Introduction

Sesame (Sesamum indicum L.) belongs to Pedaliaceae family. Among the worldwide ancient oilseed crops, sesame is one of them (Shin et al. 2016). In Pakistan, sesame cultivation area is 83 thousand hectare with an annual production and an average yield of 35.7 thousand tones and 430 kg per hectare, respectively (Economic Survey of Pakistan 2018–2019). A number of environmental factors such as rainfall fluctuation, conventional farming operations, expensive fertilizers, limited use of certified seeds, poor crop management practices, insects-pests attack all together severely affect the production and yield of the crop. Among them, a good supply of plant macro-nutrients are also necessary. Delete the line (In addition to nitrogen and potassium, phosphorus is also very important for plant growth).

Phosphorus (P) is also very important nutrient for plant growth along with potassium and nitrogen. It contributes in plant dry weight ranges from 0.3 to 0.5%. Moreover, it is associated with various plant development features such as consumption of starch and sugar, cell division and organization, photosynthesis, formation of fat, albumin and nucleus, and heredity material transmit (Lai 2002). Its deficiency results in reddish color development followed by retarded growth of the plant. However, darker green color appears under limited Phosphorus supply. Photosynthetic rate becomes lower under P-deficient conditions which reduce metabolic processes (Malhotra et al. 2018). Pakistani soils are calcareous and alkaline in nature. Therefore, sesame suffers deficiency of phosphorus and almost 80–90% soils of semiarid and arid regions are deficient in plant available phosphorus because of precipitation, adsorption and its transformation to organic forms. P-contents are 0.05% (w/w) in soil; of which just 0.1% is available to plant (Nash et al. 2014; Rodrigues et al. 2016).

Phosphorus (P) is present as an insoluble P-compound that are unavailable to plant in most of the soils. Chemical or synthetic fertilizers are used in bulk amounts to replenish the soil mineral nutrients, but the problem is that fertilizers having phosphorus either get adsorbed or precipitated in the soil (Fayiga and Nwoke 2016). According to an estimate, rock phosphate (from which mineral P fertilizers are derived) reserves of the world are becoming scarce (Jasinski 2006) and they will be exhausted within fifty to hundred years. Moreover, RP (rock phosphate) cost is increasing and its quality is decreasing day by day (Zhu et al. 2018) which means there will be restricted supply of good quality fertilizers of Phosphorus (Cordell 2008). Demand of agricultural commodities and pressure on available land resources is increasing in developing countries to fulfill food requirements (Weber et al. 2014). This pressure (called as potential phosphate crisis) is causing an increase in prices of fertilizers globally (Chowdhury et al. 2017).

In order to combat these issues, the inoculation of the microbes is now being explored throughout the world for their capability to solubilize unavailable P-sources (organic as well as inorganic) for sustainable agriculture. The free living bacteria which also inhabit in the roots of the plant and promoting the plant growth are considered as PGPR (plant growth-promoting rhizobacteria) (Mehmood et al. 2018). According to Mantelin and Touraine (2004), PGPR affect growth of the plant (either directly or indirectly) through various mechanisms. They can facilitate plant growth through atmospheric nitrogen fixation (Glick 2012), improvement in enzyme activities (Dakora and Phillips 2002), mineral solubilization, siderophore production (Khan and Ahemad 2012), nutrient cycling like potassium (K), nitrogen (N), sulfur (S) and phosphorus (P) etc. (Walker et al. 2003), decomposition of organic wastes, plant hormone synthesis and through regulation of ACC-deaminase activity which alter plant growth and development (Odoh 2017).

Enzymatic activities of microbes have a crucial role in soil-biochemical functioning including formation and degradation of organic matter, aggregation of soil structure, nutrient cycling, decomposition of wastes especially organic, In catalysis of several reactions considered necessary for various life processes in soil and in providing an early indication of soil history (Adetunji et al. 2017).

Soil phosphatases, predominantly synthesized by bacteria, help in the hydrolysis of soil organic Phosphorus and increases its available fraction (Platkowski and Telesinski 2016). Due to this breakdown, soil organic phosphorus is converted into inorganic forms and can be absorbed by roots of the plants from the soil solution (Lemanowicz et al. 2016). Microbes, as well as higher plants, are known for the production of acid phosphatase (at low pH 6.5 optimal activities can occur) (Zhu et al. 2018) but microorganisms are specific to produce alkaline phosphatase (optimal activities can occur at high pH 11) (Nannipieri et al. 2011). According to Margalef et al. (2017), both plant and microbial phosphatases regulate the release of orthophosphate ions (HPO^-₄ and H₂PO^-₄) from soil organic phosphorus effectively, with some implications that microbially produced phosphatases indicate greater efficacy in P-releasing.

