大豆根瘤内抗棉花枯萎病菌株的筛选及其防病试验

Screening endophytic bacteria from soybean root nodules to inhibit Fusarium oxysporum f. sp. Vasinfectum

摘要:

[目的] 采用优良抗病性内生菌资源来控制棉花枯萎病是一种有效的措施。本研究从大豆根瘤中筛选棉花枯萎病拮抗性内生细菌，探索其对棉花枯萎病菌丝的抑制作用和代表菌株特性，为发掘和应用防病、抗逆优良菌株提供理论基础。[方法] 采用对峙法和代谢液培养法对大豆根瘤内生细菌进行棉花枯萎病菌抑菌性筛选，显微观察法研究筛选菌株引起病原菌菌丝变化，通过菌株培养特征、理化特性和16S rDNA序列同源性分析确定菌株系统发育地位，比色法测定DD174耐盐碱性，盆栽试验验证防病效果。[结果] 经复筛和代谢液试验有5株拮抗性较强菌株，被作用病原菌菌丝畸形、细胞壁消失、自溶，菌丝基部加粗、分支增多，呈树根状；菌丝被菌苔包埋而溶解、断裂，菌丝末端球形膨大。对棉花枯萎病菌的抑制作用主要通过菌体产生胞外代谢物发挥作用。菌株DD174、DD176和DD179最相似菌株分别为Bacillus oceanisediminis H2^T(GQ292772)和B. thuringiensis ATCC 10792^T(AF290545)，菌株DD165和DD166最相似菌株均为Stenotrophomonas maltophilia LMG 958^T(X95923)。DD174能耐受6%盐浓度， pH 10生长良好，具有一定耐盐碱能力。DD174处理组防治效果达76.32%，其他防效均在62%以上，可作为棉花枯萎病的生防菌株资源。[结论] 大豆根瘤内存在棉花枯萎病内生拮抗细菌，其中有些菌株具有一定耐盐碱能力，对棉花枯萎病病原菌及病害有一定抑菌和防病作用。s

关键词:棉花枯萎病菌;根瘤内生菌;拮抗性;16S rDNA;系统发育分析

Abstract:

[Objective] Using desirable strain resources to control Fusarium wilt of cotton is an effective way. This study aimed to acquire antagonistic endophytic bacteria from soybean nodules, explore their inhibition mechanism and strain characteristics. [Methods] Confrontation and metabolic liquid culture methods were adopted to screen endophytic bacteria from soybean nodules against Fusarium oxysporum f. sp. vasinfectum. The effect of screened strains on pathogen hyphae changes were analyzed with microscopic observation method. Combined with cultural, physical and chemical characteristics, 16S rDNA sequencing results and homology analysis of screened strains, phylogenetic status were determined. Disease-control effects were demonstrated by greenhouse inoculation test. [Results] Five strains of endophytes have inhibitory effect by the second screening and metabolic liquid test. Pathogen hyphae treated with endophytes became deformity, its cell wall disappeared, autolysis, bold at the base of the mycelium, branches increased and root shape appeared. Hyphae were embeded, dissolved and fractured by lawn formed by endophytic bacteria, and presented spherical expansion of its terminal. The inhibition of endophytic bacteria against Fusarium oxysporum f. sp. vasinfectum was mainly caused by extracellular metabolites. DD174, DD176 and DD179 were similar to Bacillus oceanisediminis H2^T(GQ292772) and B. thuringiensis ATCC 10792^T(AF290545), respectively. DD165 and DD166 were similar to Stenotrophomonas maltophilia LMG 958^T (X95923). DD174 tolerated 6% salt concentration and grew well at pH 10. Control effect of treatment group with DD174 was 76.32%, those of others were above 62%, so these strains can be used as biocontrol resources against Fusarium oxysporum f. sp. vasinfectum. [Conclusion] Endophytic antagonistic bacteria inhabited soybean root nodules against Fusarium oxysporum f. sp. vasinfectum.

Key words:Fusarium oxysporum f. sp. vasinfectum;soybean nodule endophytes;antagonistic effect;16S rDNA;phylogenetic analysis