil/soil+PM mixture according to the treatments. Together this experimental setup created a high and low phosphorus resource environment in soil and allowed us to test trophic level with or without amendment on enzyme activity, and plant growth.

Afterwards, microcosms were placed in growth chamber in completely randomized design and maintained humidity (65%) and temperature (15–18°C) for optimal microbial activity [29] through watering 1-2ml distilled water with 16 hours day light and 8 hours night period, respectively.

Pre and post analyses

Phosphorus analysis

For total phosphorus analysis in samples double acid digestion method was followed. For this samples were added with (HNO3), kept overnight, heated at (80–90°C) for approximately 1hr, until dense fumes subside. After solution cooled HClO4 was added and heated 180–200°C until clear acid remains [24-31]. Free orthophosphate concentration in samples after digestion was determined following malachite green method and read under microplate reader at 620 nm [24-30]. Phosphorus in the bacterial culture after mineralization was also measured following malachite green method [24-26].

Alkaline phosphatase activity

The soil phosphatases enzyme in soil was determined by hydrolysis of an organic substrate p-nitrophenyl phosphate (pNPP) to p-nitrophenol. Sodium hydroxide is used to stop the assay and also to develop the yellow coloration (p-nitrophenol) for resultant analysis.

At the time of harvesting plant roots, samples were gently shaken, removed bulk soil and 0.3 g of soil was taken from each replicate of the treatment and diluted with 3 mL of sterile distilled water and incubated for one hour. Solution was vortexed, centrifuged at 14000 rpm, then 0.25 mL of this supernatant was taken and incubated with sodium acetate buffer containing 10 mM pNPP, re-incubated for 30 minutes at 30°C then 4 mL of 125 mM NaOH was added to allow for color not derived from p-nitrophenol released by phosphatase activity. Controls were set with each isolate sample by incubating 0.25 mL of water with 1 mL of pNPP and buffer and immediately adding 4ml of 125 mM NaOH to stop the reaction. Microplate was made with each sample replicates and production of pNPP from the color derived was determined at 405 nm [18-22].

Root and shoot dry weight

Roots and shoots were separated of each plant sample, dried in oven at 65°C for 48 hours, immediately weighed to reduce the possibility of moisture with precision balance with 0.001g accuracy [17].

Soil and poultry manure pH and available P

To measure the pH and available P in soil and manure, suspension with 1gram solid and 10 ml deionized water was made Cui et al [23] Waldrip-Dail [10] pH was determined with pH meter. For available P (orthophosphate), while reagent 1 and 2 [24] was added in respective supernatant following malachite green method for determination of available phosphorus [17-31].

Statistical analysis

Results were expressed as mean±standard deviation of different independent replicates. Data were analyzed using One-way and two-way ANOVA. Different letters were used to display significant differences. The differences between means were analyzed by factorial ANOVA followed by Tukey’s HSD post-hoc test using Statistica 7.1 (StatSoft Inc., Tulsa, OK, USA). Normality was tested using the Kolmogorov Smirnov test to meet the assumptions of ANOVA.

Results

Net available phosphorus

The net water available P was significantly affected by predator treatment regardless of the amendment. The highest available P after the addition of both bacteria and nematodes in unamended treatment (40.9 μg/g was approximately twice higher than similar amended treatment 29.9 μg/g. As a whole T1 (amended) treatment as shown in Fig 1 declined P availability 0.58 times as compared to unamended control. The unamended and amended bacteria only exhibited net P availability of 20.64 and 20.44 μg/g respectively. Which upon predation increased 2 times and 1.5 times net P respectively. The interactive effect of both amendment and predation on net available P was not significant.

Fig 1

Soil net P availability after 90 days of experiment.

C: Control, C+P: Control+wheat plant, C+P+B: Control+wheat plant+ bacterial consortia, C+P+B+N: Control+wheat plant+ bacterial consortia +nematodes: Bars are triplicate means of each treatment and standard deviation is shown by error bars. Significant difference is shown by letters (Tukey’s HSD test, P ≤ 0.05).

Alkaline phosphatase activity: At harvest, ALP activity

The ALP activity of unamended control soil (without PM, pH 5.8) was around 2.77 μmoles of pNPP/ml/hr while the addition of PM marginally reduced the ALP to 2.23 μmoles of pNPP/ml/hr at pH 6.7. However, the unamended and amended predator treatment significantly increased ALP 6.47 μmoles of pNPP/ml/hr and 1.96 μmoles of pNPP/ml/hr respectively, compared to their respective controls. Amended bacteria only treatment (+bac) decreased ALP 1.65 μmoles of pNPP/ml/hr (0.8times) at pH 7.4 compared to its control and similar unamended treatment ALP increased (1.1 times) 2.91 μmoles of pNPP/ml/hr at pH 7.2 as described in Fig 2. According to ANOVA test, soil ALP tended to increase in predator treatment compared to its control but declined from similar unamended treatment. The ALP value was not significantly affected by the interaction of factors but significantly affected by predation (+bac+nmt) in the absence of PM.

Fig 2

Soil alkaline phosphatase activity after 90 days of experiment.

C: Control, C+P: Control+wheat plant, C+P+B: control+wheat plant+ bacterial consortia, C+P+B+N:Control+wheat plant+ bacterial consortia +nematodes: Bars are triplicate means of each treatment and standard deviation is shown by error bars. Significant difference is shown by letters (Tukey’s HSD test, P ≤ 0.05).

Effects of amendment and treatments on soil net available P Vs pH and Vs ALP

At pH 7.2 and 7.6 in amended or unamended soil, the net available P content changed from 22 to 41 μg/g, with highest amount found in unamended predation treatment at pH 7.56 and second highest in similar amended treatment (29.9 μg/g) at slight increased pH 7.6. Irrespective of the addition of PM, predation treatment increased P at similar pH 7.6 but significantly higher 41 μg/g of net P was found in unamended treatment (T0). According to Fig 3A and 3B the unamended and PM amended bacteria only treatment at pH 7.25 and 7.39 showed 20.9 and 20.4 μg/g, respectively.

Fig 3

Soil net P availability versus a) soil pH change b) soil ALP activity with respect to treatments. C0: Unamended uninoculated control, Cp Poultry manure amended uninoculated control, C+P: Control+wheat plant, C+P+B: Control+wheat plant+ bacterial consortia, C+P+B+N: Control+wheat plant+ bacterial consortia +nematodes: Bars and lines are triplicate means of each treatment.

Concerning the availability of phosphorus with respect to the enzyme, a lowest soil net P availability of 3.43μg/g (2.23 μmoles of pNPP/ml/hr) under PM amended control treatment was noticed. A significantly high ALP activity 6.47 μMoles/ml/hr with consequential net available P (40.9 μg/g) was observed in unamended (+bac+nmt) treatment. PM amended plant only treatment and predation treatment showed significantly similar ALP value of 1.97 and 1.96 μmoles of pNPP/ml/hr with 20.4 and 29.9 μg/g net P, respectively. While both unamended and amended bacteria only treatment (+bac consortia) retained the available P concentration to 21μg/g which increased upon nematodes inoculation. Overall, predator treatment, whether amended or unamended, increased ALP and net P compared to their respective controls, but unamended predator treatment compared to its similar amended treatment showed the significantly highest effect of predator treatment.

Plant phosphorus concentration

Overall, Fig 4 showed a significantly higher P concentration was observed in wheat plant under +bac treatment 13490 μg/g DW/pot and + predator treatment 15404 μg/g DW/pot in the presence of PM. On the other hand, in the absence of manure amendment both inoculation treatments (+bac) and (+bac+nmt) showed the weakest influence on plant P concentration 4570.05 and 4369.01 μg/g DW/pot, respectively. The results of two-way ANOVA showed a significant interactive response (PM +bac+nmt) for plant P concentration. This result indicated the high dependence of nematodes mutualistic activity in the abiotic soil component i.e., PM substrate.