Moreover, soil microorganisms having ACC-deaminase activity; improve root elongation by lowering levels of ethylene by conversion of ACC into α-ketobutyrate and NH₃ in plant roots (Nadeem et al. 2010). This increase in root growth increases P-uptake and plant growth. Consequently, P-availability increases in the presence of phosphatase producing bacteria having ACC-deaminase activity which ultimately improves yield.

Sesame biochemical parameters (seed oil and seed protein content) are also influence by phosphorus along with growth and yield. So, a field study was carried out to assess the potential of plant growth promoting rhizobacteria linked with phosphorus availability coupled with ACC-deaminase activity for improving growth, yield and oil content of sesame.

Materials and Methods

Physiochemical characteristics of the soil like particle size, pH, CEC, EC_e, saturation percentage (%) were measured by following the standard procedures (U.S. Salinity Lab. Staff 1954; Page 1965). The complete methodology to determine the soil properties has been detailed in Rehman et al. (2015). Walkley-Black method (Walkley and Black 1934; FAO 1974) was used to determine the organic matter of the soil

Isolation, purification and screening

Bacteria were isolated from rhizosphere of sesame. Samples were taken randomly from different fields located at district Faisalabad. For the collection of the rhizospheric soil, plants were uprooted, packed in polythene bags and brought to laboratory. The roots of the plant were gently agitated to remove the soil that was loosely adhering to them.

More than a hundred rhizobacteria were isolated following the dilution plate technique (Wollum 1982) using Luria Bertani (LB) agar media. Out of hundred, 62 rhizobacteria were selected based on their fast growth in vitro for further characterization. From the sixty-two (62) bacterial isolates, nineteen isolates were found positive (+ve) for P-solubilization (both on solid and liquid media) and mineralization (phosphatase activity). Ten rhizobacteria showed the ACC-deaminase activities. These ACC-deaminase producing rhizobacterial isolates also had ability to carry out solubilization and mineralization of P. A jar trial was conducted to evaluate the performance of plant growth promoting bacterial isolates under axenic condition. Out of ten, role of five best isolates in plant growth and yield promotion was further evaluated through a pot trial (Data not shown). Moreover, a field study was also conducted to assess the activity of best bacterial isolates under natural conditions.

Characterization of bacterial strains

Qualitative P-solubilization potential of bacterial isolates was performed by following Mehta and Nautiyal (2001) on National Botanical Research Institute’s Phosphate (NBRIP) growth agar medium and halo zones around the bacterial colonies were considered as positive for P-solubilization. The solubilization index (SI) was calculated by the formula of Premono et al. (1996).

For quantitative P-solubilization test, NBPIR broth was inoculated with bacterial isolates and incubated in rotary shaker. Broth culture was harvested by centrifugation Inorganic P of the supernatant was assessed according to the method described by Fiskie and Subbarao (1925).

According to Eivazi and Tabatabai (1977), phosphatase activity (alkaline/acidic) of rhizobacteria was measured. Phosphatase enzyme acts as a catalyst in hydrolysis of colorless p-Nitrophenyl phosphate (pNPP) to form p-Nitrophenol. Activity of enzyme was directly proportional to the rate of absorbance at 405 nm wavelength.

In vitro qualitative test for ACC metabolism was performed according to the method of Shaharoona et al. (2006). PGPR strains were grown on growth medium containing ammonium sulfate (0.1 M) and ACC (3 mM). Growth of strains on both nitrogen sources was compared. Magnesium sulfate (0.1 M) was used as control. Optical density was measured at 535 nm after 0, 24, 48, 72 and 96 h of incubation. Quantitative evaluation of ACC-deaminase activity was performed following the method given by Penrose and Glick (2003). ACC deaminase breaks down ACC to α‐ketobutyrate. The amount of α-ketobutyrate (µmol) produced was determined by comparing the absorbance to a standard curve at 540 nm (Table 1).

Inoculum preparation and seed coating

Inoculum was prepared by growing the rhizobacterial strains in conical flasks having 250 mL Luria Bertani (LB) broth media by incubating at 28 ± 1ºC in the orbital shaking incubator (Firstek Scientific, Tokyo, Japan) with 120 rev. min^-1 for 48–72 h. An optical density of 0.5 was adjusted prior to seed inoculation. Seeds were surface sterilized by dipping in ethanol (95%) for few moments then washed them (3–4 times) with sterilized distilled water, followed by immersing in 5% NaClO₄ solution for 3–5 min following thorough washings with sterilized distilled water. For coating of seed, inoculum of respective strain having 10⁸–10⁹ CFU mL^-1 (inoculum to peat ratio, 1:1 v/w) and sugar solution (10%) was mixed. For un-inoculated control, seeds were coated with the autoclaved peat, sugar solution and sterilized LB broth. Inoculated seeds were dried in open air for 6–8 h under shade and then finally used for sowing.

Field trial

Experimental site: A field experiment was conducted at the Research area of Institute of Soil and Environmental sciences ISES), University of Agriculture (UAF), Faisalabad during Kharif 2017. In Pakistan, Kharif starts between March and July and it finishes at the end of autumn or beginning of winter.

