一株水稻根内生拮抗细菌SM13的分离及鉴定

Isolation and identification of an endophytic and antagonistic bacterium from rice roots

【背景】作物根内生细菌具有固氮、分泌激素、产生病原真菌抗性物质等特性,根系内生菌的分离及应用成为环境友好型防控技术研究的热点之一。近年来盐碱地水稻种植面积逐年增加,而关于盐碱地水稻根内生菌的分离与应用鲜有报道。【目的】从大庆盐碱地水稻根中分离内生细菌,筛选对植物真菌病害有拮抗作用的促生菌株,初步探讨其抑菌和促生功效,为进一步研究其抑菌和促生机理提供菌种资源。【方法】对水稻根表面灭菌后研磨涂布分离内生细菌,采用对峙培养法和改良Salkowski比色法筛选具有广谱抑菌效果并有分泌吲哚-3-乙酸(Indole-3-acetic acid,IAA)能力的菌株,通过形态鉴定、革兰氏染色、生理生化测定、16S rRNA结构基因以及srfA、ituA、fenB功能基因序列系统进化分析,确定细菌的分类地位。【结果】从水稻根部分离到一株内生细菌SM13,该菌株具有广谱性抑菌作用,对玉米新月弯孢菌、大豆菌核病菌、稻瘟病菌、禾谷镰刀菌的抑菌率分别为59.38%、78.13%、53.12%、37.50%,分泌IAA的能力为5.56±0.41μg/mL (n=6)。经形态学、生理生化试验结合系统进化分析初步鉴定SM13菌株属于枯草芽孢杆菌(Bacillus subtilis),该菌株在pH 11.0、盐浓度10%的NA培养基中生长良好,具有较高的耐盐碱性。【结论】水稻根内生菌SM13菌株具有耐盐碱性、促生和生防性能,可作为微生物农药及菌肥的材料。

[Background]Endophytic bacteria in crop roots are beneficial to fix nitrogen,secrete hormones and produce resistant substances to pathogenic fungi.The isolation and application of endophytic bacteria in roots has become one of the hotspots in environment-friendly prevention and control technology.Although the rice planting area in saline alkali soil is increasing,the isolation and application of endophytic bacteria from rice roots in saline-alkali soil are still limited.[Objective]To isolate endophytic bacteria from rice roots in saline-alkali soil in Daqing,to screen promoting-growth bacteria with antagonistic effect on plant fungal diseases,to investigate its bacteriostasis and promoting-growth effect,and to provide the bacteria resources for further studies on bacteriostasis and promoting mechanism.[Methods]Sterilized the root surface and then grinded the rice root,which were used to isolate the endophytic bacteria.Screened endophytic antagonistic strains and determined the ability of endogenous bacteria to secreting indole-3-acetic acid(IAA)by using confrontation culture method and modified Salkowski colorimetry method,respectively.The taxonomic status of the strains with broad-spectrum bacteriostatic effect was determined by using morphological identification,Gram staining,physiological and biochemical analysis,phylogenetic analysis of 16 S rRNA structural genes and srfA/ituA/fenB functional genes.[Results]The SM13 strain was an candidate of endophytic bacterium isolated from rice roots,which showed broad-spectrum bacteriostatic effect.The bacteriostasis rates for Curvularia lunata,Sclerotinia sclerotiorum(Lib.)de Bary,Magnapporthe grisea,and Fusarium graminearum Schw were59.38%,78.13%,53.12%and 37.50%respectively,and the ability to secrete IAA was 5.56±0.41μg/mL(n=6).SM13 strain was identified as Bacillus subtilis by morphological,physiological and biochemical tests and phylogenetic analysis.The strain grew well in NA medium under pH 11.0 and salt concentration10%condition,so it had higher salt tolerance and alkalinity tolerance.[Conclusion]The rice roots endophyte SM13 has the ability of tolerance to saline-alkaline,promoting-growth and biocontrol.It could be used as the material of microbial pesticides and biofertilizers.