黑龙江大豆根瘤菌及根际促共生菌株的筛选及应用

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

摘要/Abstract

摘要：

【目的】黑龙江是我国大豆的主产区，有着丰富的大豆根瘤菌资源，针对该区域筛选优质抗逆大豆根瘤菌及根际促共生菌株对于大豆的提质增产至关重要。【方法】从黑龙江采集根瘤、根际土分离纯化大豆根瘤菌、根际菌，通过rpoB、16S rRNA基因测序进行种属鉴定。在试管培养条件下进行根瘤菌菌株对盐胁迫（1.2% NaCl）、干旱胁迫（15% PEG6000）的耐受性评价。胁迫耐受根瘤菌菌株接种到黑河43，筛选高效共生菌株。代表性根瘤菌菌株及其根际促共生菌株混合菌接种到黑河43，获得高效共生根瘤菌及促共生显著的菌株。【结果】（1）分离获得大豆根瘤菌136株，其中129株慢生型根瘤菌（分属于Bradyrhizobium elkanii、B. japonicum、B. diazoefficiens、B. yuanmingense、B. liaoningense）；7株快生型根瘤菌（分属于Sinorhizobium fredii、Sinorhizobium sp.）。（2）分离获得拮抗大豆根腐病病原菌（镰刀菌）的菌株6株（B1-B6，分属于Bacillus velezensis、B. subtilis、B. licheniformis、B.cereus、B. megaterium），其中有4株（B1、B2、B4、B5）具有产IAA的能力。（3）获得耐盐或耐旱代表性大豆根瘤菌28株，其中有两株大豆根瘤菌B. japonicum GN1、B. diazoefficiens GN10对盐胁迫、干旱胁迫均具有较好抗性，并且通过共生表型的筛选确定GN10是代表菌株中最为高效的大豆根瘤菌。（4）混合接种大豆表型结果表明，B. velezensis B4相比于其余5株芽胞杆菌可以显著提升B. diazoefficiens GN10的共生表现。【结论】成功获得耐盐、耐旱且共生效率高的慢生大豆根瘤菌1株，同时获得对共生表现促进效果显著的贝莱斯芽胞杆菌1株，为高效复合根瘤菌菌剂的开发及应用提供有力支撑。

关键词: 大豆根瘤菌, 胁迫耐受, 高效共生, 根际促生菌, 共接种

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

刘克寒, 杨升辉, 黄巧云, 崔文靖. 黑龙江大豆根瘤菌及根际促共生菌株的筛选及应用[J]. 生物技术通报, 2025, 41(1): 252-262.

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.

图/表 5

图1 大豆根瘤菌的物种数和物种丰度 A：分离根瘤菌菌种的数量统计，图内数字表示菌株数；B：不同采样点菌株相对丰度

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

图2 根瘤菌对盐胁迫、干旱胁迫的耐受性及其rpoB进化树 A：根瘤菌对模拟盐胁迫、干旱胁迫的耐受性，耐受性指数阈值70%所在位置用红色虚线标识； B：基于不同根瘤菌rpoB基因构建的系统发育树，共计39条rpoB基因序列（28株候选菌株，11株参比菌株），采用Mega X最大似然法构建进化树，Bootstrap值高于60%的在分支节点用数字标明，参比菌株的rpoB 在 GenBank 的序列号在括号内备注。GN为Green Nitrogen缩写

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

图3 高效共生根瘤菌的筛选根瘤数（A）、叶绿素含量（B）、地上部干重（C）3个指标用来衡量单个根瘤菌菌株与大豆的共生表现。误差线采用SD，均值间显著差异基于ANOVA单因素分析Tukey检验用不同字母表示（α=0.05）

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）

图4 根际抗病、促生菌株及其16S rDNA序列系统发育树 A：六株拮抗大豆根腐病原菌的芽胞杆菌及其产IAA能力检测。大豆根腐病病原菌选取Fusarium oxysporum，IAA产量通过显色深浅来衡量；B：基于不同芽胞杆菌16S rRNA基因构建的系统发育树，共计25条16S rDNA序列（6株候选菌株，19株参比菌株），采用Mega X最大似然法构建进化树

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

图5 根际促共生菌与大豆根瘤菌的混合接种表现根瘤数（A）、叶绿素含量（B）及地上部干重（C）3个指标用来衡量单个根瘤菌菌株与大豆的共生表现。误差线采用SD，均值间显著差异基于ANOVA单因素分析Tukey检验用不同字母表示（α=0.05）

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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摘要

1989

最新录用	在线预览	正式出版

0	0	1989

	来源	本网站

	次数	1989
	比例	100%