Fig 4

Plant P concentration.

C: Control, C+P: Soil+wheat plant, C+P+B: Control+wheat plant+ bacterial consortia, C+P+B+N: Control+wheat plant+ bacterial consortia +nematodes: Bars are triplicate means of each treatment and standard deviation is shown by error bars. Significant difference is shown by letters (Tukey’s HSD test, P ≤ 0.05).

Plant dry biomass

After 90 days of experiment, a significantly higher plant dry biomass was observed in PM amended treatment compared to the unamended treatment Fig 5. But no significant difference was observed on plant dry biomass between the treatments. The minimum of 358mg plant dry weight (DW) was observed in unamended uninoculated control while with the bacteria only (+bac) and predator treatment a non-significant increase of 434 to 441mg DW was observed. A significant increase was found with 620 mg DW of plant, in PM amended control than unamended control. Bacteria only (+bac+plant) treatment in the presence of PM exhibited the significantly higher plant DW (702 mg) compared to the similar unamended treatment. Plant DW was significantly affected by the interaction of treatments besides individual effect of both treatments.

Fig 5

Plant total dry weight.

C: Control, C+P: control+wheat plant, C+P+B: control+wheat plant+ bacterial consortia, C+P+B+N: Control+wheat plant+ bacterial consortia +nematodes: Bars are triplicate means of each treatment and standard deviation is shown by error bars. Significant difference is shown by letters (Tukey’s HSD test, P ≤ 0.05).

Presence of P mobilizing genes

Presence of genes responsible for orthophosphate production were performed in selected bacterial strains. phnX (147) phosphonoacetaldehyde hydrolase, phoN (159) acid phosphatase and Quinoprotein glucose dehydrogenase. PhnX gene was present in Bacillus cereus, Bacillus thuringiensis and Bacillus subtillis but in Pseudomonas it was under expressed. While phoN gene was expressed and gcd was successfully expressed in the Bacillus subtillis.

Discussion

Microbial P dynamics changes in soil treatments

In this study the consequences of synergistic effect and trophic interactions among bacteria and their grazers on soil available P, plant P uptake and biomass were explored. The influence of interactions between poultry manure amendment representing input of P and trophic level was investigated, though, the trophic effects were noted both with and without PM addition but overall, results suggested that in this microcosm set-up trophic level (regardless of substrate limitation) can influence soil processes of P cycling.

During a three-month incubation, a significantly higher ALP and net soil P was noted compared to the similar PM amended treatment. No significant interaction between trophic level and PM addition was observed in this case Figs 1 and 2. Moreover, significantly similar net P concentration in the unamended sole plant and sole bacterial treatment and similar PM amended treatments was observed. The water available P concentration in sole plant treatment compared to their respective controls was increased by 3.9–6.2 times in both unamended and PM amended treatment, respectively. However, the unamended predator treatment increased 1.96–1.5 times more water available P.

Amended predator treatment improved plant dry weight and plant P concentration relative to unamended predator treatment while unamended treatment initially showed higher net available P and ALP due to nutrient starved condition. Alori et al [5] reported that water available P (Pi in soil solution) increases only when the pool of microbial biomass decreases or when the microbial biomass becomes poorer in P, Under field conditions, the size of the microbial P pool can increase or decrease depending on the soil conditions e.g., soil humidity [5-33].It has also been suggested that the contribution of nematodes and other soil fauna become significant usually under the conditions of low nutrient availability [33,34].

This suggests that the presence of predator beyond bacterial treatment may exert a stronger influence on the microbial efficiency regardless of the addition of a labile substrate i.e., PM. Bünemann et al. [35] showed that P mineralization was independent of organic matter turnover. This implies an increased plant and microbial competition for nutrient which might be the result of P starvation which may have enhanced net P mineralization by 2 to 3 times. These results of significantly higher net P and ALP in similar unamended predator treatment is an indication of the higher net organic P mineralization supplemented with release of organic P from the bacterial biomass by nematode predation i.e., microfaunal stimulation of P mineralization via the microbial loop. Also, similar net P value in all sole plant and sole bacterial treatment may be related to the increase in P cycling internally as a P starvation phenomenon. Soil extracts when treated with phosphatase enzyme released significant amounts of orthophosphate which is in support to our net water available P and ALP results, indicating organic P mineralization [35]. It was investigated that reduction in the soil organic P is related to the high phosphatase activity [36-38]. Significantly positive effect in predator treatment without PM addition may be the response of nematodes predation as indicated by Trap et al [32]. As Bonkowski [36] showed that the effect of bacterivores nematodes on soil P availability for bio assimilation was triggered by the soil microbial and plant biomass. A significantly similar net P concentration in sole bacteria treatment with and without PM addition may be attributed to the initial low P availability in both treatments so bacteria retained the P in their biomass. The higher P availability in P fertilized soil was found by Sphon and widding [33] who reported that microorganisms retained P in their biomass in soil with low P availability, whereas in P fertilized soil P turnover time from microbial biomass was shorter and water extractable P concentration in soil increased. Bacteria mineralized P from soil organic matter and calculations by Irshad & Griffith [17-34] gave evidence that bacterivores grazing results in the release of this additional P. Using labelled bacteria Irshad et al [26] reported that grazing by nematodes and protozoa increased the availability of P to plants, originating from bacterial cells but also from soil organic matter. In a research study net nutrient (P and N) availability in microcosms study with bacteria alone and nematodes showed that, inorganic P was initially immobilized in bacterial biomass, with over half of the initial P returned after 65 d in both bacteria and predation treatment. The Sphon and widding [33] in their study demonstrated that the turnover time of microbial biomass nutrients varied from 10 to 160 days, depending upon the soil carbon and P inputs [38]. In a similar study with addition of C input in the form of grass clover residue inoculation of nematodes significantly enhanced the net P availability up to 23% and Lolium perenne plant biomass up to 9% compared to control without nematodes [32].

Net P (soil solution orthophosphate) declined by PM addition in bacteria and predation treatment in part, could be attributed to the underlying factor of increasing microbial biomass due to increased C availability which may have caused high incorporation of available P in their biomass [39]. In contrast, the work of Heuck et al [33-40] showed that more C was utilized by soil MOs from organic phosphate inputs in P deficient soil, this implies that organic P mineralization occurs as result of C requirement by microbes [33-41]. This mechanism might increase P availability to plants in soils where P is limiting for plants, but not for microorganisms. Overall unamended predator treatment enhance soil measured parameters i.e. Soil net P and ALP, while the amended predator treatment significantly influenced the plant P and DW, which apparently shows the the effect of amendment increased with time. Waldrip-Dail et al [10] demonstrated a short-term initial increase in water extractable P by PM amendment, then decreased after 100 days controlled by immobilization-solubilization.

Significantly high ALP activity value in PM amended predation treatment Fig 6A (d) in current study may be associated with P starvation response as a result of P deficiency as study on organic P mineralization showed the reduction in organic P of soil when phosphatase activity was highest [37]. We observed 0.4 times decrease in the ALP activity in the PM amended predator treatment compared to the similar unamended treatment. While ALP activity in predator treatment compared to their respective controls decreased by 0.86 times and 1.96 times increase was observed with and without PM addition, respectively.

Fig 6

Schematic diagram; presentation of comparison of individual and interactive influences of soil biological drivers and PM amendment.

On available P, plant P acquisition and ALP as a result of applied treatments as a, b c and d. soil biological interactions; a plant-bacteria-nematode priming established a beneficial nutrient loop. Arrows sign (<) indicate a significant increase, Sign of equality (=) shows non-significant difference ALP enzyme activity, available P and plant P are shown as ALP and P0i. Ppi, respectively, Plant P, plants showing max height and plant number than other plants = high plant dry biomass, organisms in circles show inoculation of bacterial consortia/ nematodes and bacterial consortia.

