**4. Discussion**

The use of plant growth promoting rhizobacteria (PGPR) in combination with organic (composts, rock phosphate) and inorganic (chemical fertilizers) phosphorus sources significantly increased

the number of tillers per plant and yield components of wheat crop. The results are in conformity to the findings of Akhtar et al. [40] who recorded increase in plant height, the number of tillers, grain yield and 1000 grain weight of wheat with the use of compost and PGPR inoculation.

Maximum grain yield was obtained by the application of RPEC1 which was higher than the full dose of inorganic P fertilizers (FDP), irrespective of the PGPR seed inoculants. The observed yield increase from RPEC1 was indicative of the high P availability and greater photosynthesis as observed by an increase in chlorophyll content and dry matter production, which was maximum in RPEC1 over other treatments. Plant P availability as the key factor for maximum plant growth and higher crop production [41]. Although a full dose of P (FDP) as inorganic fertilizer (SSP) is a source of readily available phosphorus necessary for early growth of the plants, at the site of SSP application, production of the least soluble Ca-P compounds due to surface adsorption and precipitation, reduce P availability [10]. The organic acids produced due to compost might have reduced P exchange sites through chelation and released more soluble forms of plant available P [42] compared to SSP which could help increase growth and yield of wheat. Seed inoculation with *Pseudomonas* sp. increased the grain yield with fertilizer treatments, however the maximum increase was recorded from *Pseudomonas* sp. inoculation with RPEC1 followed by the inoculated FDP treatment. Microbial community in the root rhizosphere might have taken part to release fixed phosphorus through organic acids production which ultimately increased the yield of wheat. Afzal and Bano [43] reported that seed inoculation with PGPR in combination with P fertilizer increased the grain yield of wheat which was 30–40% higher than the un-inoculated P fertilizer. It was reported that organic manures and bio-fertilizers have a high impact on nutrient uptake, physiological process of wheat, and also on water holding capacity of the soil which ultimately increase grain yield of the crop [44]. Amujoyegbe et al. [45] recorded higher grain yield of maize due to the application of chicken manure in combination with microbes compared to chemical fertilizer and chicken manure alone. An association of agronomic traits with grain yield and a positive correlation of 1000 grain weight with grain yield was previously demonstrated in PGPR + manure treated plants of wheat [44].

Increase in the dry matter yield due to the application of RPEC1 compared to FDP may be due to higher vegetative growth, chlorophyll content and the maximum number of tillers during the crop growth, while mobilization of phosphorus due to dissolution of rock phosphate from RPEC1 might have taken part in the physiological processes leading to maximum biomass yield. Higher yields of mung-bean were recorded due to bio-inoculated RP enriched compost having higher citrate soluble, water soluble P and organic P, maximum microbial biomass carbon and acid phosphatase activity compared to un-inoculated composts [7]. Similarly, Nishanth and Biswas [11] prepared enriched composts with *Aspergillus awamori* inoculation and tested these on the wheat crop, which gave maximum biomass production in comparison to composts prepared without inoculants. Hossain et al. [46] reported an increase in grain and straw yield of wheat crop with the application of phosphate solubilizing bacteria (PSB) along with different levels of phosphorus. In concurrence with the present results, an increase in dry matter and grain yield of agronomic crops due to phosphate solubilizing microorganisms in combination with different P fertilizers were reported earlier by different workers [47–49].

Phosphorus plays an important role in chlorophyll production and regulation. It has been reported that the partitioning of photosynthates between leaves and reproductive organs is regulated by the availability of phosphorus to the plants [50]. Maximum increase in chlorophyll contents in flag leaves were recorded due to the application of RPEC1 followed by FDP and SPLC. Zafar et al. [51] reported an increase in chlorophyll contents by 10–89% over control in leaves of maize crop following application of P fertilizers in the form of compost and inorganic fertilizers. The PGPR in combination with compost was recorded to be stimulatory for chlorophyll production; this was confirmed for *Pseudomonas* sp. in combination with RPEC1. Seed inoculation with *Pseudomonas* sp. alone or in combination with P fertilizers, was more efficient for improving chlorophyll contents in flag leaves of wheat plants. Naseem and Bano [52] reported that the seed inoculation with *Pseudomonas* sp. and *Bacillus cereus* increased chlorophyll contents by 8–13% in leaves of wheat crop. An increase in chlorophyll contents with the application of organic manure was also recorded [53].

