云南省烟植地青枯菌RS-22的分离及其拮抗菌的筛选和鉴定

Isolation and identification of Ralstonia solanacearum-22 and its antagonistic bacteria in tobacco plantations of Yunnan province

【背景】由青枯劳尔氏菌(Ralstonia solanacearum)引起的烟草青枯病是一种重要土传病害,在我国南方烟区普遍发生。生物防控是针对烟草青枯病的一种有效防治措施,但是相关的研究报道还较少。【目的】分离云南省烟植地的青枯病原菌,筛选其拮抗菌并对其抑菌效果进行鉴定。【方法】采用平板稀释法从云南感病烟草中分离获得青枯菌,采用平板对峙法筛选青枯菌拮抗菌,筛选得到的拮抗菌通过16SrRNA基因测序比对确定菌种类型,并在实验室和大田鉴定其对青枯病的防治效果。【结果】从感病烟草茎中分离出一株强致病性青枯菌小种RS-22,该菌能侵染烟草和番茄并最终使植物死亡;筛选出12株RS-22拮抗菌,其中拮抗作用最强的是Y4;Y4被鉴定为一株解淀粉芽孢杆菌(Bacillus amyloliquefaciens),其菌体和分泌物都能抑制RS-22生长;Y4根部灌根处理能显著提高烟草和番茄对青枯菌RS-22的抗性,Y4处理能使感病烟草部分恢复正常,在云南文山州烟草种植大田施加Y4菌剂和菌剂有机肥混合物也能显著降低烟草青枯病的感病率。【结论】青枯菌RS-22具有广谱的致病性,筛选的拮抗菌Y4能显著抑制青枯菌生长,而且对青枯菌侵染植物有很好的防治效果。研究结果为进一步研究烟草青枯病的生物防控提供了新的理论依据。

[Background]Tobacco bacterial wilt disease caused by Ralstonia solanacearum is a typical soil borne bacteriosis,which widely occurs in the tobacco farms in south of China.Biological control is an effective measure to alleviate the occurrence of the tobacco wilt disease;however,the antagonistic agents still lack.[Objective]To identify the R.solanacearum isolates in tobacco plantations of Yunnan province,and to screen its antagonistic bacteria for biocontrol of tobacco wilt disease.[Methods]R.solanacearum was isolated and identified from the infected tobacco tissues from the farms in Yunnan province by the plate dilution method.Antagonistic bacteria were isolated by the plate confrontation method and the antagonism against wilt disease was investigated under laboratory and field conditions.[Results]The R.solanacearum strain RS-22 was isolated from the infected tobacco plant stem.Koch’s postulates was employed to further confirm the causal agent of the wilt disease.The antagonistic microorganism screening led to the discoveries of 12 biocontrol bacteria.Among them,the strain Y4,a Bacillus amyloliquefaciens,showed the strongest antagonistic effect to RS-22.Pretreatment of the tobacco or tomato plants by root irrigation significantly enhanced their resistance to the bacterial wilt disease.Moreover,syringe injection of Y4 could significantly promote the survival rate of the RS-22 infected N.benthamiana plants.Field application of Y4 remarkably alleviated the wilt disease in the tobacco farms in Wenshan county of Yunnan province.[Conclusion]RS-22 has a broad spectrum pathogenicity.Y4 significantly suppressed the growth of RS-22 and has excellent biocontrol effect on the RS-22 infection.This study provides a useful tool for the biological control of tobacco wilt disease.