Alternately, saturation of soil nutrient status after amendments might have potentially inhibited or delayed the enzyme activity of soils in predation treatment with PM addition [9-28]. Knowing the same effect of predation on soil ALP and net P in the treatment without PM addition can reveal that how this enzyme is introduced because of P deficiency forecasting the more available phosphorus required for the plant uptake. Fertilization study further support this possibility, a positive correlation was found between inorganic P pool size and phosphatase. Alternately, P fertilization significantly decreased phosphatase activity. A study showed increase in the phosphatase activity in rhizosphere is associated with a depletion of soil organic P which an indicative of P mineralization [2-40]. While another study demonstrated a negative correlation of phosphatase activities with available P concentration in soil [16]. Heuck et al [40] reported that high P concentration do not warrant the high phosphatase activities, but this enzyme activity can be driven by the C requirement by the microbes. An increase in ALP activity was linked with organic P mineralization in the treatment where higher trophic levels are present (i.e., bacteria and their grazers) and organic manure addition is absent [26]. This result differs from Wan et al [2] who found high phosphatase activities in the P rich organic material amended soil and also depend on the soil pH and MB, besides, inverse relation has been found between enzyme activity and P availability after fertilization in various ecosystems. In support of our results, it was reported that fertilization decreased ALP activity rather ACP [16-33]. An overall effect of grazing on thenutrient (N and P) turnover estimated by the meta-analysis was 30% of control [32]. Findings from another study suggest that when the supply P is sufficient phoD-harboring microorganisms immobilize P in biomass while mineralize organic P under P-poor condition by stimulating ALP activity, during which the rare taxa play an important role [39].

The overall impact of PM addition on the net available P and ALP was small even in the presence of biological drivers, while impact of biological drivers (predator treatment) was significant in this case and showed in unamended predator treatment Fig 6A unamended control.

Linking crop yields and P acquisition with drivers of soil ecosystem functioning

Wheat plant dry biomass yield between amended and unamended experiment varied significantly with higher in PM amended and lower in unamended treatment. Bacteria and bacterivores mutually collaborate with significant effects on the root development, but it is uncertain that how much these processes control plant response [17-34]. Similarly, Trap et al [32] in meta-analysis and Gebermikael et al [19] showed higher plant P and plant biomass in the presence of nematodes and organic amendment and nematodes respectively. Soil fauna manage plant roots, root exudates and rhizosphere microbial processes interactions and therefore affects plant growth. According to a research report, carbon exudates from roots trigger microbial growth in the rhizosphere when no P is lost by exudation resulting in the organic P mineralization and assimilation into microbial biomass [42]. Considering that most studies reported the direct nutrient effects due to the microfaunal grazing on the rhizosphere bacteria i.e., the ‘microbial loop on P mineralization and subsequently the increased growth and P transfer to the plants. We observed interaction between trophic level and PM addition similar to the changes observed for plant dry weight Fig 6B (c). Bunemann [35] reported that after organic amendment soil microbial immobilization of P prevent it from sorption and make it available for the plant uptake. We observed significant effect of PM addition with highest effect with sole bacterial treatment. The general substrate effect was primarily due to the increases in soil C pools and soil pH with PM addition because all the predator and microbe treatment without PM addition significantly reduced root P. There are increase as well as decrease in the microbial biomass as a result of predation, have been reported by [18,19] depending on the soil type, and carbon availability and various other factors [43], decline in the microbial abundance might be the reason for marginal decline in the root P uptake in predation treatment with PM addition.

Iqbal et al and Batool and Iqbal [7-12] in a study -of PSBs (Bacillus species) inoculation with the addition of P source reported more enhanced effect on root and shoot biomass and root P uptake rather P solubilization. In meta-analysis regarding the effect of bacterivores nematodes on the plant P and biomass their effect was reported up to 30% [32]. In the present study, it was observed 2.1 times more plant dry biomass in the sole bacteria treatment with the addition of PM compared to the similar unamended treatment. While predator treatment increases by 1.19 times more plant dry biomass compared to the similar unamended treatment. While shoot P was also significantly affected by the interaction of factors (drivers+substrate) with 2.5 times more shoot P acquisition in predator treatment with PM addition compared to similar unamended treatment. A strongly positive effect of Cephalobus sp. inoculated with Pseudomonas sp. or Burkholderia sp.was reported on root growth [17-44]. In a planted microcosms experiment shoot biomass and P uptake mostly increased in the presence of protozoa and nematode grazers showed strong evidence of microfaunal stimulation of P mineralization via the microbial loop as the main underlying mechanism [20]. There is another evidence of positive influence of bacterivores nematode inoculation on higher P uptake and root length of rice amended with dolomite while higher root length wasn’t linked with higher P uptake [44].

Here the individual impact of biological drivers and substrate was less pronounced but interaction of both appeared at plant growth stage as high dry plant biomass and plant P concentration Fig 6B (c & d).

pH and microbial P dynamics

The influence of PM addition, microbial trophic interaction between PSBs and their grazer nematodes, and their interaction on the soil pH were investigated and it was found a significant available P content at pH 7.6 in the predation treatment without PM addition. Soil pH may be a good indicator for the organic P mineralization as a significant correlation between phoD gene and pH and P contents in soil were reported by Wan et al [2]. Wan et al Demonstrated pH as the main driver of high P contents, organic P mineralizing microflora [2-42]. As it was reported that available P content, organic P and ALP contents were correlated under various fertilization treatments Wan et al [5], found that pH strongly effect organic P mineralization which differ from [5] study demonstrated that organic phosphorus mineralizing enzyme activities decreased as Po increased. One explanation is that soil pH can directly influence Po-mineralizing-microflora, or interaction between them [5] thus affecting Po mineralization. The average PM amended plant P concentration was across both biological treatments was approximately thrice that of unamended similar treatments.

Assimilation and chemical factors such as precipitation and sorption causing P availability are sensitive to pH change [2-6]. There are diverse reports regarding the effect of pH change on plant P availability and uptake [41]. Organic P mineralization is also pH sensitive and increase at low pH [2-6].

Considering the system under poultry manure amendment and nematodes grazing on bacterial consortia could suggest that phosphorus release from the substrate by PMBs and their grazer is need dependent. But in phosphorus deficient soil PMBs activity were strong to make P available and in ALP production (P mineralization is high). The effect of substrate and predator is less comparable to unamended predator treatment, but predator effect was also significant compared to its relative control.

ALP activity and P mobilization values by 21 bacterial strains.

(DOCX)

Click here for additional data file.

ANOVA results showing the F and P values of significant differences between the applied treatments i.e. bioinoculation and amendment and their interaction.

(DOCX)

Click here for additional data file.

Unadjusted and uncropped images underlying all gel figures.

(PDF)

Click here for additional data file.

Excel file containing raw data obtained from analysis.

(XLSX)

Click here for additional data file.

28 Oct 2021

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Additional Editor Comments:

Use the ‘title case’ in title Or replace “A” with “a” in amendment…

Figures’ overall quality is not good; they are not clear to be read; please follow the journal’s guidelines. There are too many figures and some will not affect the manuscript-quality if excluded e.g. the figure-1 and Figure-9 don’t provide much flesh to the ms, fig. 2, 3, 4, and 5 have some redundancy and can better be re-represented. Fig. 6 and 7 can also be presented as one figure with A and B panels.

Figure 4: the legend says – ‘soil net pH-change’ while the data shows about pH values. pH-change means 1-fold, 2-fold, 3-fold etc. while the author seem to mean the actual soil pH and not the net-changes; better to modify accordingly to improve clarity.

What is the purpose of Fig. 2 and 3 in the presence of Fig. 5?

Figure 6/7 root/shoot P and not root/shoot P-uptake; I think the uptake means the mechanistic accomplishment of plant from one medium (soil) into the other (plant-body) via some transporter systems while the authors’ work indicates the values/concentrations present in root/shoot. Please clarify.