PGPR alone or in combination with fertilizers showed a significant effect on IAA and GA contents of wheat flag leaves, however, maximum increase was recorded as a result of RPEC1 application followed by FDP and RPEC2. Among the PGPRs, *Pseudomonas* sp. performed better than the *Proteus* sp. Indole Acetic Acid synthesis by bacteria may have various regulatory effects in plant–bacterial interactions and significant effect on plant growth promotion [54]. Generally, phytohormones in plants plays an important role in cell division, proliferation, and differentiation, vascular tissue alteration, responses to light and gravity, general root and shoot architecture, seed and tuber germination, organ differentiation, peak predominance, ethylene synthesis, vegetative growth processes, fruit development and aging. These results are in accordance with the findings of Saharan and Nehra [55], who reported that the phytohormone production through PGPR (*Pseudomonas*, *Azotobacter*, *Azospirillum*) may contribute to growth and yield of the crop. IAA acts as a signal molecule for cell expansion, division and differentiation. Higher counts of genus *Pseudomonas* were recorded [56] in winter wheat cultivars and described the developmental phase of wheat crops as a key factor in higher population of the microbes. The GA and IAA were reported to be produced by bacterial strains such as *Bacillus* and *Pseudomonas* [57] and inoculation of wheat with *Pseudomonas* sp. gave maximum increase in growth and yield [58]. Khan et al. [59] found an increase in IAA and GA contents in leaves of wheat inoculated with *Pseudomonas* and *Bacillus* strains. Sivasankari et al. [60] isolated bacterial strains from black gram (*Vigna mungo*) rhizosphere soil and reported maximum IAA production from *Pseudomonas* sp. than *Proteus* sp.

Phosphors uptake increased with the application of RP enriched compost (RPEC1) which would be due to phosphorus in the soluble form. Higher concentration of macronutrients due to the decomposition of organic materials in the soil were recorded [61]. Incorporation of organic materials can enhance phosphorus availability in the soil solution by decreasing P sorption/fixation through chelation [62]. Phosphorus also plays an efficient role in plant photosynthesis, respiration, formation of cell membrane, glycolysis and enzymes activities [63] showing that the growth and development of all crops are dependent upon P availability [64]. The presence of P as an integral part of nucleotides, phospholipids, phosphoproteins, and coenzymes shows its importance for life [65]. An increase in P-uptake due to enriched compost in the present study was due to the maximum available P as well as total organic and readily available carbon. Sharma et al. [66] reported increased N uptake (18–38 kg ha<sup>−</sup>1), P uptake (2.7–6.6 kg ha<sup>−</sup>1), and K uptake by (16–41 kg ha<sup>−</sup>1) in the rice–wheat system when inoculated with *Pseudomonas striata*. It was reported by Nishanth and Biswas [11] that RP enriched compost inoculated with *Aspergillus awamori* can significantly enhance P uptake in wheat crop, which was recorded as 78% more efficient compared to DAP. Ghaderi et al. [67] reported 51%, 29% and 62% release of phosphorus from iron hydroxides by the application of *Pseudomonas putida*, *Pseudomonas fluorescens*, *and Pseudomonas fluorescens,* respectively. Shrivastava [10] reported that inoculation of microbes with P enriched manure show maximum P uptake in mung-bean crop compared to SSP fertilizer. The P-enriched compost in combination with effective microbes (EM) can enhance N and P uptakes of the cowpea crop [14].

Crop growth is regulated by the nutrient supply from organic or chemical fertilizer sources. Organic materials are considered to be the best source for nutrient supply to plants but with slow release until the crop maturity, which may create a delay in crop maturity or cause high nutrients content in the produce [68]. Maximum P concentration in the wheat seeds with the application of RPEC1 might be due to a slow release process resulting in P accumulation in the seeds due to mobility of the phosphorus from soil to plant process. The integrated management of P fertilizers at the root zone can increase the mobility of P from plant roots through physiological adaptive mechanisms [69]. Seed inoculation with *Pseudomonas* sp. showed an increase in seed phosphorus. According to Son et al. [70] soybean seed P content increased with inoculation of phosphate solubilizing microorganisms.

