蜡样芽孢杆菌NJSZ-13菌株对松材线虫产卵和繁殖的影响

Effect of Bacillus cereus NJSZ-13 strain on oviposition and reproduction of Bursaphelenchus xylophilus

【目的】已有研究表明蜡样芽孢杆菌(Bacillus cereus)NJSZ-13菌株对于马尾松具有促生作用,且菌悬液的促生效果最佳。为了进一步解析和评价该菌对松材线虫的生防机理,在虫液和卵液中加入NJSZ-13菌株,研究该菌株对松材线虫的杀线效果以及对其产卵、卵孵化和繁殖量的影响。【方法】首先通过离心将蜡样芽孢杆菌NJSZ-13菌株分成菌悬液、发酵液和发酵滤液,并调配成原液、5、10、100和1 000倍的稀释液,其中菌悬液和发酵液的原液浓度为3×108cfu/mL,以无菌水和NB培养基为对照。(1)用浸渍法测定NJSZ-13菌株在24 h和48 h对松材线虫的杀线效果。(2)挑取10对生长良好的雌雄4龄幼虫于1.5 mL离心管,用NJSZ-13菌株处理24 h后将虫卵从离心管冲洗到35 mm培养皿中,统计各处理的产卵量。(3)在35 mm小皿中加入1 mL无菌卵液(约含400个卵)和NJSZ-13菌株,统计12、24和36 h的孵化虫数。(4)将100μL线虫液(含500条松材线虫)与100μLNJSZ-13菌株混合接入60 mm培养皿的灰葡萄孢中,在25℃培养箱培养5 d,用贝尔曼法分离线虫并计数。【结果】(1)NJSZ-13菌株的发酵液和发酵滤液原液处理24 h后的杀线率即高达100%,而其菌悬液原液处理48 h后的杀线率仅为81.47%。(2)NJSZ-13菌株原液能极显著地抑制松材线虫产卵,随着稀释倍数的增大,这种抑制作用逐渐减小。(3)NJSZ-13菌株的10倍稀释液处理松材线虫卵,在12 h时极显著地抑制卵的孵化,在24 h时仍达到显著水平,在36 h时仅发酵液和发酵滤液有显著的抑制作用。100倍稀释液处理12 h时,发酵液和发酵滤液相对于对照有显著差异,其他处理时间点均无显著差异。(4)NJSZ-13菌株原液和5倍稀释液处理松材线虫,其繁殖力与对照组有显著差异(P<0.05)。10倍稀释液处理松材线虫时,菌悬液对线虫繁殖力无明显的影响,而发酵液和发酵滤液与对照有显著差异。【结论】蜡样芽孢杆菌NJSZ-13菌株的菌悬液、发酵液、发酵滤液均对松材线虫有杀线作用。其中,发酵液和发酵滤液的杀线效果更显著;NJSZ-13菌株能抑制松材线虫的产卵、卵孵化和繁殖,同样发酵液和发酵滤液的抑制效果较菌悬液明显。

【Objective】Previous studies have shown that Bacillus cereus NJSZ-13 strain has the best effect on Pinus massoniana growth. To further analyze and evaluate the underlying biological and control mechanism of the strain on Bursaphelenchus xylophilus, NJSZ-13 strain was added to the insect and egg liquid to study the killing effect of the strain on B. xylophilus and its oviposition, egg hatching and reproduction activities.【Method】NJSZ-13 strain was divided into suspension, fermentation broth and fermentation filtrate by centrifugation, and then mixed into the original solution, with 5, 10, 100 and 1 000 dilutions. The concentrations of suspension and fermentation broth were 3×108 cfu/mL, and sterile water and NB medium were used as the control.(1)The nematocidal effects of NJSZ-13 strain on B. xylophilus in 24 and 48 hours were determined by the immersion method.(2)Ten pairs of female and male fourth instar larvae with good growth were selected and placed in a 1.5 mL centrifuge tube. After being treated with NJSZ-13 strain for 24 h, the eggs were washed from the centrifuge tubes into 35 mm culture dishes, and the amounts of eggs laid in each treatment counted.(3)Sterile egg solutions(1 mL, containing about 400 eggs) and NJSZ-13 strains were placed into 35 mm dishes for the counting of the numbers of hatched worms at 12, 24 and 36 h.(4)The mixtures of nematode solution(100 μL)(containing 500 B. xylophilus) and 100 μL NJSZ-13 strains were put into 60 mm culture dishes of Botrytis cinerea. The nematodes were isolated and counted using the behrmann method after being cultured in a 25 ℃ incubator for five days. 【Result】(1)The nematocidal rate of NJSZ-13 was 100% after 24 h of treatment, while the killing rate of NJSZ-13 strain was only 81.47% after 48 h of treatment.(2)NJSZ-13 strain could inhibit the oviposition of B. xylophilus significantly, and the inhibition decreased with an increase in dilution ratio.(3)The 10-fold dilution of the NJSZ-13 strain inhibited the hatching of the eggs significantly at 12 h, with a significant level achieved at 24 h, only the fermentation broth and fermentation filtrate exhibited significant inhibition at 36 h. When treated with the 100-dilution for 12 h, the fermentation broth and fermentation filtrate had significant differences when compared with the control, with no significant differences at the other treatment time points.(4) There were significant differences in fecundity between NJSZ-13 strain and the control group(P < 0.05). When 10-dilution solution was used to treat B. xylophilus, the bacterial suspension had no significant effect on B. xylophilus fecundity.【Conclusion】Bacillus cereus NJSZ-13 strain had nematocidal effects on B. xylophilus. Among them, the nematocidal effects of fermentation broth and fermentation filtrate were more significant;NJSZ-13 strain could inhibit B. xylophilus oviposition, egg hatching and reproduction, and the inhibitory effects of fermentation broth and fermentation filtrate were more obvious than those of the bacterial suspension.