Combining phosphorus (P) with phosphate solubilizing bacteria (PSB) improved wheat yield and P uptake in alkaline soil

Phosphate solubilizing bacteria can reduce dependence on chemical phosphorus (P) fertilizers by mineralizing and solubilizing indigenous soil P. What’s why, we assessed the interactive effect of phosphate solubilizing bacteria (with and without PSB) and phosphorus levels (60, 90 and 120 kg P2O5 ha ) on P uptake and yield of wheat crop under field conditions. Two factorial randomized complete block design (RCBD) with three replications was used. The PSB inoculation significantly enhanced plant height (3%), 1000 grains weight (12%), grain (6%), biological (13%) and straw yield (18.5%) of wheat over control. Inoculation with PSB also significantly improved plant P concentration and uptake (26% each) over un-inoculated control. Similarly, with increasing application rates of P from 60 to 120 kg P2O5 ha the tested parameter were significantly improved except straw yield. The interactive effect of PSB and P exhibited significant effect on 1000 grains weight while the rest of parameter didn’t respond significantly. However, generally PSB inoculation with P enhanced yield attributes and improved P use efficiency over sole application of P. Thus it may be concluded that PSB should be applied with P to enhance wheat yield and P use efficiency.


Introduction
Wheat (Triticum aestivum L.) is a major cereal crop of Pakistan and used as a staple food [1]. It is estimated that by 2020 wheat demand in developing countries is going to increase by 1.6% per annum [2]. Thus improving wheat yield is very crucial for maintaining global food security. According to Food and Agriculture Organization (FAO) projection, food productivity should be increased about by 70% in-between 2005-2050 [3,4]. Adaptation of modern technologies, using good seeds, fertilizers and irrigation could be possible ways for achieving the target to ensure food security [5]. Phosphorus (P) ranks 2 nd in term of its requirement by plant and plays vital role in root development, cell division, flower, seeds and fruit formation [6]. Pakistani soils are naturally poor in P due to high pH and lime contents which leads to the limitation of crop growth. The biologically available P exist in soil are primary and secondary orthophosphate. Crop yield can be improved by optimum application of P as chemical fertilizers [7]. However, only 10-30% of the applied P is used by the plant while the rest lost in soil in many ways [8]. Phosphate solubilizing microorganisms (PSM) can be explored for their potential to enhance P availability and the crop yields [9, 10]. As they can play vital role for in enhancing plant growth under P deficient soils [11]. They are Phosphate economical, echo friendly to expensive chemical P fertilizers. PSM's inoculation enhance P availability and the crop yields by solubilizing the soil fixed and applied P [12]. Species of the genus Aspergillus, Bacillus, Pseudomonas, Penicilliumare and Rhizobium are the potential P solubilzers commonly present in alkaline soil [13]. They may produce the low molecular weight organic acids, lowering of pH in the surroundings and solubilize the insoluble phosphates. Organic acids may also act as chelating agents by discharging their functional groups thus, chelate the cations bonded to P thus, solubilize the insoluble phosphates [14]. In addition to organic acid release, the enzyme phosphatase released by PSM has a significant role in solubilization of insoluble P [15]. Siderophores, chelating compounds and mineral acids produced by PSMs and PSB has also been reported responsible for increasing P solubilization [16]. That's why, the present attempt was made to appraise the effect of PSB in enhancing P availably form phosphatic fertilizers applied at different rates on wheat crop with the following objectives.

Materials and methods Experimental site
A field experiment was conducted at the agronomic research farm of the department of Agriculture university of Swabi. The aim of this study was to evaluate the associative effect of PSB and Phosphatic fertilizer on yield and P uptake in wheat crop. The soil of experimental site was silt loamy, alkaline and calcareous and deficient in organic matter, nitrogen and phosphorus (Table 1).

Treatment combination
This attempt was made to explore the interactive role of P rates and PSB inoculation on P uptake and growth of wheat at the agronomic research farm, University of Swabi. This project was executed using factorial RCB design in triplicates. There were two factors including two types of inoculation (with PSB and without PSB) and three levels of P (60, 90 and 120 kg P2O5 ha -1 ) making a total of 6 treatment each replicated three times.