Experimental design and treatments: The statistical design was randomized complete block design (RCBD), replicated three times. There were six treatments, one treatment was kept under control (without inoculation) and the remaining treatments were five plant growth promoting rhizobacterial strains (AA-06, AA-18, AA-27, AA-39 and AA-45). The experimental area of one kanal with dimensions 100 feet × 51 feet was used for the execution of field trial. In each plot, there were four rows 1 feet apart; P × P distance 6 inch was maintained after thinning.

Crop husbandry

To prepare a fine level field two ploughings followed by 2 planking’s were carried out to ensure even seed germination. Inoculum of respective bacterial strains was prepared as has been described earlier. Sesame seeds were surface-disinfected and inoculated by using the slurry method as explained above. The sesame crop was seeded (variety, TS-3; seed rate, 250 g) with a manual hand drill.

All other agronomic practices and cultural operations were uniform during the whole crop period. As per recommendation, nitrogen (50 kg per hectare) was applied in two splits (First, at sowing time; Second, at flowering stage) whereas the recommended phosphorus (P) and potassium (K) fertilizers at the rate of 60:45 kg per hectare, respectively, were applied at the time of sowing. Weeds were controlled through hand weeding. To control the insects/pests attack (white fly and jassid), imidacloprid (Syngenta Pakistan Limited) was used. For controlling the leaf roller disease of sesame (due to sesame leaf roller moth), Karate (Syngenta Pakistan Limited) was used. Two irrigations (canal water) were applied during the full crop period (First: after 20–25 days; second: after 40–50 days).

Harvesting and data recording

At maturity, ten plants from each experimental plot were randomly selected to record the growth and yield variables: plant height, branches per plant, capsule length, capsules per plant, and seeds per capsule. The crop was harvested (after 112 days) by using meter square in each plot and shoot fresh weight was taken by using electric balance. The harvested plants were sun dried for two weeks. The dried samples were weighed on an electric balance, they were manually threshed and well cleaned to remove the inert matter and to record the seed yield. From each plot, three (3) sub-samples of 1000 seeds were taken to record the 1000-seed weight. The seed samples were further transferred to the laboratory in zip lock bags for quality analysis.

Seed oil and protein contents measurement

Crushed sesame seed sample (12 g) was mixed with N-hexane (150 mL), placed on folded filter paper and inserted in the assembled Soxhlet apparatus. Filter paper and sample weight were noted. N-hexane (solvent) was taken in a 500 mL flask and heated at 600^°C. The hot vapors of solvent (in a reflux condenser) were cooled by water flowing through the Soxhlet arrangement. In the Soxhlet portion with the folded sample, the cooled solvent was condensed back to facilitate oil extraction. Extracted sample was a combination of solvent and oil. The oil was removed, and the remaining sample was hot-pressed by using a hydraulic press which ultimately removed the remaining oil from press cake. The sample was weighed and oil yield (%) was calculated.

Nitrogen was determined by the Kjeldahl method (AOAC 1990). Total protein was measured by using a formula as described by James (1995).

Total protein (%) = N (%) × 6.25

Bacterial Identification

Rhizobacterial isolates (AA-06, AA-18, AA-27, AA-39 and AA-45) were identified by sequencing their 16S rRNA from Macrogen, Korea. Bacterial isolates were identified as Pseudomonas fluorescens (AA-06), Pseudomonas spp. (AA-18), Psychrobacter spp. (AA-27), Bacillus spp. (AA-39) and Bacillus aquimaris (AA-45).

Statistical analysis

The impact of treatments on all growth, physiological and yield parameters was studied by statistical software Statistic 8.1. Linear models were applied. For comparing the treatments, analysis of variance (ANOVA) was used. Least significant difference (LSD) test at 5% probability level test was applied to observe the difference in treatments (Montgomery 2001).

Results

Characterization

The bacterial strains were characterized for various biochemical characteristics (Table 1). P-solubilization potential (qualitative) results showed that maximum PSI (6.25) was observed with isolate AA-6 (P. fluorescens) and minimum PSI (3.94) by AA-27 (Psychrobacter spp.). Data regarding the quantitative phosphorus solubilization potential exhibited that those bacterial strains which were tested on solid media (P-solubilization) were also positive when tested on liquid media for P-solubilization. Moreover, variations in phosphate solubilization (which ranged from 525.29 to 731.63 µg mL^-1) of inorganic P-source were recorded for these strains.

The results of laboratory test for phosphatase activity revealed that strains of bacteria, present in cultured broth medium, expressed both acidic and alkaline activities of phosphatase. However, in case of inoculation with AA-18 (Pseudomonas spp.), maximum activity of acid phosphatase (41.65 µg PNP g^-1 h^-1) was recorded. The alkaline phosphatase activity ranging from 14.35 to 27.59 µg PNP g^-1 h^-1 was observed for these five rhizobacterial strains.

