枯草芽孢杆菌B006产surfactin突变株特性及其对黄瓜枯萎病的抑制能力

Characteristics of Surfactin Mutants of Bacillus subtilis B006 and Their Suppressing Ability against Cucumber Fusarium Wilt

为了明确surfactin产量不同的芽孢杆菌对黄瓜枯萎病的控制作用,本研究采用N+注入诱变共获得1250株芽孢杆菌B006的突变株,通过对黄单孢杆菌的抑菌试验筛选并获得3株产surfactin突变株B841、B73和B1020。对各突变株NB发酵液的HPLC ESI MS分析表明:突变株B841和B73的surfactin含量比野生菌株B006分别升高了15.9%和14.8%,而突变株B1020的surfactin含量比野生菌株降低了85.2%;各突变株fengycin产量未发生改变。对surfactin各组分的质谱图解析,发现突变株B841和B1020的surfactin组分发生变化,突变株B841的surfactin组分中无m/z为995的同系物,而突变株B1020的MS图谱中只发现了m/z为1037和1051的surfactin同系物;对突变株的生物学特性测定表明:突变株B73和B1020的菌落形态发生改变,生长周期缩短;3个突变株比野生菌株产芽孢能力下降,突变株B1020在含有玉米粉和豆饼粉的培养基上不产生芽孢。对各菌株添加到育苗基质中(浓度为106cfu·g 1)后,防治黄瓜枯萎病的效果测定表明:2周时,突变株B1020防效接近于野生菌株B006,达70%以上;但3周时,防效下降到36.2%;突变株B841防效始终低于野生菌株B006,只有29.1%。本研究发现芽孢杆菌B006及其突变株在NA培养液中的surfactin产量的高低与其在育苗基质中的防病效果无直接的相关性,对其在自然土中的防病效果值得进一步研究。

B. subtilis B006 could effectively suppress cucumber wilt caused by Fusarium oxysporum f. sp. cucumerinum. In order to understand the suppressing ability of Bacillus strain B006 and its surfactin-mutants against cucumber wilt disease, we implanted strain B006 by nitrogen ions. Mutants B841, B73 and B 1020 were selected from 1250 implanted strains. Surfactin analysis with HPLC-ESI-MS showed that surfactin amount excreted by strain B841 and B73 in nutrient broth increased by 15.9% and 14.8% respectively, in comparison with their parental strain B006, while that from strain B 1020 decreased by 85.2%. Amounts of fengycin produced by the three mutants were similar with strain B006. Mass spectrum analysis of surfactin showed that its constitution changed, as indicated by absence of m/z at 995 from B841 and only m/z at 1037 and 1051 were detected from mutant B 1020, in comparison with the mass spectrum of B006 with m/z at 995, 1009, 1023, 1037 and 1051. Determination of biological characteristics of the mutants indicated that colony appearance of mutant B73 and B 1020 changed and their growth period shortened, and spore-forming rates reduced on NA and a spore-forming medium consisted of corn and soybean cake powders. Mutant B1020 couldn＇t produce endospores on the spore-forming medium. The control efficacy of the mutants were tested in greenhouse by mixing the cell suspension with substrate at 106 cfu.g-1. Mutant B1020 could suppress cucumber wilt disease effectively similar with B006 at 2 weeks, with the control efficacy over 70%, but at 3 weeks with a lower efficacy of 36.2%. Mutant B841 didn＇t show good suppressing ability, the control efficacy was only 29.1% at 3 weeks. The results indicated that the amounts of surfactin produced in nutrient broth by Bacillus strains was not positive to their control efficacies against cucumber Fusarium wilt in nursery substrate. Further study will be conducted in natural soil.