Figure 8: Plant total dry weight per pot; how did you normalize this? Were there same number of plants in each pot for all treatments? What was the normalization/standardization of plant health before the treatment application?

Figure 9: For molecular gene analysis: on the basis of PCR and gel electrophoresis bands, how can you claim the gene expression?? Manuscript’s sub-section “Successful identification of organic P mineralizing genes” looks inappropriate; the gel pictures simply mean the presence or absence of the potential genes and is not the gene expression. Neither were the amplified pcr-products sequenced, so cannot be said as identification either. Moreover, the authors used 20-30 years old primer systems; it would be surprising to know if there is not primer-improvement (for better specificity) during these years?? And, lastly, what is the role of Quinoprotein glucose dehydrogenase in organic P-mineralization?

Figure 10 is quite complicated, and its legend is even more. As this diagram is the summary of their whole work, the authors should improve it to make it comprehensive as well as clear to understand.

Table-2: replace bacteria name codes with ‘bacterial codes’. What does the column 3 and 5 mean?

As per Table-3, total soil P and soil organic P are the same that means this soil does not have any inorganic P; what do the authors say in this regard??

As per ms data, Soil net available P and soil ALP is always less in manure-amended treatments than the control treatments (Fig 2,3,4,5) ; does it mean that the organic farm manure application are not good while the farmers are frequently using this strategy as fertilization?? On contrary, the authors found enhanced root/shoot P in manure-amended treatments than the control treatments (Fig 6, 7). I recommend the authors to validate these observations and to make the appropriate discussion accordingly.

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Reviewer #1: Yes

Reviewer #2: Partly

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Reviewer #1: Yes

Reviewer #2: Yes

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Reviewer #1: Yes

Reviewer #2: Yes

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Reviewer #2: Yes

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5. Review Comments to the Author

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Reviewer #1: The current study examined the potential effects of co-inoculation of phosphorus solubilizing bacteria and their grazer nematodes on soil P availability in presence of poultry manure. The subject of this article is coherent with the scope of Plos ONE. Given the depletion of the limited P-fertilizer resources, scientific understanding the interaction of soil microfauna to improve the soil P fertility is important. The manuscript is well written. However, the manuscript requires revisions before it could be accepted for publication.

Specific comments:

Need to define first PSB.

L122: “A synthetic solid medium” is the starting sentence of L123.

Material and method

A huge amount of information is presented in the mat&met section. Some sections could be presented as a supplementary information to simplify the paper.

L110-L121: The soil properties should be presented with emphasis on the soil type (P-deficient or not, alkaline or acidic soil).

L136: PKO???

L168-L169: What criteria did you choose to select the 6 efficient strains of the 21 based on pH, ALP activity and P mineralization? Please report the results even as a supplementary information.

L287-L289: Can you provide the characteristic/succinct history of the field where soil was sampled (e.g. previous crop).

L302-L3016: Summarize the tested treatments of the experimental design in table to better help readers. Specify the duration of the experiment. How did you collected the soil samples for subsequent analyses (rhizosphere or bulk for each pot).

In statistical analysis section: provide sufficient information for data handling: software, the tested treatment with the statistical approach used (here one and two-way anova). The test of normality of the data and the homogeneity of variance.

Results:

L376-L381: Give the Anova result either in the text or in a separate table. Describe the T1 treatment here. How did you calculate the soil net P availability in Fig 2? May be it’s better to add this information in mat&met section.

I think it’s better to present the result for each tested parameters first before going in a specific description in your text: bacteria treatment, Nematode treatment, Poultry treatment, and interactions, and should be supported by Anova results. The same also with ALP results in L388-L395, and with pH and soil net available P (L402-L406).

It is interesting to see the different response of soil net available P with soil net pH. Increasing soil pH from 5.75 to 7.5 (control to Control+wheat plant+ bacterial consortia +nematodes) increased soil P from 0.0.4 to 4 µg.g-1) in unamended treatment , while increasing soil pH from 6.5 to 7.5 increased soil P from 0.0.2 to 2.8 µg.g-1). Focusing in nematode treatment impact, how about the response of increasing soil P to soil pH between the two treatments C+P+B vs C+P+B+N with or without PM addition?

The same remarks with the net P liberation vs ALP activity.

The clear effect of Nematode under PM amendment or no organic amendment could be describe and general conclusion can be drawn from this.

I suggest that authors presents the correlation between the measured parameters as ALP, pH, soil P, and plant biomass and P uptake.

Discussion:

I suggest to clearly state the nematode predator effect in presence of PM and without PM on the measured parameters before discussing the underlying mechanism in L507.

L580: Fig 9???

L584: Fig 8 instead of Fig 9.

In the section “Microbial P dynamics changes in soil treatments”, L507-L555, authors stated that the predation treatment declined Net P and ALP activity with PM addition in contrast with those without PM amendment; this is related to the immobilization of available P in microbial biomass. Does it suggest that predator effect is OM-dependent in soil? Through which mechanism?

Total P uptake (P root + P shoot) could be added to clearly show the contrasting effect of treatment. Could you try to calculate the P uptake efficiency (PUPE as the ability of crops to uptake P from the soil) = P uptake/soil P available (Moll et al. (1982)) to verify the predator , PM treatment effects.

Do you have any results on soil available P & ALP activity measurement with time (incubation of microcosm study)? This could be interested to be related to the higher plant P uptake & biomass in PM amendment. Is it possible that after high P uptake, soil could be depleted in available P? Because here soil P & ALP seems to be negatively correlated with plant P uptake.

I consider the data from different experiments including strains screening and microcosm experiment should be highly valuable and hopefully presented. I would like to encourage the authors to present clearly the data analysis in the result section and to resubmit the revised manuscript.

I hope the specific comments may also help authors for the revision.

Reviewer #2: # Review report: Ahmed et al.,

The submitted manuscript with the entitled "Organic Amendment plus inoculum drivers: who drives more P nutrition for wheat plant fitness in small duration soil experiment" is a very nice research article with up-to-date information submitted by Dr. Usman Irshad as a corresponding author. The research article describes the importance of biological drivers that significantly enhanced the soil ALP and available P and thus enhanced dry biomass and P uptake. However, reviewer has few suggestions to improve the impact of research article to the readers. Comments, and suggestion to the author are the following:

Title: Organic Amendment plus inoculum drivers: who drives more P nutrition for wheat plant

fitness in small duration soil experiment

The title of this manuscript does not match the results. Kindly, check it and reframe it accordingly.

Abstract: It is too lengthy, and sentences are repeated. Kindly make it short and put the main findings here.

Line 30-What is WEP?

Note-Key word are missing.

Introduction: The introduction of this manuscript is up to date. However, the authors used much time etc. Instead of etc. put more information that is required to strengthen your manuscript. Kindly do not use etc words.

Line 37- act as major sink and source (What does it mean?) of key abiotic components such as carbon

Line 42- development and growth of plant (Reference?). Agriculture soils are strongly depleted in available P for plants (How??), despite being virtually sufficient in total P 50-65% [3]

Line 53- Organic amendments of??

Line 55-Biomass of??

Line 56- other essential ecological functions such as?

Line 61- large amount of P in their biomass (Kindly write the proper sentences. I did not get the point here)

Line 64- Depending on the soil and fertilizer input the release of P from microbial biomass-How??

Question 1: Why did you choose only poultry manure?

Question 2: What was the concentration of P in soil before adding PM? And what was the content of P in PM?

Question 3: How did you decide the experiments duration for 3-months? Kindly, mention the specific reason.

Line 90-91: These microbial growths sequester large amounts of available nutrients such as P, C and N, e.g., nematodes while grazing sequester 50–70% of the prey carbon for biomass production [19]. Kindly rewrite the whole sentence to make it clear for audience.