Qualitative ACC-deaminase activity was measured based on the optical density of bacterial growth on ACC (1-aminocyclopropane-1-carboxylate) as a substrate. The maximum and minimum OD values were noted due to inoculation with bacterial strain AA-27 (Psychrobacter) and AA-45 (B. aquimaris), respectively. When ACC-deaminase activity was recorded quantitatively for these strains, varying efficacy degrees for ACC-deaminase activity (ranging from 334 to 355 nmol α-ketobutyrate g^-1 biomass h^-1) were observed.

Growth parameters

Inoculation with PGPR strains proved significantly efficient in improving the height of the sesame plant in the field. Upon inoculation with AA-45 (B. aquimaris), the maximum height of the plant was recorded. While, in case of inoculation with strain AA-27 (Psychrobacter spp.), 11% increase in height of plant was observed than uninoculated control. There was a significant difference in plant height due to the effect of inoculation over control but statistically at par with each other. Results showed that the fresh weight of shoot was significantly increased due to bacterial inoculation. The highest fresh weight of shoot (25%) was observed with the inoculation of AA-06 (P. fluorescens) than the control treatment while the lowest (5%) in response to strain AA-39 (Bacillus spp.) inoculation when compared with uninoculated control.

Table 1: Characterization of bacterial strains

Fig. 1: Effect of PGPR on capsule length, capsules per plant, seeds per capsule and 1000

Grain weight of sesame crop under field trial (Average of three replicates)

Similarly, rhizobacterial inoculation significantly enhanced the shoot dry weight over non-inoculation. Increase in shoot dry weight (16%) was noted upon inoculation with AA-06 (P. fluorescens) than control and 14% increase was found for AA-18 (Pseudomonas spp.) strain. A significant effect of bacterial inoculation on the number of branches per plant compared to uninoculated control was observed. Among the rhizobacterial strains, AA-06 (P. fluorescens) was found to be more effective and caused a 43% increase in branches per plant compared with control treatment (Table 2).

Study revealed that a significant increase in capsule length was observed upon bacterial inoculation in comparison with uninoculated control. Maximum increase in capsule length by 52% was recorded for strain AA-27 (Psychrobacter spp.) while the minimum response (13%) was observed upon inoculation with AA-39 (Bacillus spp.) in comparison with uninoculated control. The results exhibited that almost all rhizobacterial strains produced higher numbers of capsules plant^-1 than uninoculated control. Inoculation with AA-18 (Pseudomonas spp.) caused 48% increase in capsule number per plant than the control treatment. Bacterial strain AA-39 (Bacillus spp.) resulted in a minimum increase in capsules plant^-1 in comparison to all other strains. However, it was still significant than uninoculated control (13% more).

Data regarding effect of inoculation on seeds per capsule was showed that seeds per capsule was increased (47%) upon inoculation with AA-27 (Psychrobacter spp.) followed by AA-06 (P. fluorescens) that caused up to 38% increase in the number of seeds per capsule than uninoculated control. While, upon inoculation with rhizobacterial strain AA-39 (Bacillus spp.), the least effect (up to 12% increase in seed number per capsule) was obtained over uninoculated control. The data indicated that upon inoculation with AA-27 (Psychrobacter spp.), a maximum 1000 grain weight was recorded and that was 12% more than control. The effect of different bacterial strains was at par with each other statistically but significantly different from control (Fig. 1).

Table 2: Effect of PGPR with enzymatic activity on growth parameters of sesame crop under field conditions (average of three replicates)

Means sharing the same letter (s) are statistically non-significant at 5% probability level (n-5). LSD shows least significant difference

Table 3: Effect of PGPR with enzymatic activity on seed/biological/straw and harvest index of sesame crop under field conditions (average of three replicates)

Means sharing the same letter (s) are statistically non-significant at 5% probability level (n-5). LSD shows least significant difference

Yield parameters

Regarding seed yield, the increase in seed yield by inoculation with rhizobacteria (with enzymatic activity) ranged from 8 to 37% over control while the strain AA-06 (P. fluorescens) resulted in 37% more seed yield (maximum) than control. Upon inoculation with bacterial strain AA-06 (P. fluorescens), a maximum increase of almost 25% in biological yield was observed than control treatment. However, AA-39 (Bacillus sp.) inoculation caused just a 5% increase in biological yield than control.

Moreover, inoculation with bacterial strains also enhanced straw yield of sesame and the maximum yield was recorded in response to inoculation with AA-06 (P. fluorescens) (24% over control). The next effective strain was AA-18 (Pseudomonas sp.) that caused a 21% increase in straw yield over control. On the other hand, in the harvest index, the minimum increase was recorded upon inoculation with AA-39 (Bacillus spp.) compared with other strains but it was still significantly better than control treatment (Table 3).

