番茄青枯病拮抗菌株B-6的诱变选育

Mutagenesis Breeding of the Antagonistic Bacterium Strain B-6 against Tomato Bacterial Wilt

青枯病是由青枯劳尔氏菌(Ralstonia solanacearum)侵染引起,是设施番茄生产上一类重要的土传性病害,给种植户造成巨大的经济损失。筛选高效突变生防菌株成为目前青枯病防控的重要手段之一。为了给生防菌剂的进一步开发应用提供依据,研究以筛选出具有抑菌活性的铜绿假单胞菌(Pseudomonas aeruginosa)B-6为出发菌株,采用紫外线和亚硝酸钠复合诱变处理,计算致死率和正突变率确定最佳诱变条件,利用平板初筛和摇瓶发酵复筛获得高效突变菌株,测定高效突变菌株抑菌活性的遗传稳定性,并与出发菌株进行生长曲线的对比。结果表明,在紫外照射时长为3 min和亚硝酸钠浓度为0.045 mol/L复合诱变条件下,获得1株抑菌活性最强的高效突变菌株UH-9;该菌株对番茄青枯病菌抑菌圈平均直径达到28.6 mm,抑菌活性与出发菌株相比提高了86.5%,菌株在传代培养7次后抑菌活性无显著变化,具有良好的遗传稳定性。复合诱变处理比单一紫外诱变处理及亚硝酸钠诱变处理抑菌活性分别提高39.1%和25.4%,复合诱变比单一诱变抑菌活性更好。通过对比菌株UH-9和B-6的生长曲线可知,生长时期相同,且13 h后UH-9的OD600高于B-6。

Bacterial wilt caused by Ralstonia solanacearum is an important soil-borne disease in the production of protected tomatoes,which can result in huge economic losses for growers.Screening out mutant strains with high control efficiency has become one of the important methods.To provide a basis for the further development and application of biocontrol agents,in this study,Pseudomonas aeruginosa B-6 with antibacterial activity was selected as the starting strain and mutated by the UV-NaNO2 compound.The optimal mutation conditions were determined by the lethality rate and positive mutation rate,and the highefficiency mutant strains were first screened on the plate and re-screened by fermentation in shake flasks.The genetic stability of the antibacterial activity of the high-efficiency mutant strain was determined and the growth curve was compared with that of the starting strain.The results showed that a mutant strain UH-9 with high efficiency and the strongest antibacterial activity was selected after the combined treatment of 3 min of ultraviolet irradiation and 0.045 mol/L of sodium nitrite concentration.The average diameter of the inhibition zone against Ralstonia solanacearum reached up to 28.6 mm,and the activity increased by 86.5%compared with that of the starting strain.After subculture for seven generations,the mutant strain UH-9 was proved to be genetically stable since the antibacterial activity had no significant difference.The antibacterial activity of composite mutagenesis treatment was 39.1%and 25.4%higher than that of single UV mutagenesis treatment and sodium nitrite mutagenesis treatment,respectively.The antibacterial activity of composite mutagenesis was better than that of single mutagenesis.By comparing the growth curves of strain UH-9 and strain B-6,the growth period was the same,and the OD600 of strain UH-9 was higher than that of strain B-6 after 13 h.