Materials and methods:

Comments-I would like to suggest to the authors kindly make it short and crisp with only required information. Materials and methods are too lengthy as compared to results sections. You can shift unnecessary part of this section to the supplementary sections.

Note-Kindly make the italic all the scientific names.

Line 113- The soil samples were collected from the upper soil layer (depth 113 0-15cm) in triplicates, A substantial P turnover occur in the surface layers of soil and microbial number reduce along soil depth [21]-Why did you write this statement in materials and methods section?

Line 119- while the remaining portion of soil was mixed with?

Line 118- Isolation of phosphorus solubilizing bacteria from? And how did you isolate?

Line 120- soil initial pH, water available phosphorus and total phosphorus. All determinations and results were done in triplicate (Kindly make it clear). Line 122- Isolation, screening and identification of PSBs on A (Why this is capital?) synthetic solid medium

Question 4-How did you control the growth of other microorganisms?

Line 165- with each isolate sample by incubating 0.25 mL of water with 1 mL (Space) of pNPP and buffer and

Line 166- immediately adding 4 mL (space) of 125 (Space) mM NaOH to stop the reaction.

Line 169- 6 efficient strains out of 21 were selected for further analys (remove extra space)es. This statement is not supported by your table no-2.

Line 177- Phosphorus mineralizing gene expression using q-PCR: In bacteria phoN, phnX gene (full form?) encoding enzymes are responsible for organic P mineralization and gcd (full form?) for inorganic P solubilization i.e.,

Note-Figure2-Kindly prepare a new diagram with proper formatting. You must have to keep the size of figures 300 dpi.

Line 227- The synergistic activity by combination of 6, 4, 3 and 2 of 6 or 4?? selected isolates

with high available

Figure 1: This is not providing a clear message to the readers; therefore, I would like to ask to make a table for figure 1.

Results:

Note-Kindly, prepare all the figures with proper formation to keep at least 300 dpi size.

Figure 9-You should replace this figure with other. As I can see clearly shadow instead of bands. I am not able to see the proper bands.

Note-In each results heading, you should write outcome of the results (kind of 1 line result). For example, do not write Plant dry biomass. You can write treatment of X and Y enhanced dry biomass.

Note-Conclusion of this manuscript is missing. Kindly write it.

Once you address all the comments and queries, after that, I will recommend this manuscript for further process.

**********

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Reviewer #1: No

Reviewer #2: No

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Submitted filename: Revision Plos ONE.docx

Click here for additional data file.

2 Mar 2022

Responses to Reviewers/Editors Comments

First of all, we are thankful to Editors and reviewers for valuable suggestion and correction in our manuscript. We incorporated all the suggestion and comments and please find the detail of each improvement here.

General Comments

Q1. Use the ‘title case’ in title Or replace “A” with “a” in amendment…

Answer: Thanks for highlighting this issue with title. We have incorporated your suggestion as

‘Organic amendment plus inoculum drivers: who drives more P nutrition for wheat plant fitness in small duration soil experiment’.

Q2. Figures’ overall quality is not good; they are not clear to be read; please follow the journal’s guidelines. There are too many figures and some will not affect the manuscript-quality if excluded e.g. the figure-1 and Figure-9 don’t provide much flesh to the ms, fig. 2, 3, 4, and 5 have some redundancy and can better be re-represented. Fig. 6 and 7 can also be presented as one figure with A and B panels.

Figure 4: the legend says – ‘soil net pH-change’ while the data shows about pH values. pH-change means 1-fold, 2-fold, 3-fold etc. while the author seem to mean the actual soil pH and not the net-changes; better to modify accordingly to improve clarity.

Answer: We agree with editors’ observation and we revised all figures as per Journals criteria (saved more than 600 dpi). In current version figures numbers reduced from 10 to 6, further as per Editor’s suggestion figure 1 and 9 removed from the revised manuscript. Figures 2, 3, 4 and 5 are represented in high quality as fig. 1, 2, 3a & 3b in current revised manuscript. Figures 6 and 7 are combined together as one figure (fig. 4 now) as per reviewers and Editor remarks.

Figure 4 now represented as figure 3a modified regarding pH change. In current version it is showing actual pH change rather than net pH change as suggested by Editor.

Q3. What is the purpose of Fig. 2 and 3 in the presence of Fig. 5?

Answer: We have reduced and changed the presented figures by taking in account all the comments. We still feel that figures 2 and 3 representing only one parameter data respectively in older version. While figure 5 currently presented as fig. 3b representing the data of two parameters which are dependent on each other. We tried to show the trend of these parameters in graph that how two parameters respond against applied amendments and treatments. We wish to show their trends either in case of amended experiments or unamended together putting both parameters.

Q4. Figure 6/7 root/shoot P and not root/shoot P-uptake; I think the uptake means the mechanistic accomplishment of plant from one medium (soil) into the other (plant-body) via some transporter systems while the authors’ work indicates the values/concentrations present in root/shoot. Please clarify.

Answer: Thanks for nice suggestion we strongly agree with Editor’s suggestion and changed our statement from root/shoot P uptake to ‘Total Plant P’ in graphs.

Q5. Figure 8: Plant total dry weight per pot; how did you normalize this? Were there same number of plants in each pot for all treatments? What was the normalization/standardization of plant health before the treatment application?

Answer: Yes sir, we normalized by same numbers of plants per pot after germination by pulling out extra wheat seedlings. As per plant health concerns, the almost similar height and number of leaves of different plants were measured and considered as their health status before treatment application. Further visual observation based on color were monitored and found approximately similar in all pots.

Q6. Figure 9: For molecular gene analysis: on the basis of PCR and gel electrophoresis bands, how can you claim the gene expression?? Manuscript’s sub-section “Successful identification of organic P mineralizing genes” looks inappropriate; the gel pictures simply mean the presence or absence of the potential genes and is not the gene expression. Neither were the amplified pcr-products sequenced, so cannot be said as identification either. Moreover, the authors used 20-30 years old primer systems; it would be surprising to know if there is not primer-improvement (for better specificity) during these years?? And, lastly, what is the role of Quinoprotein glucose dehydrogenase in organic P-mineralization?

Answer: Yes, we do agree with reviewer that we did not study gene expression, we studied the presence and absence of genes through PCR. So, we corrected the mentioned results as per Editor suggestion in revised manuscript. As the studied bacteria are already sequenced and we designed specific primers for studied genes, therefore, sequencing is not necessary for identification. Moreover, we checked the primers sequence in studied genes and these primers sequences were present and these are already optimized, that’s why, we used these primers as well as these primers are still in practice, and we took help from a recent paper published in ‘Journal of Microbiological Methods’ 5 years ago. The presence of gcd gene in our setup indicated that it’s not only P-mineralization phenomenon happening in experiment. The other related P fixation after mineralization phenomenon also happening in same system, so as per literature, people use this gcd as an indication of microbial P turnover mechanism. Further our inoculated consortia possess not only mineralizers but also P solubilizers having gcd genes.

Q7. Figure 10 is quite complicated, and its legend is even more. As this diagram is the summary of their whole work, the authors should improve it to make it comprehensive as well as clear to understand.

Answer: We have simplified the figure 10 as suggested which is represented as figure 6 in revised manuscript. We tried to remove the data presented and highlighted the mechanism by the use of arrow signs.

Q8. Table-2: replace bacteria name codes with ‘bacterial codes’. What does the column 3 and 5 mean?

As per Table-3, total soil P and soil organic P are the same that means this soil does not have any inorganic P; what do the authors say in this regard??

Answer: The bacterial names were replaced with bacterial codes in revised manuscript. In Table 2 column 3 indicates alkaline phosphatase produced by bacteria when grown in liquid culture and column 5 indicated the Pi release in same medium from organic P sources (phytate sodium salt). We have completed the headings and now they are more explicit in table 1 of revised manuscript. Thanks for indicating this technical point we have provided the standard deviation data with means in current manuscript which showed huge variation in available/water soluble P in soil. Further we have changed the organic P heading of the table as ‘unavailable P’.