Fig. 2: Effect of PGPR on seed oil content, seed oil yield, seed protein and seed protein

Yield of sesame crop under field trial (Average of three replicates)

Biochemical parameters

Field study revealed an increase in seed oil content upon inoculation with bacterial strains than control treatment. Maximum oil content was observed in plants inoculated with AA-27 (Psychrobacter spp.) strain as compared to control. While minimum increase (3%) was observed upon inoculation with AA-39 (Bacillus spp.) as compared to non-inoculated plants. Regarding the seed oil yield parameter, significant results were obtained for all rhizobacteria compared to control but the bacterial strain AA-06 (P. fluorescens) was the most effective strain for increasing the oil yield (47% more than un-inoculated control).

It was observed that upon inoculation with rhizobacterial strains, a significant improvement in seed protein content was recorded. Maximum increase in protein content was obtained upon inoculation with strain AA-06 (P. fluorescens) (almost 23%) as compared to control. Whereas minimum increase was noted upon inoculation with AA-27 (Psychrobacter spp.) that resulted in 10% increase in protein content over control. Rhizobacterial strain AA-06 (P. fluorescens) inoculation resulted in increased seed protein yield by 62% higher than control (Fig. 2).

Discussion

Diverse plant-soil microbe interaction takes place in the rhizosphere which is the most active region of the soil. Rhizospheric bacteria have been well documented for their numerous beneficial effects on growth and plant production through various mechanisms (direct or indirect) (Vejan et al. 2016). The present experiment revealed the efficacy of PGPR in improving the sesame growth, yield and oil content through their ability to solubilize and mineralize phosphorus coupled with ACC-deaminase activity under field conditions.

Our in vitro tests showed that almost all the P-solubilizers with their phosphatase activity were mineralizing/solubilizing organic and inorganic phosphorus which is supported by Susila et al. (2016) who reported that bacteria can have mineralization and solubilization (inorganic phosphorus) activities. Similary, Alori et al. (2017) reported that under in vitro conditions, isolated rhizobacteria from different crops that solubilize the inorganic phosphate were also able to produce phosphatase enzyme. Therefore, the higher phosphorus solubilization may be due the enhanced production of phosphatase enzymes produced by bacterial strains. In vitro results revealed both acid and alkaline phosphatase activities for most of the rhizobacteria. For example, many researcher have studied the rhizobacterial inoculation effect on the production of phosphatases activity and their results indicated that the bacterial strain which was grown on organic phosphorus compounds (as a source of phosphate), has the capability to produce acid phosphatase as well as an alkaline phosphatase in a liquid medium having organic phosphorus as a substrate (Behera et al. 2017; Sang et al. 2018).

In present experiment, the bacterial strains exhibited ACC-deaminase activity with varying degrees both qualitatively and quantitatively. The obtained results were similar to those recorded by Shameer and Prasad (2018) that some of the rhizobacteria that solubilize phosphate also have the ACC-deaminase activity which promotes plant growth.

The results of our field experiment indicated that all the rhizobacterial strains improved the growth, yield and biochemical parameters of sesame as compared with control treatment that might be due to the greater microbial activities which resulted in solubilization of the fixed forms of phosphorus (inorganic P) in the soil or they might have mineralized the organic matter to release organically bound phosphorus (Richardson and Simpson 2011; Anand et al. 2016; Bechtaoui et al. 2020). Supply of phosphorus is usually related to increased root proliferation and density leading to more exploration and supply of water and nutrients to the plant roots that ultimately increased the growth and yield and as a result more grain and dry matter yield is ensured (Kang et al. 2014). In the same way, Nkaa et al. (2014) reported that with the application of rhizobacterial strains that have potential to solubilize inorganic P as well as mineralize organic P compounds, cowpea (Vigna unguiculata L.) grain numbers pod^-1, leaves number plant^-1 and seed yield ha^-1 were increased. In another study, field inoculation with phosphate solubilizing bacteria which enabled rapeseed growth promotion and crop yield (seed and biological yield) increased, minimizing the use of chemical fertilizers and contributing to the development of sustainable agriculture (Valetti et al. 2018; Tahir et al. 2018).

In the present study, the improvement in plant growth may also be associated with the ACC-deaminase activity ability of bacterial strains that can help the plant for phosphorus acquisition indirectly by enhancing root growth. However, an increase in root growth may be helpful in improving P-uptake. Mukhtar et al. (2020) found that 54% phosphorus solubilizing strains were positive in ACC-deaminase activity.

Results of field trial revealed that the microbial inoculation significantly improved the oil and protein content of the sesame plant. This improvement might be due to production of phosphatase enzyme by microorganisms which assisted in solubilizing the insoluble organic and inorganic P-compounds (Omer and Abd-Elnaby 2017). Phosphorus availability to the plants play an important role in root growth, formation and translocation of carbohydrates, resistance against disease pathogens and crop maturation that ultimately causes an increase in seeds capsule^-1, seed yield, capsules number plant^-1, sesame protein and oil content (El-Fawy and El-Said 2018).