Experimental procedure
The inoculums of PSB inoculum composed of Mycobacterium, Pseudomonas, Rhizobia, Burkholderia, Pantoea and Bacillus were obtained from Department of Microbiology, Hazara University, Mansehra. The seed of wheat verity Serin was inoculated with PSB as per proposed treatment via using stander inoculation procedure. The plots were treated with 60, 90 and 120 Kg P2O5 ha -1 according to the treatment using SSP fertilizer as a source. Inoculated and un-inoculated wheat seed were sown at the rate of 120 Kg ha -1 in 30 cm apart rows while keeping a plant to plant 15 cm in a plot of size 3 x 5 m 2 . Uniform dosage of N and K2O was applied @ 120 and 60 Kg ha -1 through urea and sulphate of Potash (SOP) fertilizers. Normal recommended cultural practices were adopted during the growth period. Data were recorded throughout the growing season under field conditions. Agronomic parameter Plant height (cm) was measured as mean of five randomly selecting plants from each experimental unit at the peak of physiological maturity from base to the tip of spike. The plants from central two rows were harvested, sun dried threshed and weighed in each treatment plot and the yield was converted into kg ha -1. Thousand grains were randomly counted from the harvest of each treatment plot and their weight was measured by sensitive electronic balance. The plants from central two rows were harvested and sun dried in each treatment plot and was weighed as such for P TDM yield. The yield was then converted into kg ha -1. Straw yield of each treatment plot was measured by subtracting grain yield from biological yield (TMD) of each respective plot and was converted into kg ha -1.

Plant analysis
The wheat plant samples obtained from each experimental plot at harvest was processed for P concentration and uptake. A 0.5g oven dried plant sample was taken into 150 ml flask and treated with 10 ml concentrated nitric acid for 24 hrs. Then, 4 ml HClO4 was added followed by gentle heating till the emergence of white fumes for obtaining 3 ml of the colorless aliquot. The aliquot was then transferred to 100ml volumetric flask and P was determined by spectrophotometer by using stander procedure of Richards [17]. The total P uptake by wheat was taken as a product of total P and total dry matter yield.

Statistical analysis
The replicated data collected on each parameter was subjected to analysis of variance (ANOVA) of two factor complete randomized block design according to the procedure of Steel and Torrie [18]. In case of significant results the data were further subjected to least significant difference (LSD) test at 5% level of probability for obtaining variations among treatments for various parameters.

Results and discussion Plant height
Result for the effect of P, PSB and their interaction present in (Table 2). Upon analysis of various result demonstrated that P rates and PSB inoculation significantly affected plant height while their interactive effect was non-significant. It was exhibited by the result that PSB inoculation improved wheat plant height over un inoculated treatment. Application of P at the rate of 120 kg P2O5 ha -1 produced taller plants (93 cm), which was considerably taller than that of 90 kg ha -1 where the lowest plant height was recorded at 60 kg ha -1 . These result demonstrated that PSB inoculation along with P fertilization can further enhance the plant height of wheat and thus can pertain to increased production. Our results are in agreement to [19] who reported that P applied at the rate of 90 kg P2O5 ha -1 had significantly increased plant height (93.63cm).

Grain weight
Result about the effect of P and PSB and their interaction are mentioned in (Table 3). Upon analysis of various result indicated that P, PSB inoculation and their interaction significantly affected 1000 grains weight (g) of wheat. PSB inoculation improved wheat 1000 grain weight over un inoculated treatment. A 120 kg P2O5 ha -1 produced 1000 grain (47.91 g) which was appreciably heavier than that of 90 kg ha -1 whereas, the lighter grains was observed for 60 kg ha -1 . The interaction effect of PSB and P indicated that P applied @ 120 kg P2O5 ha -1 along with PSB produced heaver 1000 grains which was statistically more than the rest of treatment combinations while the lighters grains were noted for 60 kg P2O5 ha -1 without PSB produced. P applied at the rate of 60 kg ha -1 with PSB produced statistically higher than 1000 grain weight to that of 90 kg ha -1 P without PSB and similar to 120 kg ha -1 P without PSB. These result demonstrated that PSB inoculation can reduce or may minimize dependence on chemical P fertilizer up to 100% under exist soil and climatic conditions and may increase P use efficiency when applied with phosphatic fertilizers. These findings are in confirmation to those of [19] who reported a significant increase in 1000 grains weight (46.80g) over control at that 90 kg P2O5 ha -1 .

