Enhancing the chickpea-Mesorhizobium symbiosis using beneficial rhizobacteria

Abstract

Chickpea is a major pulse crop grown in the Australian cropping system. It can fix a substantial amount of nitrogen (N) when it forms a symbiotic association with highly effective Mesorhizobium spp. Phosphorus (P) is an important nutrient for efficient chickpea- Mesorhizobium symbiosis. Chickpea exudes large amounts of carboxylates that can mobilise P from sparingly soluble P sources. Additionally, a number of bacteria associated with plant roots are capable of solubilising P, and these bacteria are generally called phosphate solubilising bacteria (PSB). Such bacteria that are able to promote plant growth more generally are designated as plant growth-promoting rhizobacteria (PGPR). However, PSB do not always enhance the chickpea-Mesorhizobium symbiosis under different P conditions. Additionally, the responsible plant growth-promoting (PGP) mechanisms have not always enhanced P solubilisation. Therefore, this study was investigated whether efficient PSB could enhance the chickpea-Mesorhizobium symbiosis in a widely varied P condition. Firstly, this study was tested whether pre-screening methods result in efficient PSB, if selected efficient P solubilisers had the ability of PSB to solubilise P from a wide array of P sources, and if the expression of PGP characteristics can directly and indirectly affect the plant P nutrition. Additionally, plantrelated factors that may affect chickpea P nutrition were investigated. Accordingly, this study was hypothesised that the presence of high carboxylate concentrations and acidic pH in the chickpea rhizosphere may affect the efficiency of PSB. Seventy-four soil samples collected from major agricultural lands across Australia were used in this research. Major soil chemical and physical properties were examined. A total of 743 isolates of Bacillus- and Pseudomonas-like bacteria were isolated using taxonomically selective methods of extraction. Based on 16S rRNA sequences, these isolates were closely related to diverse species of Bacillus, Pseudomonas and Burkholderia spp. (formerly classified as Pseudomonas spp.). Of 743 isolates, 616 (83%) were able to produce IAA in the presence of L-tryptophan. 1-aminocyclopropane-1-carboxylate (ACC) deaminase activity was detected in 57 isolates (7.7%) in vitro. All isolates were further tested for their ability to promote seedling growth. Most of these isolates (71%) were able to promote seedling root elongation. The effect of isolates on seedling growth predicted their effect on nodulation and growth of chickpea after dual inoculation in aseptic conditions. The effect of rhizobacteria on seedling root growth and chickpea-Mesorhizobium symbiosis was associated with their capacity to produce IAA and ACC deaminase. The synthesis of IAA along with ACC deaminase activity by rhizobacteria gave an added advantage by promoting the ability of rhizobia to form an efficient chickpea-Mesorhizobium symbiosis as measured by biomass production and nodulation. Most of the isolates able to express both PGP traits belonged to the genus Burkholderia. Hence, the use of a plant-based first-stage screening strategy in combination with assays for in vitro production of IAA and ACC deaminase enabled the identification of efficient PGPR that were able to enhance the legume-rhizobia symbiosis. The influence of soil, climatic and crop management variables on the occurrence of PGPR and their PGP characteristics was examined. IAA production by isolates and their beneficial effects on chickpea root elongation were associated positively with soil copper, manganese and zinc concentrations and the aridity index, and negatively with soil carbon (C), N, C/N ratio, Ca and P at the sampling sites from which the PGPR were isolated. Additionally, the P solubilisation activity of the isolates was also negatively correlated with C/N ratio, N, P, C and magnesium content of the soils. However, none of the investigated soil environment variables were correlated with the potential of the isolates to express ACC deaminase. A greater proportion of IAA producing PGPR had a greater ability to withstand metal ion toxicity and water stress. Therefore, these findings have potential application in designing a strategy for the development of efficient PGPR that have ecological traits and plant growth-promoting mechanisms that may increase chickpea production. An additional 98 novel strains of P solubilising bacteria were isolated through enrichment in media with AlO₄P, Ca₃(PO₄)₂ or FeO₄P. Following enrichment, the proportion of PSB among total culturable bacteria was significantly increased compared with the PSB population in the original soils. These isolates were assigned into twenty-five bacteria genera based on 16S rRNA sequences. The majority of these isolates belonged to Burkholderia, Variovorax, Leifsonia, Pantoea and Rhizobium. These isolates had a greater P solubilisation index (PSI) than those obtained using the taxonomically selective method. From both isolation methods, seven isolates, namely Peribacillus simplex 37F, Bacillus megaterium 8F, Pseudomonas fluorescens 27F, Bacillus pumilus 98F, Bacillus cepacia 126F, Burkholderia sp. 12F and Burkholderia cenocepacia 127F were proportionally selected, based on their PSI values, to investigate their P solubilisation ability in liquid media. Generally, isolates obtained following enrichment were able to solubilise between 1.2 to 2.8, 1.2 to 3.1 and 1.5 to 4.5-fold Pi from AlO₄P, Ca₃(PO₄)₂ or FeO₄P than those obtained using the taxonomically selective method. The P solubilising efficiency was related to citrate and α-ketoglutarate production in vitro. Enriching rhizobacteria by culture in conditions with sparingly soluble P increased the likelihood of isolating elite PSB from bulk soils and plant rhizospheres. The relationship between the phosphate solubilising ability of plant growth-promoting rhizobacteria and their ability to produce ACC deaminase was analysed. Five isolates, namely B. megaterium 8F, B. pumilus 98F, B. cepacia 126F, Burkholderia sp. 12F and B. cenocepacia 