In line with our findings, Kutlu et al. (2019) described that microbially inoculated plants exhibited more oil content than uninoculated control. Okon and Labandera-Gonzales (1994) also stated that inoculation of bacteria that produce phosphatase along with ACC-deaminase activity resulted in an increasing plant biomass, oil and protein content in a pot trial as a result of enhanced plant P acquisition indirectly by increasing root growth. The study showed that bacteria possessing multi-traits like P solubilization, phosphatase production along with ACC-deaminase activity may be more efficient PGPR in improving growth, yield parameters and oil content in sesame crop.

Similarly, efficacy of multi-traits PGPR was revealed by Minaxi et al. (2012), rhizobacterial strain (Bacillus spp.) having numerous beneficial characters including IAA, ammonia production, ACC-deaminase activity, antifungal activity and P-solubilization. In this study, it was observed that bacteria exhibiting plant growth promoting characters in axenic conditions were more prominent to improve the growth and yield parameters of sesame under field conditions which could likely be linked with multi-traits functions of PGPR.

Conclusion

The PGPR having potential to solubilize the organic and inorganic P compounds in addition to ACC-deaminase activity isolated from rhizospheric soil, can be helpful in improving sesame growth, development and yield through the mobilization of P (from both inorganic and organic P-sources) by organic acids and phosphatase production. It was depicted from this study that multifaceted bacteria may be an efficient and reliable biological product for the improvement in crop growth and yield under field conditions.

Author Contributions

Faiza designed and carried out the experiments. She performed the analysis, processed the experimental data, interpreting the results, and wrote the manuscript. Hafiz Naeem Asghar devised the study, the main conceptual ideas, and proof outline, He contributed to the design and implementation of the research. He also aided in analysis, results interpretation, and the writing of the manuscript. Prof. Dr. Zahir Ahmad Zahir and Dr. Muhammad Shahid provided critical feedback and helped shape the research, analysis and manuscript.

References

Adetunji AT, FB Lewu, R Mulidzi, B Ncube (2017). The biological activities of β-glucosidase, phosphatase and urease as soil quality indicators: A review. J Soil Sci Plant Nutr 17:794–807

Alori ET, BR Glick, OO Babalola (2017). Microbial phosphorus solubilization and its potential for use in sustainable agriculture. Front Microbiol 8; Article 971

Anand K, B Kumari, MA Mallick (2016). Phosphate solubilizing microbes: An effective and alternative approach as biofertilizers. J Pharm Sci 8:37‒40

AOAC - Association of Official Analytical Chemists (1990). Official Methods of Analysis: Changes in Official Methods of Analysis Made at the Annual Meeting, Vol. 15. Arlington, Virginia, USA

Bechtaoui N, A Raklami, L Benidire, AI Tahiri, M Göttfert, K Oufdou (2020). Effects of PGPR co-inoculation on growth, phosphorus nutrition and phosphatase/phytase activities of faba bean under different phosphorus availability conditions. Pol J Environ Stud 29:1557‒1565

Behera BC, H Yadav, SK Singh, RR Mishra, BK Sethi, SK Dutta, HN Thattoi (2017). Phosphate solubilization and acid phosphatase activity of Serratia spp. isolated from mangrove soil of Mahanadi river delta, Odisha, India. J Genet Eng Biotechnol 15:169‒178

Chowdhury RB, GA Moore, AJ Weatherley, M Arora (2017). Key sustainability challenges for the global phosphorus resources, their implications for global food security, and options for mitigation. J Clean Prod 140:945‒963

Cordell D (2008). 8 reasons why we need to rethink the management of phosphorus resources in the global food system. In: The Story of P Information Sheet 1. Linköping University, Sweden

Dakora FD, DA Phillips (2002). Root exudates as mediators of mineral acquisition in low-nutrient environments. Plant Soil 245:35‒47

Economic Survey of Pakistan (2018–2019). Ministry of Finance, Agriculture, Federal Bureau of Statistics, Islamabad, Pakistan

Eivazi F, MA Tabatabai (1997). Phosphatases in soils. Soil Biol Biochem 9:167‒172

El-Fawy MM, MAA El-Said (2018). Effect of foliar application of some zinc and phosphorus sources on controlling helminthosporium leaf spot disease and production of sesame. J Plant Prot Pathol 9:201‒207

FAO (1974). The euphrates pilot irrigation project: Methods of soil analysis, p:74. In: Gabed Soil Laboratory (A Laboratory Manual), Food and Agriculture Organization, Rome, Italy

Fayiga AO, OC Nwoke (2016). Phosphate rock: Crigin, importance, environmental impacts, and future roles. Environ Rev 24:403‒415

Fiskie CH, Y Subbarow (1925). A colorimetric determination of phosphorus. J Biol Chem 66:375‒400

