Partial substitution of exogenously applied phosphatic fertilizers by phosphate solubilizing bacteria in maize under calcareous soil

Experimental site

This study was executed at the agronomic research farm (34.02071349555321, 71.48139963048956) of The University of Agriculture Peshawar (UAP) in 2021-22. The soil of the experimental site before the experiment was non saline (0.56 d Sm⁻¹), alkaline (7.86) and calcareous (15.4% lime) in nature, and silt loam in texture (clay 11%, silt 57.6% and sand 30.9%), low in organic matter (0.91%) and AB-DTPA extractable P (3.63 mg kg⁻¹).

Experimental material

The farm yard manure (FYM) containing 1.6% N, 0.54% P and 0.91% K was purchased from local dairy form. The rock phosphate containing 11.35% P was obtained from Hattar Industrial State, Haripur. The PSB inoculum having a bacterial load of 10⁹ cells g⁻¹ was purchased from National Agricultural Research Center (NARC) Islamabad. It was composed of Azotobacter (17%), Bacillus (22%), endosymbiotic rhizobia (16%), Enterobacter (9%), Flavobacterium (13%), Pseudomonas (9%) and Thiobacillus (6%) (Adnan et al., 2022) obtained from National Agricultural Research Center (NARC) Islamabad.

Treatments detail

The experiment was consisted of two factors including two levels of PSB (with and without PSB) and eight phosphorus sources combinations (control, 100% FYM, 100% RP, 75% FYM + 25% SSP, 75% RP + 25% SSP, 50% FYM+50% SSP, 50% RP + 50% SSP and 100% SSP) added at the rate of 100 kg P₂O₅ ha⁻¹.

Experimental procedure

This field study was arranged in factorial (two) split plot randomized complete block design (RCBD) with three replications. PSB was applied to main plots at the rate of 2 kg ha⁻¹ through seed inoculation while, different combinations of P sources into subplot with a size of 3*4 m². The FYM and RP were analyzed for their P content and were applied a month before sowing of maize crop. The basal dose of 140 kg N ha⁻¹ and 80 kg K₂O ha⁻¹ (inclusive of N and K to be received from FYM) were added to each sub plot as urea and sulphate of potash (SOP) respectively. Urea was supplemented in two doses half each at sowing and knee height stage. PSB was applied at the rate of 2 kg ha⁻¹ via seed inoculation techniques. The inoculated seed of cultivar Jalal was sown at the rate of 30 kg seed ha⁻¹ while maintaining the recommended row to row (75 cm) and plant to plant (25 cm) distances. Recommended irrigation schedule was followed as per crop requirements subject to weather condition. Weeds were controlled by using recommended chemical herbicides. Uniform standard cultural practices recommended for field experiment were adopted throughout the growing season.

Agronomic parameters

Plant height was measured from the base to the top by selecting ten plants randomly in each sub plot at maturity stage. Thousand grains were counted from grain harvested in each treatment plot and were weighed using electronic balance. The whole above the ground plants was harvested, air dried and weighed with field balance and transformed into kg ha⁻¹ as follows: ${\displaystyle \text{Total dry matter}\left(\mathrm{kg}{\mathrm{ha}}^{-1}\right)=\frac{\text{Dry matter yield of}\mathrm{a}\text{subplot in kgs}}{\text{Subplot Size}{\mathrm{m}}^2}\times 10,000{\mathrm{m}}^2.}$

For grain yield, the cobs were husked from the plants harvested from each subplot, dried and shelled, and changed to kg ha⁻¹ as follows: ${\displaystyle \text{Grain yield}\left(\mathrm{kg}{\mathrm{ha}}^{-1}\right)=\frac{\text{Grain}\text{yield of}\mathrm{a}\text{subplot in kgs}}{\text{Subplot Size}{\mathrm{m}}^2}\times 10,000{\mathrm{m}}^2.}$

Soil analysis

The soil of the experimental plot was physico-chemically characterized (pH, EC, texture, organic matter, lime and AB-DTPA extractable P contents) before the experiment. While, the post-harvest soil analysis were performed for AB-DTPA extractable P and organic matter contents. The pH of the composite soil sample collected from experimental site was measured in 1:5 soil water suspensions by pH meter (Mclean, 1983). Soil EC (1:5 soil water extract) was measured by electrical conductivity meter (Rhoades, 1982). The texture of soil sample collected from experimental site before the experiment was measured by hydrometer method (Koehler, Moudre & McNeal, 1984). The soil organic matter (SOM) was quantified by adopting the procedure of Nelson & Sommers (1983). Post-harvest P concentration in soil was measured by Soltanpour & Schwab (1977) protocol. Lime content in samples was quantified by acid neutralization method (Leo, 1963).

