Isolation and Application of Soybean Rhizobia and Symbiosis-promoting Rhizobacteria from Heilongjiang Province

doi:10.13560/j.cnki.biotech.bull.1985.2024-0564

Abstract

Abstract:

【Objective】Heilongjiang province is the main area of producing soybeans in China, with rich resources of soybean-nodulating rhizobia. Selecting high-quality stress-resistant soybean-nodulating rhizobia and rhizobacteria for soybean quality and yield improvement in this region was of great importance.【Method】In this experiment, rhizobia and symbiosis-promoting rhizobacteria were isolated and purified from root nodules and rhizosphere soil in Heilongjiang province, respectively. Species identification was conducted by rpoB and 16S rRNA gene sequencing. The tolerances of rhizobial strains to salt stress（1.2% NaCl）and drought stress（15% PEG6000）were evaluated in tube culture conditions. Highly efficient symbiotic rhizobial strains were selected by inoculating with stress tolerant rhizobial strains to soybean HH43. Representative rhizobial strains and symbiosis-promoting rhizobacterial strains were co-inoculated to HH43 to obtain highly-efficient symbiotic rhizobial strains and rhizobacterial strains with significant symbiosis-promoting effects.【Result】1）A total of 136 rhizobial strains were isolated, of which 129 were slow growing rhizobia（belonging to Bradyrhizobium elkanii, B. japonicum, B. diazoefficiens, B. yuanmingense and B. liaoningense）and 7 were fast-growing rhizobia（belonging to Sinorhizobium fredii and Sinorhizobium sp.）. 2）Six rhizobacterial strains that inhibited the pathogenic fungus of soybean root rot（Fusarium solani）were isolated（B1-B6, belonging to Bacillus velezensis, B. subtilis, B. licheniformis, B. cereus and B. megaterium）and four of them（B1, B2, B4 and B5）had the ability of producing IAA. 3）A total of 28 representative rhizobial strains with the tolerance to salt or drought were obtained, among which two strains, B. japonicum GN1 and B. diazoefficiens GN10, demonstrated dual resistance to salt and drought stress. Additionally, GN10 was determined to be the most efficient symbiotic strain among all representative strains through screening of their relative symbiotic phenotypes. 4）The results of co-inoculation phenotype of soybean indicated that B. velezensis B4 significantly enhanced the symbiotic performance of B. diazoefficiens GN10 compared to other tested bacillus strains.【Conclusion】We successfully obtaine one slow-growing soybean-nodulating rhizobial strain with dual-tolerance to salt and drought and one B. velezensis strain B4 that significantly enhance the symbiotic performance, providing strong support for the development and application of high-efficiency composite rhizobia inoculants.

Key words: soybean-nodulating rhizobia, tolerance to stress, efficient symbiosis, growth-promoting rhizobacteria, co-inoculation

LIU Ke-han, YANG Sheng-hui, HUANG Qiao-yun, CUI Wen-jing. Isolation and Application of Soybean Rhizobia and Symbiosis-promoting Rhizobacteria from Heilongjiang Province[J]. Biotechnology Bulletin, 2025, 41(1): 252-262.

Figures/Tables 5

Fig. 1 Species number and relative abundance of isolated soybean-nodulating rhizobia A: Statistics of the species number of isolated soybean-nodulating rhizobia, the number in the Figure indicate the number of a strain. B: The relative abundance of isolates in different sampling sites

Fig. 2 Tolerances of isolated rhizobia strains to salt and drought and its rpoB phylogenetic tree A: Tolerances of isolated strains to simulated salt and drought. The index threshold（70%）of tolerance to stress is indicated by red dotted line. B: Phylogenetic tree was constructed based on rpoB genes of different rhizobia, totally 39 rpoB sequences（（28 candidates, 11 reference strains）.）by Mega X maximum likelihood software. Bootstrap values above 60% are shown at the branch points. Accession numbers of the reference sequences from GenBank are given in brackets. GN is the abbreviation of Green Nitrogen

Fig. 3 Screening of highly efficient symbiotic rhizobial strains Nodule number（A）, leaf chlorophyll content（B）, shoot dry weight（C）are measured to evaluate the effects of the symbiotic performance of individual rhizobia on soybean. Error bars are for SD. Significant differences among means are indicated by different letters based on ANOVA followed by Tukey's test（α = 0.05）

Fig. 4 Disease-resistant and growth-promoting rhizobacteria and its phylogenetic tree of 16S rDNA A: Six bacillus strains antagonizing soybean root rot pathogen and producing IAA test. Fusarium oxysporum was a selected soybean root rot pathogen, IAA production was measured by color intensity. B: Construction of phylogenetic tree based on 16S rRNA genes of different Bacillus strains. Maximum likelihood phylogenetic trees of 25（6 candidates, 19 reference strains）16S rDNA sequences was constructed by Mega X maximum likelihood software

Fig. 5 Performance of co-inoculation of symbiosis-promoting rhizobacteria and soybean-nodulating rhizobia Nodule number（A）, leaf chlorophyll content（B）and shoot dry weight（C）were measured to evaluate the effect of the symbiotic performance of individual rhizobia on soybean. Error bars are for SD. Significant differences among means are indicated by different letters based on ANOVA followed by Tukey's test（α = 0.05）

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