Glick BR (2012). Plant Growth-promoting Bacteria: Mechanisms and Applications. Hindawi Publishing Corporation, London, UK

James CS (1995). Analytical Chemistry of Foods. Springer, The Netherlands

Jasinski SM (2006). Phosphate Rock, Statistics and Information. US Geological Survey 988, National Center, Reston, Virginia, USA

Kang LY, SC Yue, SQ Li (2014). Effects of phosphorus application in different soil layers on root growth, yield, and water-use efficiency of winter wheat grown under semi-arid conditions. J Integr Agric 13:2028‒2039

Khan MS, M Ahemad (2012). Effect of fungicides on plant growth promoting activities of phosphate solubilizing Pseudomonas putida isolated from mustard (Brassica compestris L.) rhizosphere. Chemosphere 86:945‒950

Kutlu M, R Cakmakci, A Hosseinpour, H Karagöz (2019). The use of plant growth promoting rhizobacteria (PGPR)’s effect on essential oil rate, essential oil content, some morphological parameters and nutrient uptake of Turkish oregano. Appl Ecol Environ Res 17:1641‒1653

Lai L (2002). Phosphatic biofertilizers, pp:221‒224. Agrotech Publ. Academy, Udaipur, India

Lemanowicz J, A Bartkowiak, B Breza-Boruta (2016). Changes in phosphorus content, phosphatase activity and some physicochemical and microbiological parameters of soil within the range of impact of illegal dumping sites in Bydgoszcz (Poland). Environ Earth Sci 75:510–523

Malhotra H, Vandana, S Sharma, R Pandey (2018). Phosphorus nutrition: Plant growth in response to deficiency and excess. In: Plant Nutients and Abiotic Stress Tolerance, pp:177‒190. Hasanuzzaman M, M Fujita, H Oku, K Nahar, B Hawylak-Nowak (Eds.). Springer, Singapore

Mantelin S, B Touraine (2004). Plant growth promoting bacteria and nitrate availability: Impacts on root development and nitrate uptake. J Exp Bot 55:27‒34

Margalef O, J Sardans, M Fernández-Martínez, R Molowny-Horas, IA Janssens, P Ciais, D Goll, A Richter, M Obersteiner, D Asensio, J Peñuelas (2017). Global patterns of phosphatase activity in natural soils. Sci Rep 7; Article 1337

Mehmood U, M Inam-ul-Haq, M Saeed, A Altaf, F Azam, S Hayat (2018). A brief review on plant growth promoting Rhizobacteria (PGPR): A key role in plant growth promotion. Plant Prot 2:77‒82

Mehta S, CS Nautiyal (2001). An efficient method for qualitative screening of phosphate-solubilizing bacteria. Curr Microbiol 43:51‒56

Minaxi LN, RC Yadav, J Saxena (2012). Characterization of multifaceted Bacillus spp. RM-2 for its use as plant growth promoting bioinoculant for crops grown in semi-arid deserts. Appl Soil Ecol 59:124‒135

Montgomery DC (2001). Design and Analysis of Experiment, 5^th Edition. By John Wiley & Sons, New York, USA

Mukhtar S, M Zareen, Z Khaliq, S Mehnaz, KA Malik (2020). Phylogenetic analysis of halophyte‐associated rhizobacteria and effect of halotolerant and halophilic phosphate‐solubilizing biofertilizers on maize growth under salinity stress conditions. J Appl Microbiol 128:556‒573

Nadeem SM, I Hussain, M Naveed, HN Ashgar, ZA Zahir, M Arshad (2010). Performance of plant growth promoting rhizobacteria containing ACC-deaminase activity for improving growth of maize under salt-stressed conditions. Pak J Agric Sci 43:114‒121

Nannipieri P, L Giagnoni, L Landi, G Renella (2011). Role of phosphatase enzymes in soil. In: Phosphorus in Action, pp:215‒243. Springer, Berlin, Heidelberg University, Germany

Nash DM, PM Haygarth, BL Turner, LM Condron, RW McDowell, AE Richardson, M Watkins, MW Heaven (2014). Using organic phosphorus to sustain pasture productivity: A perspective. Geoderma 221:11‒19

Nkaa F, OW Nwokeocha, O Ihuoma (2014). Effect of phosphorus fertilizer on growth and yield of cowpea (Vigna unguiculata L.). J Pharm Biol Sci 9:74‒82

Odoh CK (2017). Plant growth promoting rhizobacteria (PGPR): A bioprotectant bioinoculant for sustainable agrobiology. A review. Intl J Adv Res Biol Sci 4:123‒142

Okon Y, CA Labandera-Gonzales (1994). Agronomic applications of Azospirillum: An evaluation of 20 years worldwide field inoculation. Soil Biol Biochem 26:1591‒1601

Omer A, A Abd-Elnaby (2017). Effect of phosphate dissolving bacteria on physiological behavior of some sesame cultivars under saline conditions at Sahle Eltina-North Sinai. Alex Sci Exchang J 38:687‒698