Q9. As per ms data, Soil net available P and soil ALP is always less in manure-amended treatments than the control treatments (Fig 2,3,4,5); does it mean that the organic farm manure application are not good while the farmers are frequently using this strategy as fertilization?? On contrary, the authors found enhanced root/shoot P in manure-amended treatments than the control treatments (Fig 6, 7). I recommend the authors to validate these observations and to make the appropriate discussion accordingly.

Answer: Thank you for the interest. Your consideration and valuable advice made a huge improvement in our research paper. Yes, as it is evident from research literature that ALP activity by MOs is high under the nutrient starved condition. Furthermore, manure has many binding sites which upon application in soil convert the available P into complex compound (unavailable P) therefore the release of Pi in soil is need based. Farmers are using livestock manure but biodiversity enrichment is very necessary, so the application of biological drivers is key point to better get a positive output from added manure. We were not able to sample the experiment on temporal basis due to the less soil quantity used in our experimental system. That might show us the point where we have prominent changes in soil available Pi before it gets fixed with manure. There could be more Pi availability on the course of experimental duration which released efficiently only upon need, so manure applied soil contributed more P to plant compared to unamended treatment.

Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. Is the manuscript technically sound, and do the data support the conclusions?

The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented.

Reviewer #1: Yes

Reviewer #2: Partly

Answer: Thanks for your valuable and encouraging feedback

________________________________________

2. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: Yes

Reviewer #2: Yes

Answer: We appreciate your response and consideration.

________________________________________

3. Have the authors made all data underlying the findings in their manuscript fully available?

The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.

Reviewer #1: Yes

Reviewer #2: Yes

Answer: Thanks for your positive feedback and encouragement.

________________________________________

4. Is the manuscript presented in an intelligible fashion and written in standard English?

PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.

Reviewer #1: Yes

Reviewer #2: Yes

Answer: Thanks for your attention and positive feedback.

________________________________________

5. Review Comments to the Author

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #1: The current study examined the potential effects of co-inoculation of phosphorus solubilizing bacteria and their grazer nematodes on soil P availability in presence of poultry manure. The subject of this article is coherent with the scope of Plos ONE. Given the depletion of the limited P-fertilizer resources, scientific understanding the interaction of soil microfauna to improve the soil P fertility is important. The manuscript is well written. However, the manuscript requires revisions before it could be accepted for publication.

Specific comments:

Q1. Need to define first PSB.

Answer: Thank you to point out this deficiency. We have defined and expanded the abbreviated “PSBs” (as phosphate solubilizing bacteria) in the revised manuscript in introduction section from line 70 to line 73 and abstract section in line 14.

Q2. L122: “A synthetic solid medium” is the starting sentence of L123.

Answer: Dear reviewer thank you again for highlighting the improvements we need to do. We have corrected this formatting mistake presented in L120 of revised manuscript.

Q2. Material and method A huge amount of information is presented in the mat&met section. Some sections could be presented as a supplementary information to simplify the paper.

Answer: Respected reviewer thank you for valuable advice. We have worked on mat and met section and squeezed certain sections especially isolation, experimental design (231-243), compatibility test (196-203), phosphorus analysis (259-266), ALP (267-279) and DNA extraction (167-185). Now it is squeezed (previous version line 110 to 372) to revised version from line 111 to line 294.

Q3. L110-L121: The soil properties should be presented with emphasis on the soil type (P-deficient or not, alkaline or acidic soil).

Answer: The soil used in experiment was loamy clay with deficiency in available plant P possessing slight alkaline properties. The same presented with previous crop history of soil in section sampling and processing of soil and poultry manure of Materials and Methods.

Q4. L136: PKO???

Answer: Dear reviewer Thank you for highlighting the required improvement. In the revised manuscript the abbreviated PKO is now represented as Pikovskaya.

Q5. L168-L169: What criteria did you choose to select the 6 efficient strains of the 21 based on pH, ALP activity and P mineralization? Please report the results even as a supplementary information.

Answer: Respected reviewer thank you for your valuable comment.

We have selected the 4 efficient strains out of 21 on the basis of maximum ALP and Pi release from phytate source when grown in media with phytate as sole P source. Then these strains further tested for compatibility against each other to make consortia. Results regarding ALP and Pi release of 21 strains is provided as supplementary data as S table 3.

Q.4. L287-L289: Can you provide the characteristic/succinct history of the field where soil was sampled (e.g. previous crop).

Answer: Dear reviewer after your valuable advice we have incorporated the desired information in material and method section in L236.

Q5. L302-L3016: Summarize the tested treatments of the experimental design in table to better help readers. Specify the duration of the experiment. How did you collected the soil samples for subsequent analyses (rhizosphere or bulk for each pot).

Answer: Respected reviewer thanks for the suggestion. We have summarized and simplified the experimental design test treatments in mat and met section; L232 to 257 in revised manuscript previous 288-321. We have specified the duration of the experiment as 90 days in abstract as well. For soil initial analyses we have collected bulk soil, while for final analyses rhizosphere samples were taken.

Q5. In statistical analysis section: provide sufficient information for data handling: software, the tested treatment with the statistical approach used (here one and two-way anova). The test of normality of the data and the homogeneity of variance.

Answer: Unless otherwise stated, the results are given as mean ± standard deviation (n 3). The differences between means were analyzed by factorial ANOVA followed by Tukey’s HSD post-hoc test using Statistica 7.1 (StatSoft Inc., Tulsa, OK, USA). Normality was tested using the Kolmogorov Smirnov test to meet the assumptions of ANOVA.

Results:

Q6. L376-L381: Give the Anova result either in the text or in a separate table. Describe the T1 treatment here. How did you calculate the soil net P availability in Fig 2? May be it’s better to add this information in mat&met section.

Answer: Dear reviewer thanks for your advice and suggestion. We have provided the ANOVA results as supplementary data as Supplementary Table 2. We have calculated the soil net P availability by subsequent subtraction of inoculation treatment from previously un inoculated treatment, which was due to the effect of inoculation. T1 treatment is set after poultry manure addition at 5% by weight compared to T0 without amendment. This information is also added in revised manuscript where found appropriate.

Q7. I think it’s better to present the result for each tested parameters first before going in a specific description in your text: bacteria treatment, Nematode treatment, Poultry treatment, and interactions, and should be supported by Anova results. The same also with ALP results in L388-L395, and with pH and soil net available P (L402-L406).

Answer: Dear reviewer we thank you for your valuable advice and suggestion. In every section of the result where we got significant results, we highlighted it by proper statement; each subheadings of result section.

Q8. It is interesting to see the different response of soil net available P with soil net pH. Increasing soil pH from 5.75 to 7.5 (control to Control+wheat plant+ bacterial consortia +nematodes) increased soil P from 0.0.4 to 4 µg.g-1) in unamended treatment , while increasing soil pH from 6.5 to 7.5 increased soil P from 0.0.2 to 2.8 µg.g-1). Focusing in nematode treatment impact, how about the response of increasing soil P to soil pH between the two treatments C+P+B vs C+P+B+N with or without PM addition? The same remarks with the net P liberation vs ALP activity.

Answer: Thanks for the encouragement. Yes, the net soil P change from sole bacteria treatment to predator treatment in both amended and unamended treatments. This result is stated in the result section in revised manuscript. Similar increasing trend was observed in case of soil ALP, before and after nematode inoculation in both unamended and amended bacteria only treatments.

Q9. The clear effect of Nematode under PM amendment or no organic amendment could be describe and general conclusion can be drawn from this.

