Mikrobiol. Z. 2020; 82(2):38-50.
Phenotypic and Genotypic Properties of Bradyrhizobia Nodulating Leguminous Plants
of the Glycine, Vigna and Lupinus Genera
Institute of Agricultural Microbiology and Agro-Industrial Manufacture, NAAS of Ukraine
97 Shevchenko Str., Chernihiv, 14027, Ukraine
Objective. To study phenotypic and genotypic features of nodule bacteria – microsymbionts of leguminous plants of the Glycine, Vigna and Lupinus genera. Methods. Serological affinity of 12 rhizobia strains was determined in agglutination reactions. Host specificity of strains was studied in vegetation experiments. RFLP analysis of rpoB gene was performed using MspI, HaeIII and NdeII restrictases. Sequencing of the 16S-23S rDNA intergenic spacer of rhizobia was performed on ABI 3130 Genetic Analyzer. Results. It was established that studied rhizobia strains differ significantly in serological properties and belong to 7 serogroups: КВ11, М8, 1967, 46, В1, В2 and 367а. Microsymbionts of soybean, cowpea and mung bean form the group of cross inoculation, however, are unable to infect lupine. Alternatively, lupine rhizobia B. lupini 367а and Bradyrhizobium sp. ЛД4, enter into symbiotic interactions with lupine, however, they do not nodulate soybean. B. japonicum 631 strain is capable of symbiosis with leguminous plants of different tribes: Phaseoleae and Lupineae. Based on RFLP analysis of rpoB gene, rhizobia were grouped into four clusters with following microsymbionts: I – cowpea, II – mung bean and soybean, III – slow-growing soybean nodule bacteria, IV – lupine rhizobia and intensive-growing soybean rhizobia. 16S-23S rDNA sequencing confirmed pertinence of soybean rhizobia to the B. japonicum species (USDA 4, USDA 6, USDA 123 genetic groups). This species also included mung bean microsymbionts (USDA 4 group). Bradyrhizobium sp. В11 isolate was obtained for the first time from cowpea nodules, and it was identified as a new B. diazoefficiens species (USDA 110 group). ITS regions of B. lupini 367а and Bradyrhizobium sp. ЛД4 rhizobia were found to be identical. B. japonicum 631 strain has a 100% similarity with B. lupini 367a strain according to 16S-23S rDNA, and with high degree of probability can be included to B. lupini species. Conclusions. Serological heterogeneity of rhizobia from root nodules of plants of the Glycine, Vigna and Lupinus genera has been demonstrated. It was established that microsymbionts of soybean, cowpea and mung bean belong to one group of cross inoculation. 16S-23S rDNA sequencing allowed to classify the studied strains as different genetic groups and identify them as B. japonicum, B. diazoefficiens and B. lupini. The serological grouping of nodule bacteria was found to coincide with the genetic (rpoB gene and ITS region) grouping, and their host specificity was related to species affiliation.
Keywords: Bradyrhizobium japonicum, B. diazoefficiens, B. lupini, RFLP analysis, 16S-23S rDNA, rpoB gene, soybean, cowpea, mung bean, lupine.
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