Page A (1965). Methods of soil analysis. Part 2. Chemical and microbiological properties. Agronomy Monograph 9.2. ASA, SSSA, Madison, Wisconsin, USA

Penrose DM, BR Glick (2003). Methods for isolating and characterizing ACC deaminase containing plant growth-promoting rhizobacteria. Physiol Plantarum 118:10‒15

Płatkowski M, A Telesiński (2016). Response of soil phosphatases to glyphosate and its formulations–roundup (laboratory conditions). Plant Soil Environ 62:286‒292

Premono ME, AM Moawad, PLG Vlek (1996) Effect of phosphate-solubilizing Pseudomonas putida on the growth of maize and its survival in the rhizosphere. Indonesian J Crop Sci 11:13‒23

Rehman MZ, M Rizwan, A Ghafoor, A Naeem, S Ali, M Sabir, MF Qayyum (2015). Effect of inorganic amendments for in situ stabilization of cadmium in contaminated soils and its phyto-availability to wheat and rice under rotation. Environ Sci Pollut Res 22:16897‒16906

Richardson AE, RJ Simpson (2011). Soil microorganisms mediating phosphorus availability update on microbial phosphorus. Plant Physiol 156:989‒996

Rodrigues M, PS Pavinato, PJA Withers, APB Teles, WFB Herrera (2016). Legacy phosphorus and no tillage agriculture in tropical oxisols of the Brazilian savanna. Sci Tot Environ 542:1050‒1061

Sang MK, JJ Jeong, J Kim, KD Kim (2018). Growth promotion and root colonisation in pepper plants by phosphate‐solubilising Chryseobacterium spp. strain ISE14 that suppresses Phytophthora blight. Ann Appl Biol 172:208‒223

Shaharoona B, M Arshad, ZA Zahir (2006). Effect of plant growth promoting rhizobacteria containing ACC-deaminase on maize (Zea mays L.) growth under axenic conditions and on nodulation in mung bean (Vigna radiata L.). Lett Appl Microbiol 42:155‒159

Shameer S, TNVKV Prasad (2018). Plant growth promoting rhizobacteria for sustainable agricultural practices with special reference to biotic and abiotic stresses. Plant Growth Regul 84:603‒615

Shin BR, HW Song, JG Lee, HJ Yoon, MS Chung, YS Kim (2016). Comparison of the contents of benzo (a) pyrene, sesamol and sesamolin, and volatiles in sesame oils according to origins of sesame seeds. Appl Biol Chem 59:129‒141

Susila KD, IM Sudana, NP Ristiati, IM Adnyana (2016). Phosphatase activity and phosphate solubility by phosphate solubilizing rhizobacteria in volcanic soils of Pancasari, Bali. Intl. J Biosci Biotechnol 4:39–48

Tahir M, U Khalid, M Ijaz, GM Shah, MA Naeem, M Shahid, K Mahmood, N Ahmad, F Kareem (2018). Combined application of bio-organic phosphate and phosphorus solubilizing bacteria (Bacillus strain MWT 14) improve the performance of bread wheat with low fertilizer input under an arid climate. Braz J Microbiol 49:15‒24

U.S. Salinity Lab. Staff (1954). Diagnosis and improvement of saline and alkaline soils. United States Salinity Laboratory, US Department of Agriculture, Washington, USA

Valetti L, L Iriarte, A Fabra (2018). Growth promotion of rapeseed (Brassica napus L.) associated with the inoculation of phosphate solubilizing bacteria. Appl Soil Ecol 132:1‒10

Vejan P, R Abdullah, T Khadiran, S Ismail, AN Boyce (2016). Role of plant growth promoting rhizobacteria in agricultural sustainability–a review. Molecules 21:573-589

Walker TS, HP Bais, E Grotewold, JM Vivanco (2003). Root exudation and rhizosphere biology. Plant Physiol 132:44‒51

Walkley A, IA Black (1934). Examination of a rapid method for determination of organic carbon in soils-effect of variation in digestion conditions and inorganic soil constituents. Soil Sci 63:251‒257

Weber O, J Delince, Y Duan, L Maene, T McDaniels, M Mew, U Schneidewind, G Steiner (2014). Trade and finance as cross-cutting issues in the global phosphate and fertilizer market. In: Sustainable Phosphorus Management, pp:275‒299. Springer, Dordrecht, The Netherlands

Wollum AG (1982). Cultural methods for soil microorganisms. In: Methods of Soil Analysis: Chemical and Microbial Properties, pp:718‒802. Page AL, RH Miller, DR Keeney (Eds.). ASA and SSSA Publ. Madison Wisconsin, USA

Zhu J, M Li, M Whelan (2018). Phosphorus activators contribute to legacy phosphorus availability in agricultural soils: A review. Sci Tot Environ 612:522‒537