Plant analysis

The whole above the ground plant samples randomly collected from each sub plot at maturity were examined for P concentration by spectrophotometer using the protocol of Richards (1954). Phosphorus uptake by maize plant in each plot was measured by using the following expression: ${\displaystyle \mathrm{P}\text{uptake}\left(\mathrm{kg}{\mathrm{ha}}^{-1}\right)=\frac{\text{Plant}\mathrm{P}\text{content}}{100}\times \text{Biomass in kg}{\mathrm{ha}}^{-1}.}$

The agronomic phosphorus use efficiency (APUE) was measured as follows: ${\displaystyle \text{APUE}\left(\mathrm{kg}{\mathrm{kg}}^{-1}\right)=\frac{\left(\text{Grain yield of fertilized plot}-\text{Grain yield of control plot}\right)}{\text{Fertilizer}\mathrm{P}\text{applied}}.}$

While, the apparent phosphorus recovery (APR) was measured by the following formula: ${\displaystyle \mathrm{APR}\left(\text{\%}\right)=\frac{\left(\mathrm{P}\text{uptake in fertilized plot}-\mathrm{P}\text{uptake in control plot}\right)}{\text{Fertilizer}\mathrm{P}\text{applied}}\times 100.}$

Economic analysis

The profitability of maize crop in response to applied treatments was carried out by the procedure out lined by CIMMYT (1988).

Statistical analysis

The collected data were run for analysis of variance (ANOVA) suited for two factorial split plot RCBD using Statistix 8.1 (Steel & Torrie, 1980). In case of significant F test, the means were further compared by least significant difference (LSD) test (α = 0.05).

Yield and yield component of maize

Maize plant height was significantly improved by using phosphate solubilizing bacteria (PSB) and P sources (Table 1). Application of PSB was better by 5.6% over no PSB for plant height. Taller plants were observed for plots either treated with 50% P as RP or FYM with 50% P as SSP. Significantly taller plants were recorded when SSP was integrated with other P sources especially RP and FYM at 50:50. In phosphorus sources combination, plant height increased from 143.5 cm to 186.2 cm with 50 FYM + 50% SSP. The increasing trend in plant height was observed at each increment of SSP (%) with the combine application of FYM and RP. The interaction effect of PSB and P sources for plant height was non-significant. However, the increasing trend in plant height was observed at each combination of SSP with FYM. It was shown that 50% FYM + 50% SSP proved better than any other combination with PSB. Application of 50% FYM + 50% SSP with PSB was better by 32.9% over absolute control.

PSB inoculation significantly improved thousand grain weight of maize over without PSB (Table 1). The heavier thousand grains were found in plots treated with PSB (222.5 g) as compared to control (200.5 g), resulting 11.0% improvement in 1,000 grain weight in response to PSB inoculation. Similarly, significant change was found in thousand grain weight among the plots either treated with 50% FYM or RP with 50% of SSP fertilizers. The heavier thousand grain weight 236.8 g was recorded in plots treated with 50% FYM + 50% SSP which as at par to 50% RP + 50% SSP. The lighter (179.3 g) 1,000 grains were observed under control. Plots treated with 50% FYM + 50% SSP maximally increased maize 1,000 grain weight by 32.1% over P plot. The significant interaction of PSB and P sources for 1,000 grain weight indicated that 50% FYM + 50% SSP with PSB was the most appropriate combination representing a maximum increase of 49.1% over absolute control (Fig. 1).

Experimental data clearly revealed that PSB inoculation and P sources considerably improved dry matter, grain yield and stover yield of maize (Table 2). The significant interactive effect was also observed for PSB and P sources on the aforesaid yield and yield components of maize. Sole PSB application significantly improved dry matter, grain yield and stover yield of 7.82%, 10.51% and 6.77% respectively over PSB control (where no PSB was applied). Similarly, data revealed that P application irrespective of their sources improved dry matter, grain yield and stover yield of maize. Specifically, maximum dry matter of 28.92% was observed where P was applied 50% from FYM + 50% from SSP. This was followed by 50% from RP + 50% SSP, 100% from SSP and 75% from FYM + 25% from SSP having an increase of 26.11%, 23.45% and 14.86% over control. Lower dry matter yield was observed for P control. The interaction between PSB × P sources indicates that, greater dry matter yield was obtained when PSB and P as 50% from FYM + 50% from SSP were applied in combination (Fig. 2) exhibiting a maximum increase of 43.5% over absolute control.