Organic and inorganic amendments have a great impact on soil properties [71]; however, while the application of fertilizer increases P availability at all crop growth stages compared to control treatment, the RP compost showed maximum P availability at later stages of wheat crop growth [11]. The increase in post-harvest soil P availability with the application of RP enriched compost may be due to mineralization of both RPEC and soil organic P, and chelation of P through ligand exchange reactions to reduce P fixation throughout the crop growth stages. The ligand exchange reactions can increase P mobilization through organic and phosphate anions adsorption with Fe and Al sites [72]. Slow release of P through mineralization of organic P fraction from enriched compost was reported previously [73]. Organic acids produced by phosphate solubilizing microorganisms are sources of H<sup>+</sup> ions which help mineralize tri-calcium phosphate of RP to mono-calcium phosphate; the available form of phosphorus for better plant growth [74].

The application of compost treatments showed significantly higher nitrate-nitrogen contents in post-harvest soil compared to control. Higher nitrate nitrogen content from compost treated plots would be due to reduced nitrate leaching from the soil [75]. Sommers and Giordano [76] stated that all inorganic N in soil amended with Municipal Solid Waste (MSW) compost was available for plant uptake, but 5 to 75% of the organic N will be mineralized within 1 year after application. The findings are in accordance with the results of Baziramakenga et al. [77] who reported an increase in inorganic nitrogen (NO3-N) contents of snap-bean post-harvest soil with the application of compost of de-inking paper residues and poultry manure. The reason for higher NO3-N contents due to the application of composts is attributed to the formation of phospho-protein due to the interaction with rock phosphate from enriched compost, which is less susceptible to volatilization. The proteins are decomposed by soil bacteria and change into ammonium that is further nitrified by nitrifying bacteria. This form of nitrogen from compost is slowly available to plants having fewer chances of loss through volatilization. The escape of ammonia from soil decreases if the nitrogen source is compost, organic manure or green manure [78]. The presence of phosphate preserves the nitrogen resulting in a decrease in the number of denitrifying bacteria [79]. The slow release process of nutrients from enriched compost might be another reason for higher nitrate-nitrogen contents than inorganic fertilizers (FDP) in post-harvest soil. Adeli et al. [80] reported higher residual soil NO3-N contents after cotton crops with the application of poultry manure compared to inorganic fertilizers. Seed inoculation with PGPR (*Pseudomonas* sp. and *Proteus* sp.) showed an increase in post-harvest nitrate-nitrogen contents. Canbolat et al. [81] also reported increase in soil post-harvest nitrogen contents with application of *Pseudomonas putida* compared to the control on barley crop.

Extractable potassium contents increased in post-harvest soil with the application of compost compared to inorganic P fertilizer (FDP). A significantly higher concentration of K with the application of enriched compost compared to FDP and control might be due to the higher water-soluble potassium present in the enriched composts. Stratoon et al. [82] reported that K in composts remains in water soluble forms and thus does not need to be mineralized before becoming available to plants. The increase in soil extractable K by rock phosphate enriched compost (RPEC) may be related to the direct addition to the available K pool of the soils, and to the reduction of K fixation and increase the release of K from the soil solid phase due to the interaction of organic matter and/or soil microorganisms with K-bearing minerals [77]. It was revealed that potassium in manure and compost is highly plant-available and can be used similar to K fertilizer application [83].