127F were selected based on their potential for P solubilisation and their ability to express ACC deaminase. Generally, ACC deaminase had no role in AlO4P and FeO4P solubilisation. However, the amount of ACC deaminase produced by PSB was significantly associated with the liberation of Pi from Ca₃(PO₄)₂ when ACC was the sole N source. Ca₃(PO₄)₂ solubilisation was associated with the extent of acidification of the medium. Additionally, α-ketobutyrate by itself was able to solubilise significant amounts of Pi from AlO₄P, Ca₃(PO₄)₂ and FeO₄P. Conversely, the P solubilisation potential of PSB was independent of their ability to express ACC deaminase activity when (NH4)2SO4 was the sole N source. The ability of efficient PSB isolates, namely Burkholderia sp. 12F, P. fluorescens 27F and B. cenocepacia 127F, selected based on their ability to solubilise P from different rock phosphates (as above), was investigated. Results showed the highest potential of Burkholderia sp. 12F in P solubilisation from Boucraa, Togo, Sechura and Weng Fu rock phosphates. All bacterial isolates poorly solubilised Phalaborwa, Peru or Vietnam rock phosphate. The solubilisation of these P sources by the PSB was not related to the amount of available and total P, nor to the concentration of Al³⁺, Fe²⁺ and Cd³⁺ in the rock phosphate. Additionally, their solubility in carboxylates was varied and higher solubility was recorded in di- and tri-carboxylates than for mono-carboxylates added separately. The variation in P solubilising activities between Burkholderia sp. 12F, B. cenocepacia 127F and P. fluorescens 27F was not associated with acidification of their culture media. The highest P solubilising activity of Burkholderia sp. 12F was related to its ability to produce citrate, malate and maleate during mineral phosphate solubilisation. The effect of IAA and its precursor L-tryptophan on the P solubilising activity of rhizobacteria, namely B. pumilus 98F, B. cenocepacia 127F, Burkholderia sp. 12F and P. fluorescens 27F, was investigated. The ability to produce IAA was related to the improved potential of PSB to solubilise P from rock phosphate. The addition of L-tryptophan to growth media improved the P solubilising activity of PSB that were able to produce IAA. A remarkable effect of this precursor on P solubilisation was observed for B. cenocepacia 127F and Burkholderia sp. 12F, that produced 41.9 and 54.3 μg mL⁻¹ IAA, respectively. Additionally, the potential of Burkholderia sp. 12F to solubilise rock phosphate was increased with increasing IAA concentration in the media. This effect was connected to the reduction of pH and release of high concentrations of carboxylates, comprising α-ketoglutarate, cis-aconitate, citrate, malate and succinate. IAA solution by itself was able to liberate Pi only between 1.45 to 3.00 μg Pi L⁻¹ from rock phosphate. Therefore, increased production of organic acids rather than IAA production per se may be the possible mechanism by which IAA ultimately resulted in the improved capacity of PSB in P solubilisation. Based on the above experiments, Burkholderia sp. 12F was selected to examine its P solubilising activity in chickpea root exudates and its effect on the chickpea-Mesorhizobium symbiosis. The ability of the root exudates obtained from six chickpea cultivars to mobilise P was tested in the presence and absence of PSB. In particular, the root exudates were able to solubilise Togo rock phosphate but not Peru rock phosphate in vitro. In this case, the amount of solubilised P by root exudates was not related to the extent of acidity in the root exudates before and after incubation. The presence of PSB significantly increased the amount of solubilised P in all root exudates from both rock phosphates. The efficiency of Burkholderia sp. 12F to alter the chickpea-Mesorhziobium symbiosis was tested using six cultivars. In this experiment, four P sources including Peru and Togo rock phosphate, K₂HPO₄ and the control check (no added P) were used. Inoculation of PSB significantly increased shoot and root biomass production, and nodulation of chickpea cvv. Genesis-863, PBA-Striker and PBA-Slasher but did not significantly affect Ambar, Genesis- 079 and Genesis-090. Increased nodulation and growth of chickpea following inoculation of PSB were not always explained by increasing P nutrition. PSB inoculation significantly increased the P concentration in the rhizosphere of plants fertilised with Togo and Peru rock phosphate. In this case, P uptake was associated with P concentration in the rhizosphere extract. Additionally, P uptake by plants fertilised with K₂HPO₄ was increased following inoculation with PSB. In this case, the PSB did not affect the P concentration in the rhizosphere but improved root biomass. An increased P concentration in the rhizosphere following PSB inoculation was not related to the extent of acidity in the rhizosphere. More acidity in rhizosphere was instead associated with more nodulation. This may suggest that more nodulation may have a positive feedback effect on further solubilisation of P, which acidified the rhizosphere. In conclusion, the selection of PSB following enrichment and selection of rhizosphere isolates from the genera Bacillus, Pseudomonas and Burkholderia provided efficient isolates able to solubilise P from diverse P sources. Plant-based screening of these isolates indicated the possible PGP traits (ACC deaminase and IAA production) that could affect chickpea growth and nodulation. These traits affected the P solubilising activity of efficient PSB in vitro as well. Although chickpea releases a large amount of carboxylate and has an acidic rhizosphere pH, inoculation of the PSB Burkholderia sp. 12F increased the nodulation, growth and P uptake of some cultivars of chickpea. This PSB isolate did not affect the cultivars that by themselves produce relatively high carboxylate concentrations. Enhancing the chickpea-Mesorhizobium symbiosis was mediated by multiple PGP traits, including P solubilisation and possibly IAA and ACC deaminase production. Future research is considered in the final part of this document.