Maximum grain yield of 42.61% was observed where P was applied 50% from FYM + 50% from SSP. This was followed by 50% from RP + 50% SSP, 100% from SSP and 75% from FYM + 25% from SSP, having an increase of 35.22%, 33% and 30.47% over control. The performance of 50% P each from RP and SSP, 100% from SSP and 75% from FYM + 25% from SSP were at par however, their effect was significant when compared to remaining treatment combinations. Lower grain yield was observed for P control treatment. The interaction between PSB × P sources indicates that, maximum grain yield was produced when PSB and P as 50% from FYM + 50% from SSP were applied in combination (Fig. 3) representing a 67.4% increase over absolute control.

The treatment receiving P as 50% from FYM + 50% from SSP recorded maximum stover yield of 24.17% over control treatment. This was followed by 50% from RP + 50% SSP, 100% from SSP and 75% from FYM + 25% from SSP, having an increase of 22.96%, 20.16% and 9.49% over control. Phosphorus application as 50% each from FYM and SSP was statistically comparable to 50% each from RP and SSP but those were significantly different from the rest of P sources. Lower stover yield was observed for P control treatment. The interaction between PSB × P sources indicates that, maximum stover yield was produced when PSB and P as 50% from FYM + 50% from SSP were applied in combination (Fig. 4) resulting 35.7% increase over absolute control.

Phosphorus concentration (%) and uptake (kg ha⁻¹)

Data regarding inoculation of PSB together with different sources of P on maize P concentration and uptake is presented in Table 3. The combined inoculation of PSB with different P sources in increasing the P concentration and uptake by maize was highly significant (p ≤ 0.05). Compared to un-inoculated PSB, the maximum plant P concentration of 0.210% having a 10.1% increase over control was observed in the PSB inoculated treatments. Similarly, the high value of P (19.4 kg ha⁻¹) taken by maize plants with 18.6% increase over control was recorded in the PSB applied treatments in comparison to the treatments without PSB. The minimum data for both maize plants P concentration (0.191%) and uptake (16.4 kg ha⁻¹) was found in the treatments without PSB inoculation.

Furthermore, significant (p ≤ 0.05) increase in plant P concentration and uptake by maize plants was also recorded in all the treatments where different P sources were applied in combination with different doses of SSP (Table 3). It is obvious from the data that compared to control the maximum value of both P concentrations (0.247%) and uptake (24.6 kg ha⁻¹) by maize plants was recorded in the treatments where 50% FYM was supplemented together with 50% SSP, respectively. It was followed by the treatment where 100% sole SSP was applied. Similarly, in comparison to control the lowest P concentration of 0.159% with a 16.5% increase over control by maize plants was depicted in the treatments having 100% FYM without any dose of SSP. The minimum P uptake (17.3 kg ha⁻¹) was observed under 75% RP together with 25% SSP. Overall, the role of PSB in inoculated treatments as well as the combination of different sources of P with SSP on P uptake and concentration by maize plants was significant in comparison to control and the treatments without PSB.

The combined effect of PSB and P supplements on maize P uptake is summarized in Fig. 5. The inoculation of PSB together with different rates of SSP and P sources was found non-significant (p ≤ 0.05) in improving plant P concentration but was significant for plant P uptake (Fig. 5). The highest P content (101.1%) and uptake (188.7 kg ha⁻¹) in maize were under 50% SSP + 50% FYM along with PSB inoculation, corresponding to 95% high P content and 170% P uptake in plants over absolute control, respectively. These results were closed to the treatments where 100% SSP was applied alone and in combination with PSB. The un-inoculated PSB treatments were also improved in the presence of different P sources alone and in combination with various rates of SSP. During the interactive effects of various sources and rates of P fertilizers in combination with different rates of SSP, the minimum increase in P concentration of 7.5% and uptake of 18.8 kg ha⁻¹ by maize plants was observed in control, followed by the treatments having sole 100% FYM and RP, respectively. Overall, the recorded trend of PSB inoculation in combination with P sources and SSP in enhancing P concentration and uptake was positive and significant towards maize plants.

Likewise, the statistical analysis of the interactive effects of both PSB with different P sources on maize plants uptake is demonstrated in Fig. 5. Compared to un-inoculated treatments, the interaction of PSB with various P sources and rates along with SSP was effective and significant (p ≤ 0.05) in enhancing the P uptake (kg ha⁻¹) by maize plants. The maximum P uptake (26.5 kg ha⁻¹) in maize was recorded in the treatments, provided the interaction of PSB in combination with 50% SSP and 50% FYM. It was almost equivalent to the treatments having 100% supplemented SSP alone (23 kg ha⁻¹) and 50% RP combined with 50% SSP (21.5 kg ha⁻¹). The minimum P uptake and improvement in maize plants was found in all those treatments having different P sources and various rates of SSP without PSB inoculation. Overall, Compared to control and supplemented 100% FYM and/or 100% RP alone treatments, significant linear increase in P uptake by all maize plants was depicted in the treatments inoculated with and even without PSB along with various sources of organic P fertilizers and different rates of SSP.