Soil enzymes (alkaline phosphatase and acid phosphatase) play a vital role in conversion of fixed soil phosphorus to plant available form [7]. The increase in alkaline phosphatase activities with the application of RP enriched compost in the present study may be due to the availability of organic C which consequently increased the soil phosphatase activity [84] and the compost might have provided considerable carbon and nitrogen for maximum growth of microbes. It has been emphasized that C and N are interlinked with P mineralization by microbes [85] and Shrivastava et al. [10] concluded that the availability of metabolizable C plays a significant role to increase soil phosphatase activity with the application of P enriched manure on mungbean crop. Soil enzymes such as acid and alkaline phosphatases help to increase mineralization of P0 to Pi by creating a strong relation between bio-available and unavailable P in the soil [86]. Some researchers [87,88] believe that there is an inverse relationship between available P and phosphatases due to negative feedback of phosphate ions on PHO genes, that suppress phosphatase synthesis by microbes [89]. However, phosphatase activity was not affected by the use of rock phosphate as a phosphate source in RP enriched compost [9] showing long persistence and least biodegradation of enzymes with the application of compost. But Pascual et al. [90] endorsed the decrease in phosphatase activities with time span due to exhaustion of biodegradable substrates by microbial activity.

Microbial biomass is an important factor assessing soil quality and its ability to provide energy for nutrient recycling and transformation in the soils [91]. Kiani et al. [92] documented the microbial biomass responses to different land management systems including fertilizer addition and organic amendment application and identified suitable soil quality indicators. The microbial biomass carbon (MBC) acts as substrate supplying entity for microbial communities in soil [93]. In the present study, maximum MBC with the application of RP enriched compost compared to poultry litter compost is due to higher percentage of existing microbial biomass carbon, mineralizable nitrogen and water-soluble carbon in the former compost. The results are in conformity to the findings of Meena et al. [94] who reported an increase in soil microbial biomass carbon with the application of enriched compost compared to ordinary compost as well as inorganic fertilizer. Previously Ayed et al. [95] found an increase in microbial biomass carbon with the application of compost compared to inorganic chemical fertilizer and control in wheat crop.

The maximum microbial biomass phosphorus (MBP) with the application of RPEC and inorganic P fertilizer could be due to the transformation of labile and nonlabile inorganic phosphorus to the organic pool through microbial activity of compost. As Leytem et al. [96] reported the assimilation of various fractions of P into microbial biomass which ultimately provides available P for plants since most organic phosphorus in microbial cells is hydrolysable. Microbial biomass phosphorus may also help in calcareous soils by providing plant available P with application of manure [97] by the mechanism in which P is immobilized and transformed to labile P, which is safe from fixation and transmitted to available P [98]. The results in the present study showed less microbial biomass P with the addition of a full recommended dose of inorganic P fertilizer, indicating that soil existing organic carbon was limited to support microbial growth and activity. Minimum microbial biomass was recorded with the addition of high P inorganic fertilizer to the soil in an incubation study.

#### **5. Conclusions**

The present research revealed that enrichment of rock phosphate with poultry litter and PGPR during the process of composting improves nutrient availability and biological properties of the compost. Application of RP enriched compost in field experiment increased yield and yield components of the wheat crop compared to the full recommended dose of inorganic fertilizer and control. Moreover, seed inoculation with PGPR showed significant results to improve the agronomic effectiveness of RP enriched compost. Chemical (availability of phosphorus) and biological (microbial biomass C & P, alkaline and acid phosphatase activities) properties of post-harvest soil improved with the application of RP enriched compost. It can be concluded that RP enriched compost may be an alternative to chemical fertilizer to improve the growth and yield of the crop.

**Author Contributions:** Conceptualization, M.B., A.B. and N.K.; Methodology, M.B., M.K., K.M.D., and N.K.; Formal analysis, M.B., M.K., S.N. and N.K.; Software, M.B., K.M.D. and N.K.; Data curation, S.N., K.M.D. and A.M.; Validation, M.B., A.B., A.H., A.M. and N.K., Investigation, M.B., A.B., S.B., A.H. and N.K., Resources, M.B., M.K., A.B., S.N., A.M. and N.K., writing—original draft preparation, M.B., M.K., A.B. and N.K.; writing—review and editing, M.B., M.K., A.B., A.H., A.S. and N.K.; supervision, M.B., A.B. and N.K. All authors have read and agreed to the published version of the manuscript.

**Funding:** This research received no external funding.

**Acknowledgments:** The author is thankful to Higher Education Commission Islamabad Pakistan and Pakistan Agriculture Research Council, Islamabad, Pakistan for the assistance to conduct this research.

**Conflicts of Interest:** The authors declare no conflict of interest.