Answer: Dear reviewer thank you again for your valuable comment. Yes predator treatment affect was observed in both amended and unamended treatment. The predator effect was more noticeable at start, but plant health shows the pronounced amendment effect.

Q10. I suggest that authors presents the correlation between the measured parameters as ALP, pH, soil P, and plant biomass and P uptake.

Answer: Dear reviewer thank you very much for your comment and valuable suggestion. We have tried a correlation analyses between the tested parameters, but there was less correlation found due to more treatments. But we tried to show the link between to measured parameters in the form of trend as shown in figure 3a and 3b in revised manuscript. This correlation analysis could have been possible if the duration of the experiment was longer.

Discussion:

Q11. I suggest to clearly state the nematode predator effect in presence of PM and without PM on the measured parameters before discussing the underlying mechanism in L507.

Answer: Respected reviewer thank you for the valuable suggestion. Yes, we have clearly mentioned the predator effect with and without amendment in discussion.

Q12. L580: Fig 9???

Q13. L584: Fig 8 instead of Fig 9.

Answer: Dear reviewer thank you for highlighting the required corrections. We have provided the figure reference with correct number in revised manuscript in discussion section.

Q13. In the section “Microbial P dynamics changes in soil treatments”, L507-L555, authors stated that the predation treatment declined Net P and ALP activity with PM addition in contrast with those without PM amendment; this is related to the immobilization of available P in microbial biomass. Does it suggest that predator effect is OM-dependent in soil? Through which mechanism?

Answer: Thank you for the valuable comment. Yes, it is stated the decrease in net available P in predator treatment with amendment compared to similar unamended treatment. It is due to that manure has many binding sites which upon application in soil convert the available P into complex compound (unavailable P) therefore the release of Pi in soil is need based. Yes, and for second part the predation effects depend upon microbial biomass which is dependent upon amendment.

That is why predation is indirectly OM dependent.

Q14. Total P uptake (P root + P shoot) could be added to clearly show the contrasting effect of treatment. Could you try to calculate the P uptake efficiency (PUPE as the ability of crops to uptake P from the soil) = P uptake/soil P available (Moll et al. (1982)) to verify the predator , PM treatment effects.

Do you have any results on soil available P & ALP activity measurement with time (incubation of microcosm study)? This could be interested to be related to the higher plant P uptake & biomass in PM amendment. Is it possible that after high P uptake, soil could be depleted in available P? Because here soil P & ALP seems to be negatively correlated with plant P uptake.

Answer: Respected reviewer thank you very much for the valuable suggestions.

We tried to calculate PUPE according to your suggestion. The data we obtained showed significant increase in PUPE on amended site of inoculation treatments. But as per Editor suggestion we need to show P concentration in plants not uptake by plants so in revised manuscript we are providing Total plant P not plant P uptake.

Due to the short duration of the experiment and less soil volume used we were only able to analyze the soil ALP and P at initial and final stages.

Yes, it is possible. We have observed high plant P concentration in amended treatment than its comparable unamended treatment with less net available P.

I consider the data from different experiments including strains screening and microcosm experiment should be highly valuable and hopefully presented. I would like to encourage the authors to present clearly the data analysis in the result section and to resubmit the revised manuscript.

I hope the specific comments may also help authors for the revision.

Answer: Thanks for your valuable feedback. We really appreciate your contribution for improvement of manuscript.

Reviewer #2: # Review report: Ahmed et al.,

The submitted manuscript with the entitled ‘Organic Amendment plus inoculum drivers: who drives more P nutrition for wheat plant fitness in small duration soil experiment’ is a very nice research article with up-to-date information submitted by Dr. Usman Irshad as a corresponding author. The research article describes the importance of biological drivers that significantly enhanced the soil ALP and available P and thus enhanced dry biomass and P uptake. However, reviewer has few suggestions to improve the impact of research article to the readers. Comments, and suggestion to the author are the following:

Title: Organic Amendment plus inoculum drivers: who drives more P nutrition for wheat plant fitness in small duration soil experiment

The title of this manuscript does not match the results. Kindly, check it and reframe it accordingly.

Answer: Dear reviewer thank you for the comment and suggestion. We have tried to incorporate all the suggestion from the respected reviewers. Now the manuscript contents reflect the title more closely in revised version.

Abstract: It is too lengthy, and sentences are repeated. Kindly make it short and put the main findings here.

Answer: Respected reviewer thank you for highlighting the required improvement. We have majorly revised our manuscript. Abstract is modified in the revised manuscript according to your suggestions.

Line 30-What is WEP?

Answer: Dear reviewer thank you. Water extractable P. As we used the term available P in the whole manuscript, so we replaced this in the revised manuscript with available P.

Note-Key word are missing.

Answer: Dear reviewer thank you for the comment. Key words are present. In revised manuscript we tried to represent them in better way.

Introduction: The introduction of this manuscript is up to date. However, the authors used much time etc. Instead of etc. put more information that is required to strengthen your manuscript. Kindly do not use etc words.

Answer: Dear reviewer thank you for your comment. We have removed the word etc used in introduction section in the revised manuscript.

Line 37- act as major sink and source (What does it mean?) of key abiotic components such as Carbon

Answer: Key abiotic components are chemical elements such as phosphorus, nitrogen and potassium, these are present in various organic and inorganic forms and convert in other forms such as bioassimilation, when they get absorbed by living microorgansims, MOs are sink here. But when they are released by certain processes or stimuli such as nematodes when prey on bacteria the bacterial phosphorus is released as excretion product from nematodes, this is source of phosphorus.

Line 42- development and growth of plant (Reference?). Agriculture soils are strongly depleted in available P for plants (How??), despite being virtually sufficient in total P 50-65% [3]

Answer: Dear reviewer thank you for highlighting the required improvements and comments.

Yes, we rephrased the sentence to make it comprehensible also provided the reference L38. Agriculture soil are depleted in available P due to; high application of fertilizers, Soil type such as alkaline soil binds P with Ca to form Ca3(PO4)2 and in acidic soil it binds with Iron and Al to from strengite and variscite, , which plant cannot directly asses but in the form of mono and ortho hydrogen phosphate. This means phosphorus is sufficiently present in soil but plants cannot use this complex form of P which is fixed. This sentence is rephrased in revised manuscript.

Line 53- Organic amendments of??

Answer: Respected reviewer thank you. We have completed the sentence in revised manuscript L52.

Line 55-Biomass of??

Dear reviewer thank you for highlighting the required improvement. We have corrected the sentence as” microbial biomass and their diversity” L53 in revised manuscript.

Line 56- other essential ecological functions such as?’

Answer:Dear reviewer thank you we have corrected the sentence as “extracellular enzyme activities e.g. phospahatase enzyme production” L54 in revised manuscript.

Line 61- large amount of P in their biomass (Kindly write the proper sentences. I did not get the point here)

Answer: Dear reviewer thank you for the suggestion. Yes we rephrased the sentence and split in two sentences for better understanding.

Line 64- Depending on the soil and fertilizer input the release of P from microbial biomass-How??

Answer: Yes, Soil MOs adjust according to the soil bioavailability of nutrients. They retain or release P from their biomass not to disturb the CNP ratio of their biomass, so depending upon the fertilizer input more carbon or phosphorus is added to which MOs adapt, more DNA is formed and microorganisms do not retain P in their biomass for longer time.

Question 1: Why did you choose only poultry manure?

Answer: Dear reviewer thank you for the suggestion and comment. We have used other amendments, but we got contradictory results between these amendments based upon their nutrients stoichiometry differences. And the presentation of all amendments data in one manuscript is not possible so here we are presenting the data only from poultry manure.

Question 2: What was the concentration of P in soil before adding PM? And what was the content of P in PM?

Answer: Dear reviewer thank you for the comment. Yes we have analyzed total and available P in soil and PM. We have provided the table 3 in revised manuscript comprising initial analyses of soil and PM regarding P contents.

