蜡样芽孢杆菌NJSZ-13菌株防治松材线虫病研究

【目的】松材线虫病是一种由松材线虫对赤松、马尾松、黑松等易感树种寄生取食继而引起树种死亡的病害,其发病速度快、易传播,传统的物理、化学防治成本高,且化学防治对环境易造成不可逆的污染,目前还没有较好的防治方法。为寻找安全有效的生物防治方法,笔者探究蜡样芽孢杆菌在温室下对马尾松盆栽苗松材线虫病的防治效果。【方法】采用人工皮接法将2 000、4 000和6 000条松材线虫AMA3虫株分别接种到2年生马尾松幼苗上,测定发生松材线虫病症状的临界值;蜡样芽孢杆菌NJSZ-13菌株是从南京湿地松上分离出的具有杀线虫作用的松树内生细菌,在接种松材线虫AMA3虫株的前7天、当天和接种后第7天用灌根法灌入蜡样芽孢杆菌NJSZ-13菌株菌粉水悬液,并设置对照试验:将发酵液、菌悬液和发酵滤液稀释10倍后,各取20 mL灌注到2年生马尾松根部,其中发酵液和菌悬液的菌体浓度为4×10⁸ cfu/mL,以无菌水和培养基处理为对照,比较蜡样芽孢杆菌不同发酵产物对松材线虫病的防治效果;以灌根法分别施入20 mL菌体浓度为4×10⁸、4×10⁷、2×10⁷ cfu/mL的蜡样芽孢杆菌NJSZ-13菌株菌粉水悬液;将蜡样芽孢杆菌以菌粉水悬液灌根法和菌粉涂抹法两种方式处理2年生马尾松幼苗。从施菌浓度、施菌方法和施菌时间等3个方面对经过低温喷雾干燥制备的蜡样芽孢杆菌菌粉进行防治松材线虫病试验,观察各处理下马尾松的发病情况。【结果】2年生马尾松接种松材线虫AMA3虫株后发病的临界值为4 000条。采用灌根法施入蜡样芽孢杆菌NJSZ-13菌株的发酵液和无菌发酵滤液后不能对松材线虫病起到预防作用,施蜡样芽孢杆菌菌悬液对松材线虫病有一定的预防效果(33%)。蜡样芽孢杆菌对马尾松松材线虫病的预防效果要大于治疗效果,健康马尾松在接种线虫之前以灌根法施入蜡样芽孢杆菌的预防效果会更好。将蜡样芽孢杆菌菌粉直接涂抹松枝,与菌粉水悬液灌根的方法相比,两者对松材线虫病的防治效果相当。同一菌体浓度下蜡样芽孢杆菌菌粉水悬液和发酵菌悬液对松材线虫病的预防效果相当;施用蜡样芽孢杆菌菌粉水悬液对松材线虫病的防治效果随菌体浓度的增加有所提高,当菌体浓度达到一定值以后,增加浓度也不会提高防治效果。【结论】在蜡样芽孢杆菌NJSZ-13菌株防治2年生马尾松幼苗松材线虫病前,先筛选2年生马尾松接种松材线虫的临界值,能有效避免因接种松材线虫浓度过大而使真实防治效果降低的缺点。蜡样芽孢杆菌NJSZ-13菌株菌悬液和经低温喷雾干燥制得的菌粉对松材线虫病均有一定的预防效果,在马尾松未感染松材线虫的健康状态施用蜡样芽孢杆菌NJSZ-13菌株效果更好,通过菌粉涂抹法处理松枝可以对松材线虫病产生预防效果。

A study on the biocontrol of pine wilt disease by Bacillus cereus NJSZ-13

【Objective】The pine wilt disease caused by parasitic Bursaphelenchus xylophilus occurs in susceptible tree species such as Pinus densiflora, P. massoniana and P. thunbergii. There is no optimal control treatment available owing to the fast onset and spread of the disease, high costs of physical and chemical control methods, and environmental pollution issues. To determine safe and effective biological control methods, this study explored the prevention and control of the disease by Bacillus cereus NJSZ-13 using potted P. massoniana seedlings in a greenhouse. 【Method】Different number of Bursaphelenchus xylophilus AMA3 (2 000, 4 000 and 6 000) were inoculated on two-year-old P. massoniana seedlings to determine the critical value of symptoms of the pine wilt disease. Bacillus cereus NJSZ-13 is a nematocidal endophyte that was isolated from P. elliottii in Nanjing. To understand the effect of B. cereus NJSZ-13 on the pine wilt disease, the roots of two-year-old P. massoniana were inoculated with a 20 mL suspension of 4×10 ⁸ cfu/mL of B. cereus NJSZ-13 as the treatment and with sterile water and an agar medium as the control. Twenty milliliters of bacterial powder suspension of 4×10 ⁸,4×10⁷ and 2×10⁷ cfu/mL of B. cereus NJSZ-13 were applied by the root filling method. Two-year-old P. massoniana seedlings were treated under two conditions, namely root filling with powder suspension and application of bacterial powder. Bacillus cereus NJSZ-13 and B. xylophilus AMA3 were inoculated with the P. massoniana roots in different orders, namely together, before 7 d, and after 7 d, and the control group was set up. The bacterial concentration, inoculation method, and time were included in the evaluation of the biocontrol effect. 【Result】The critical point of B. xylophilus inoculation in P. massoniana was 4 000. The fermented culture and sterilized culture of B. cereus NJSZ-13 had no effect on the pine wilt disease development. The biocontrol effect of the B. cereus NJSZ-13 suspension on the pine wilt disease was 33%. At the same concentration, the preventive effect of B. cereus NJSZ-13 did not differ between the powder and bacterial suspensions. The biocontrol effect of B. cereus NJSZ-13 did not improve when the bacterial suspension concentration reached a certain level. The biocontrol effect of B. cereus NJSZ-13 was significantly higher than that of the control. The control effect of B. cereus NJSZ-13 powder application was the same as that of the root filling method. 【Conclusion】The critical point of B. xylophilus inoculation in two-year-old P. massoniana can effectively avoid the disadvantage of an excessive concentration of B. xylophilus inoculation, which can reduce the control effect. The B. cereus NJSZ-13 bacterial suspension and powder had control effects on the pine wilt disease. It is better to apply B. cereus NJSZ-13 when P. massoniana is still healthy and uninfected. The preventive effect of B. cereus NJSZ-13 can be achieved by smearing the bacteria on pine branches.