Grain yield (kg ha -1 )
Result regarding the response of wheat grain yield P, PSB and their interaction is presented in (Table 4). Upon analysis of various result indicated that P and PSB inoculation had significant effect on grain yield while the effect of their interaction was nonsignificant. It was exhibited by the result that PSB inoculation improved wheat yield over un inoculated control treatment. Application of phosphorous at the rate of 120 kg P2O5 ha -1 produced higher grain yield (2615 kg ha -1 ) which was appreciably higher than that of obtained (2541kg ha -1 ) at 90 kg ha -1 where the lowest yield (2347 kg ha -1 ) was observed for 60 kg ha -1. The interaction P and PSB was non-significant in this case, however it indicated that P applied at the rate of 120 kg ha -1 along with PSB produced relatively higher yield than the rest of treatment combinations. These result also demonstrated that PSB inoculation can reduce or may minimize dependence on chemical P fertilizer up to some extent under exist soil and climatic conditions. According to Khan et al. [10] Wheat grain yield was appreciably improved (by 22%) from 2920 kg ha -1 to 3560 kg ha -1 with application of 90 kg P2O5 ha -1 over control.

Biological yield
Result regarding the effect of PSB, P and their interaction on biological yield is present in (Table 5). Upon analysis of various result indicated that P and PSB inoculation significantly affected biological yield of wheat while their interaction was observed non-significant. It was exhibited by the result that PSB inoculation improved wheat biological yield over un-inoculated treatment. Phosphorous applied at the rate of 120 kg P2O5 ha -1 resulted maximum biological yield of 7129 kg ha -1 which was statistically similar to that of 90 kg P2O5 ha -1 where the lowest was observed for 60 kg P2O5 ha -1 which was similar to that obtained from plots treated with 90 while lower than that of 120 kg P2O5 ha -1 . These result demonstrated that PSB inoculation may improve BY of wheat can reduce or may minimize dependence on chemical P fertilizer up to a great extent under exist soil and climatic conditions. The increase in BY may be attributed to the fact that PSB increase nutrient and water use efficiency and P utilization.

Straw yield
Upon analysis of various result indicated that PSB inoculation had significant effect while P rates and P* PSB showed non-significant effect on straw yield kg ha -1 of wheat ( Table  6). The PSB inoculation significantly enhanced wheat straw yield over un inoculated control treatment. Higher application of P (120 kg ha -1 ) resulted straw yield to 4513 kg ha -1 whereas the lowest (3748 kg ha -1 ) was observed for 60 kg ha -1. The interaction effect of PSB and P indicated that P applied at the rate of 120 kg ha -1 along with PSB produced higher straw yield than the rest of treatment combinations. The increase in straw yield over control in PSB inoculated plots may be due to taller plants. These result demonstrated that PSB inoculation along with P fertilization may improve straw yield of wheat comparatively better than sole application of P.

Wheat phosphorus concentration (%)
Analysis of variance exhibited that inoculation and P application rates significantly affected P concentration of wheat while non-significant effect was observed in case of their interaction (Table  7). Inoculation significantly improved wheat P concatenation by 25.3% over un-inoculated control. Similarly, P concentration was gradually increased with increasing level of P. However, the performance of 120 kg P2O5 ha -1 was at par to 90 but significantly better than 60 kg P2O5 ha -1 . PSB improve P concentration and uptake by plant by enhancing P solubalization through soil acidification and organic and mineral acids (nitric and sulphuric acids) production [20][21][22]. Jilani et al. [23] and Yazdani et al. [24] reported that PSB inoculation may reduce P application as external sources by 50%. Zaida et al. [25] reported significant improvement in P accessibility for plants through PSB inoculation.

Conclusion and recommendations
Our findings imply that, PSB inoculation and P fertilization and their interaction significantly improved plant height, 1000 grains weight, grain yield and P uptake in wheat. With increasing rates of P from 60 to 120 kg P2O5 ha -1 wheat yield and P uptake were improved, however this increase was more prominent in inoculated treatments than un-inoculated control. Thus, it can be deduce that PSB inoculation may reduce the use of chemical P fertilizer up to 45 kg ha -1 but further studies should be conducted to confirm these results at a variety of soils on other cereals