生防菌XNB-02的鉴定及其对烟草赤星病的防病机理

Identification of the biological control bacterium XNB-02 and its actionmechanism in prevention of the tobacco brown spot

为获得防控烟草赤星病的优异生防资源,自青海省西宁市土壤样品中分离纯化获得细菌菌株,筛选对烟草赤星病菌Alternaria alternata有较好抑制作用的菌株,测定其对其他6种植物病原菌的抑制作用,采用形态学、生理生化特性和分子生物学对筛选的生防菌株进行鉴定,初步探究该菌株对烟草赤星病的抑菌机理、室内防效和促生作用,并测定该菌株发酵液的稳定性、体内生防相关基因及其对烟草体内抗病基因的影响。结果显示:经分离纯化共获得了5株菌株,其中生防菌株XNB-02对烟草赤星病菌有较好的拮抗效果,抑菌圈半径为10.33 mm;对其他6种植物病原菌也有较好的抑制作用。经鉴定生防菌株XNB-02为贝莱斯芽胞杆菌Bacillus velezensis。生防菌株XNB-02能促进烟草幼苗的生长,当其发酵液浓度为1×10~9CFU/mL时对烟草赤星病的防效达到61.82%。生防菌株XNB-02胞内粗提物能抑制烟草赤星病菌菌丝生长和孢子萌发,能改变菌丝和孢子的形态。温度低于90℃、pH 3~13、光照7 d、紫外照射240 min和蛋白酶K处理后,生防菌株XNB-02对烟草赤星病菌的抑制作用无明显变化。接种生防菌株XNB-02后,烟草中抗病基因被激活。生防菌株XNB-02体内存在表面活性素、伊枯草菌和芬原素等生防相关基因。表明生防菌株XNB-02具有开发为烟草赤星病生防制剂的潜力。

In order to obtain the biocontrol bacterial strains with excellent effect on the prevention and control of tobacco brown spot disease,bacterial strains were isolated and purified from soil samples in Xining City,Qinghai Province.Bacterial strains with good inhibitory effects on tobacco brown spot disease were screened,and their inhibitory effects on six other plant pathogens were determined.The morphological,physiological and biochemical characteristics were analyzed using molecular biological technology to identify the screened strains.The inhibitory mechanism was preliminarily investigated,and the greenhouse pot plant disease prevention test was performed and biocontrol efficacy of this strain on tobacco brown spot was tested.The stability of the fermentation broth of the strain,the biocontrolrelated genes in the strain,and the effect of the strain on the disease resistance genes in tobacco were in-vestigated.The results showed that five strains were obtained after isolation and purification,among which the biocontrol strain XNB-02 had a good inhibitory effect on Alternaria alternata with a radius of inhibition of 10.33 mm,and also had a good inhibitory effect on six other plant pathogens.The strain XNB-02 was identified as Bacillus velezensis.The strain XNB-02 could promote the growth of tobacco seedlings,and the prevention effect of XNB-02 against tobacco can reach 61.82%when the concentration of its suspension was 1×109 CFU/mL.The intracellular crude extract of the biocontrol strain XNB-02 could inhibit the growth of mycelium and spore germination of A.alternata,and change the morphology of mycelium and spores.The inhibitory effect of the biocontainment strain XNB-02 on A.alternata did not change significantly after treatment at temperatures lower than 90℃,pH 3-13,light exposure for seven days,UV irradiation for 240 min and proteinase K.Disease resistance genes were activated in tobacco after inoculation with the biocontrol strain XNB-02.The genes related to biosynthesis of lipopeptide antibacterial substances such as surface activein,ichthyobacillus and fenugreek were present in the biocontrol strain XNB-02.These results suggest that the biocontrol strain XNB-02 has the potential to be developed as a biocontrol agent against tobacco brown spot.