Question 3: How did you decide the experiments duration for 3-months? Kindly, mention the specific reason.

Answer: Dear reviewer thank you for the valued comments. Our decision to continue the experiment for days was based on the objectives and desired outcomes. Further, we designed a pot experiment on short scale and in small pots, that restrain us to continue the experiment for longer time. Most importantly the effect of microbial inoculation and amendment became evident in the decided duration of the experiment and the effect could be measured well. Due to small capacity of system, it was harvested after 90 days.

Line 90-91: These microbial growths sequester large amounts of available nutrients such as P, C and N, e.g., nematodes while grazing sequester 50–70% of the prey carbon for biomass production [19]. Kindly rewrite the whole sentence to make it clear for audience.

Answer: Dear reviewer thank you for the valued suggestion. Yes we rephrased the sentence to make it understandable in revised version.

Materials and methods:

Comments-I would like to suggest to the authors kindly make it short and crisp with only required information. Materials and methods are too lengthy as compared to results sections. You can shift unnecessary part of this section to the supplementary sections.

Answer: Dear reviewer thank you for your valuable advice. we have shortened our mat and met section and retained only required information. In revised version now shortened to L111-L 294.

Note-Kindly make the italic all the scientific names.

Answer: Dear reviewer thank you for your suggestion. We have changed all the scientific names to italic font in revised MS.

Line 113- The soil samples were collected from the upper soil layer (depth 113 0-15cm) in triplicates, A substantial P turnover occur in the surface layers of soil and microbial number reduce along soil depth [21]-Why did you write this statement in materials and methods section?

Answer: Dear reviewer thank you. We agree with your comment, so we have removed the sentence from mat and met section and incorporated in introduction section.

Line 119- while the remaining portion of soil was mixed with?

Answer: Dear reviewer thank you for highlighting the required improvement. While improving mat and met section according to the respected reviewer’s suggestions we have rephrased and removed the in comprehensible sentence.

Line 118- Isolation of phosphorus solubilizing bacteria from? And how did you isolate?

Answer: Dear reviewer thank you for the comment. Isolation of PSBs was done from forest rhizosphere soil. We have given a detailed procedure for isolation of PSBs (phosphorus solubilizing bacteria) in the revised manuscript.

Line 120- soil initial pH, water available phosphorus and total phosphorus. All determinations and results were done in triplicate (Kindly make it clear). Line 122- Isolation, screening and identification of PSBs on A (Why this is capital?) synthetic solid medium

Answer: Dear reviewer thank you for the comment. Yes, sentence in the revised manuscript was rephrased. This formatting mistake has been removed in revised manuscript.

Question 4-How did you control the growth of other microorganisms?

Answer: Dear reviewer thank you for your comment.

We did not control the growth of soil indigenous microorganisms:

1. We were comparing the organic or plant unavailable P addition as nutrition to MOs (amended) to unamended as close to the natural system.

2. We added treatments to make it close to the natural soil environment because naturally farmers do not control the microbial growth before adding amendment.

3. On the other hand there were research reports that killing the indigenous flora to control the growth via sterilization of soil can increase the available nutrients from their biomass which can bias the desired results.

Line 165- with each isolate sample by incubating 0.25 mL of water with 1 mL (Space) of pNPP and buffer and Line 166- immediately adding 4 mL (space) of 125 (Space) mM NaOH to stop the reaction. Line 169- 6 efficient strains out of 21 were selected for further analyses (remove extra space). This statement is not supported by your table no-2.

Answer: Respected reviewer thank you for highlighting the required improvements. So we have corrected the formatting mistakes in revised manuscript.

Dear reviewer thank you for highlighting this inappropriate statement. We have simplified the use of strains from 6 to 4 in revised manuscript in mat and met section, now it is supported by table 2.

Line 177- Phosphorus mineralizing gene expression using q-PCR: In bacteria phoN, phnX gene (full form?) encoding enzymes are responsible for organic P mineralization and gcd (full form?) for inorganic P solubilization i.e.,

Answer: Dear reviewer thank you for the comment. The full forms of primers used are written in revised manuscripts in result section and Table 2.

Note-Figure2-Kindly prepare a new diagram with proper formatting. You must have to keep the size of figures 300 dpi.

Answer: Respected reviewer thank you very much for the valuable suggestion. Yes, after incorporating the reviewer’s all comments we have revised all the figures accordingly and presented in better format.

Line 227- The synergistic activity by combination of 6, 4, 3 and 2 of 6 or 4?? Selected isolates with high available

Answer: Respected reviewer thank you for highlighting the issue. We have simplified the statement to be comprehensible in mat and met section of revised MS. This sentence was explaining the compatibility test between the strains. Test was performed between 2, 3, 4 isolates.

Figure 1: This is not providing a clear message to the readers; therefore, I would like to ask to make a table for figure 1.

Answer: Dear reviewer thank you for the suggestion. According to the reviewers comments we found it unnecessary and removed it.

Results:

Note-Kindly, prepare all the figures with proper formation to keep at least 300 dpi size.

Answer: Dear reviewer thank you for your valuable advice. Yes after incorporating the reviewer’s all comments we have revised all the figures accordingly and presented in better format.

Figure 9-You should replace this figure with other. As I can see clearly shadow instead of bands. I am not able to see the proper bands.

Answer: Dear reviewer thank you for your valuable suggestion. Yes after incorporating the reviewer’s all comments we removed it from the revised MS and mentioned our experiment in results and mat sections.

Note-In each results heading, you should write outcome of the results (kind of 1 line result). For example, do not write Plant dry biomass. You can write treatment of X and Y enhanced dry biomass.

Answer: Dear reviewer thank you for your valuable suggestion. Yes in every section of result where we found significant results, we highlighted them as per your suggestion.

Note-Conclusion of this manuscript is missing. Kindly write it.

Answer: Conclusion is provided in revised manuscript as per Journal format it is embedded after discussion not as separate section..

Once you address all the comments and queries, after that, I will recommend this manuscript for

further process.

Answer: We addressed all suggestion and revised manuscript and hopeful for better understanding of current version.

Submitted filename: Response to Reviewers.docx

Click here for additional data file.

18 Mar 2022

Organic amendment plus inoculum drivers: who drives more P nutrition for wheat plant fitness in small duration soil experiment

PONE-D-21-30854R1

Dear Dr. Irshad,

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Reviewer's Responses to Questions

Comments to the Author

1. If the authors have adequately addressed your comments raised in a previous round of review and you feel that this manuscript is now acceptable for publication, you may indicate that here to bypass the “Comments to the Author” section, enter your conflict of interest statement in the “Confidential to Editor” section, and submit your "Accept" recommendation.

Reviewer #1: All comments have been addressed

Reviewer #2: All comments have been addressed

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2. Is the manuscript technically sound, and do the data support the conclusions?

The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented.

Reviewer #1: Yes

Reviewer #2: Yes

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3. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: Yes

Reviewer #2: Yes

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4. Have the authors made all data underlying the findings in their manuscript fully available?

The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.

Reviewer #1: Yes

Reviewer #2: Yes

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5. Is the manuscript presented in an intelligible fashion and written in standard English?

PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.

Reviewer #1: Yes

Reviewer #2: Yes

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6. Review Comments to the Author

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #1: I would like to thank the authors for the improvement of the manuscript after revision. I saw that the authors adressed all comments and suggestions of the reviewer by incorporating them and updating in the text. Thus, I am fully satisfied of authors responses to reviewer's comments. I think that the manuscript meets the journal's requirement now, and could be considered for publication.

Reviewer #2: (No Response)

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7. PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files.

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Reviewer #1: Yes: Andriamananjara Andry

Reviewer #2: No

1 Apr 2022

PONE-D-21-30854R1

Organic amendment plus inoculum drivers: who drives more P nutrition for wheat plant fitness in small duration soil experiment

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