Symbiotic effectiveness, phylogenetic diversity and ecological adaptation of chickpea rhizobia isolated from Australian and Myanmar soils

Abstract

The presence of rhizobia has significant impact on the nodulation, nitrogen fixation and productivity of chickpea. However, inoculation practices for chickpea differ globally, which may impact the genetics, physiology and utility of rhizobia. Nodulation and symbiotic effectiveness (SE) of 80 rhizobial strains isolated from across Australian cropping regions were evaluated in a glasshouse experiment. Tolerance of these rhizobial strains to pH, temperature, antibiotics, heavy metals and NaCl were examined on supplemented YMA plates in vitro. In addition, the phylogenetic diversity among Australian chickpea rhizobia were investigated by sequencing of 16S-23S rRNA, atpD, recA, nodC and nifH genes. Phylogenetic analysis of 16- 23S rRNA IGS, atpD and recA genes revealed that most Australian strains belonged to Mesorhizobium ciceri (68%), with most of the remaining strains closely associated with M. temperatum, M. huakuii and M. tianshanense. Although the strains were diverse in 16S-23S rRNA IGS-based phylogeny, they shared similar symbiosis genes with common chickpea symbionts. Inoculation of chickpea with 80 strains collected from Australia revealed that variation in SE% among isolated strains was correlated with phylogenetic relatedness to the commercial inoculant strain Mesorhizobium ciceri CC1192. Strain A47 collected from Queensland gave the highest shoot biomass and two strains (A78 and A79) from Western Australia grew under acidic conditions (pH 4.4), indicating the potential adaptation of these strains to different environmental conditions. Some isolated strains had equal or superior SE relative to inoculant strain CC1192. The incongruence between core and symbiosis gene phylogenies of rhizobial strains in this study suggests the potential occurrence of HGT of symbiosis genes in Australian soils. A total of 120 Mesorhizobium strains were isolated from 103 soils sampled from fields in the central dry zone (CDZ) of Myanmar and evaluated their infectivity and effectiveness in a pot experiment. Nodulation and SE varied considerably among these strains and the majority (about 90%) provided high shoot dry weight, which was comparable with CC1192. Strain M082 had the highest SE, followed by M009 in a pot experiment and were considered as potential strains to be evaluated under field conditions. Some strains showed potential tolerance of low pH (e.g., M094 and M113), high temperatures (M.021, M075) and 3% NaCl (w/v) such as M062 and M107. The 16-23S rDNA IGS phylogeny confirmed that all Myanmar strains were members of the genus Mesorhizobium and most Myanmar strains were most closely related to M. gobiense, M. muleiense, M. silamurunense, M. tamadayense and M. temperatum. Three of the four main species groups Mesorhizobium; M. gobiense, M. huakuii and M. muleiense, were distributed throughout Myanmar but M. temperatum was not found in the Southwestern area of Magway. Nearly 70% of Myanmar strains were most closely related to Indian strain IC-2058 (CA-181), which is also most closely related to M. gobiense, while none of Myanmar strains were closely related to the cognate chickpea rhizobial species M. ciceri and M. mediterraneum. However, Myanmar strains shared shared similar nodC and nifH gene sequences with chickpea symbionts. Detailed sequence analysis of the nodC and nifH found that the strains in Myanmar were slightly divergent from the group of cognate chickpea rhizobia and were more closely related to symbiotic genes ofM. muleiense and the Indian strain IC-2058-CA181. Mutation (substitution) in the nodC protein had no significant effect on nodulation and SE of the test strains. Comparative analysis of 16S-23S rDNA sequences of strains from Australia and Myanmar revealed that there was little overlap in species found in the two countries. The only species found in both Myanmar and Australia were M. tamadayense and M. silumurunense. The isolated Australian and Myanmar strains showed similar adaptive traits, and the adaptation traits were phylogenetically related within each country. The genetic discrepancy between Australian and Myanmar strains was not only due to inoculation history but to adaptation to soil conditions and crop management over time in each country, and there has been virtually no loss of symbiotic efficiency of Myanmar strains relative to Australian commercial inoculant CC1192, in pot experiments. Twelve strains were selected based on their improved SE for chickpea in pot experiments, and acid tolerance in laboratory experiments, and were then tested in five separate field experiments. In low pH soils, the strains collected in Australia were vastly superior compared with those from Myanmar, however in most cases they did not perform better than CC1192. Myanmar strains had lower SE in Australian field conditions compared with performance under glasshouse conditions. As an exception, two Myanmar strains (M022 and M065) provided significantly higher SDW than CC1192 in the low rainfall and high pH sandy soil environment in Loxton. A strong positive association was found between survival of rhizobia on seed and nodulation. Two Australian strains (A21 and A47) and CC1192 showed higher survival on seed at 3 to 24 hours after inoculation and better nodulation across experimental sites. Strain A47 improved symbiotic N2 fixation and strain A78 showed some tolerance to strongly acidic soil (pH 4.18). The inconsistent performance of Myanmar strains in this study may be due to genetic divergence from common inoculant species, or they simply are not able to survive as an inoculant (on the seed) or in Australian soils. This may not be the case in Myanmar soils, and strains isolated from Myanmar should be further tested in Myanmar soil conditions. Future research in this area should consider the testing of these strains across multiple variable field environments to further evaluate their potential as inoculants to improve chickpea performance.