紫花苜蓿根腐病生防菌的筛选、鉴定及发酵条件优化

Screening,identification and optimization of fermentation conditions of biocontrol fungi for alfalfa root rot

【目的】筛选获得对紫花苜蓿根腐病病原菌具有拮抗作用的菌株,并对其发酵条件进行优化,为紫花苜蓿根腐病生物防治提供菌株资源。【方法】以青藏高原的紫花苜蓿根际土壤为研究对象,以紫花苜蓿根腐病5种病原菌为靶标,利用稀释涂布法分离细菌;利用平板对峙法筛选对苜蓿根腐病病原菌具有拮抗作用的菌株;利用平板对峙法和菌丝生长速率法测定拮抗菌株对5种苜蓿根腐病病原菌的抑菌活性;通过形态学观察、生理生化测定和16S r DNA序列分析确定拮抗菌株的分类地位;利用单因素试验和正交试验对拮抗菌株发酵条件进行优化。【结果】从紫花苜蓿根际土壤中共分离纯化出92株细菌,从中筛选出16株对供试病原菌具有较强拮抗作用的菌株,其中菌株LJ3-1的抑菌效果最强,且具有广谱抑菌活性。对菌株LJ3-1发酵滤液的抑菌活性测定结果显示,其发酵滤液对供试病原菌的抑制率为44.9%~77.4%。结合菌落形态特征、生理生化测定和16S rDNA序列分析结果,将菌株LJ3-1鉴定为枯草芽胞杆菌(Bacillus subtilis)。对菌株LJ3-1的发酵条件优化结果显示,其最适发酵培养基组分为3%葡萄糖、1%酵母浸粉和0.5%NaCl;最适发酵条件为初始pH 6.5~7.0、培养温度35℃、250 mL锥形瓶装液量80 mL、接种量3%、培养时间36 h、转速180 r/min。在优化后的发酵培养条件下,菌株LJ3-1对燕麦镰刀菌(Fusarium avenaceum)的抑菌活性由76.5%提高至89.3%。【结论】菌株LJ3-1对紫花苜蓿根腐病5种病原菌均具有良好的拮抗作用,可作为研制苜蓿根腐病生物防治菌剂的候选菌株。

【Objective】To screen and obtain the strains with antagonistic effects on the pathogens of alfalfa root rot,and optimize fermentation conditions,so as to provide strain resources for the biological control of alfalfa root rot.【Method】The rhizosphere soil of alfalfa in Qinghai-Tibet Plateau was taken as the research object,and 5 pathogens of alfalfa root rot were used as the target to isolate bacteria by dilution coating method.The strains with antagonistic effects on alfalfa root rot pathogens were screened by plate confrontation method.The antibacterial activities of the antagonistic strains against 5 kinds of pathogens of alfalfa root rot were measured by plate confrontation method and mycelial growth rate method.Morphological observation,physiological and biochemical measurements and 16S rDNA sequence analysis were used to determine the taxonomic status of the antagonistic strains.The fermentation conditions of antagonistic strains were optimized by single factor test and orthogonal test.【Result】92 strains of bacteria were isolated and purified from the rhizosphere soil of alfalfa,among which 16 strains with strong antagonistic effects against the tested pathogens were screened,among which strain LJ3-1 showed the strongest antifungal effect and broad spectrum antifungal activity.The antifungal activity of the filtrate of strain LJ3-1 showed that the inhibitory rate of the filtrate was 44.9%-77.4%.Strain LJ3-1was identified as Bacillus subtilis based on colony morphological characteristics,physiological and biochemical measurements and 16S rDNA sequence analysis.The optimized fermentation conditions of strain LJ3-1 showed that the optimal fermentation medium was 3% glucose,1% yeast extract and 0.5% NaCl.The optimal fermentation conditions were as follows:initial pH 6.5-7.0,incubation temperature 35 ℃,250 mL conical bottle volume 80 mL,inoculum volume 3%,incubation time 36 h and rotation speed 180 r/min.Under the optimized fermentation conditions,the antifungal activity of strain LJ3-1 against Fusarium avenaceum increased from 76.5% to 89.3%.【Conclusion】Strain LJ3-1 has good antagonistic effects against the 5 pathogens of alfalfa root rot,and it can be used as a candidate strain for the development of biological control agents of alfalfa root rot.