Post-harvest extractable P (mg kg⁻¹) and organic matter (%) in soil

Analysis of the data showed a non-significant effect of PSB on soil extractable P (Table 4). However, inoculation improved soil extractable P by 3.1% over control. Soil organic matter (SOM) was shown maximum in those plot to which no PSB was applied. In case of different sources of phosphorus, soil extractable P was significantly affected and maximum soil P was noticed in those plots to which 100% phosphorus was applied only from SSP source, while this was significantly at par with the combine applications of 100% FYM with no SSP supplementation followed by 50% FYM and 50% SSP source. The minimum soil P was recorded at control treatment. The application of SSP have shown maximum increase of 41.2% followed by 100% sole application of FYM 39.9% and 50% FYM+50% SSP have increase of 39.2%. In case of soil organic matter, various combined sources of P significantly affect the soil organic matter. The maximum SOM was observed in those plots to which only 100% of FYM was applied, which was followed by the combine application of 75% FYM and 25% SSP.

The interaction between bio-fertilizers with various phosphorus sources on post-harvest soil AB-DTPA extractable P are indicated in Fig. 6. Integration of PSB with phosphorus source showed significant impact on soil fertility and nutrients availability, The maximum soil extractable P (3.07 mg kg⁻¹) was recorded in those plots to which 100% FYM in combination with PSB was applied representing a maximum increase (46%) over absolute control.

Phosphorous Use efficiency (PUE)

Analysis of variance exhibited a significant influence of PSB and P sources over Agronomic P efficiency (APE-Table 5) and apparent P recovery (APR-Table 6) in maize. PSB inoculation significantly improved APE from 5.2 to 7.7 (kg kg⁻¹) and APR from 6.81 to 10.48 (%) when compared to without PSB inoculated plots. Among the P sources the maximum APE (10.2 kg kg⁻¹) and APR (15.79%) were observed in plots where P was supplemented 50% each from FYM and SSP which was followed by plots treated with 100% SSP and 50% RP + 50% SSP while, the lowest APE and APR were observed in control plots. With respect to APE the sources could be ranked as 50% each FYM and SSP (10.2 kg kg⁻¹) >50% each RP and SSP (8.7 kg kg⁻¹) ≥SSP (8.2 kg kg⁻¹) ≥ 75% FYM + 25% SSP (7.8 kg kg⁻¹) >75% RP +25% SSP (5.5 kg kg⁻¹) >100% FYM (5.3 kg kg⁻¹) >100 RP (4.0 kg kg⁻¹) >control (1.7 kg kg⁻¹) when averaged across the PSB inoculation (Table 5). The similar trend was observed for sources in APR as well (Table 6).

The interaction effect of PSB and P sources was significant for APR (Table 6) while, non-significant for APE. The maximum APE (19.60%) was observed in plots treated with 100 kg P₂O₅ as 50% each from FYM and SSP along with PSB inoculation which was followed by 50% each from RP and SSP + PSB (15.10%) while the lowest APE was observed for plots solely treated with RP (1.66%). The performance of solely applied PSB was even superior than sole RP treated plots. Furthermore, The APE of 100% FYM with PSB was significantly better than 100% RP with and without PSB and sole application of FYM, and was at par to plots treated with 75%P as FYM and 25% as SSP. PSB were effective in enhancing APE regardless of the sources used however, its role was more pronounced when P was either supplemented as FYM or RP compared to SSP. SSP was observed as a most recoverable source without PSB inoculation.

Economic analysis

Data concerning value cost analysis of the study (Table 7) showed a maximum yield return of PKR 150767 ha⁻¹ with a value cost ratio (VCR) of 3.1 for plots amended with 100 kg P₂O₅ ha⁻¹ as 50% each from FYM and SSP along with PSB. The VCR value of P applied as 50% each from FYM and SSP along with PSB was similar to those of 75% FYM + 25% SSP + PSB (Rs. 137186 ha⁻¹) and 100% FYM (Rs. 120648 ha⁻¹) however their net return was lower than 50% P as FYM + 50% SSP + PSB while the lowest VCR was recorded for absolute control plots. These observations have proven that under PSB inoculation the organic manure applied alone or in integration to mineral fertilizers are comparatively more economical than those of solely applied mineral fertilizers either with or without PSB inoculation. PSB inoculation improved VCR value regardless of the P sources used however, it impact was more pronounced in organic sources than chemical fertilizers. Even under control P the PSB inoculation improved VCR value from 2.54 to 2.98.