枯草芽胞杆菌R31影响巴西蕉根系活性氧产生及对枯萎病的防治效果

Inoculation Concentration of Bacillus subtilis Strain R31 Influenced the Roots Reactive Oxygen Species Generation of Banana(Musa spp.AAA Cavendish cv.Baxi) and Its Biological Control Effect on Fusarium Wilt

研究枯草芽胞杆菌R31接种浓度对香蕉根系活性氧产生、R31自身根系定殖和枯萎病防治效果的影响。首先检测了不同浓度R31接种对巴西蕉根系Mn-SOD活性和过氧化氢含量的影响,并研究了过氧化氢对不同细胞浓度R31生物被膜形成的影响,然后利用R31-gfp观察了不同浓度R31接种在香蕉根表定殖的差异,最后利用大田试验验证了不同浓度R31可湿性粉剂的枯萎病防效。研究结果显示,1.0×10~6和1.0×10~7 cfu/mL R31菌体灌根使巴西蕉根系Mn-SOD活性分别在0.76~5.99和0.81~6.55 U/g显著波动,根系过氧化氢含量显著增加,而1.0×10~8 cfu/mL R31菌体接种的根系Mn-SOD活性在4~5 U/g波动,不激发根系过氧化氢爆发。80和120μmol/mL以上过氧化氢分别抑制1.0×10~5和1.0×10~6 cfu/mL R31在24 h内形成薄皮,但分别显著促进2种浓度R31在72 h形成薄皮。40~240μmol/mL过氧化氢对1.0×10~7和1.0×10~8 cfu/mL R31 24 h的薄皮形成无影响,但显著促进2种浓度R31 72 h的薄皮形成。1.0×10~6 cfu/mL R31-gfp接种巴西蕉后第5 d才可以观察到根表产生荧光,而1.0×10~7和1.0×10~8 cfu/mL R31-gfp接种处理在第3 d即可观察到根表产生明显荧光。利用R31可湿性粉剂进行田间试验,以2.0×10~6和2.0×10~7 cfu/mL2000 mL灌根处理新植巴西蕉,施用4次后,枯萎病防效分别为35.41%和72.96%。R31低浓度接种能够激发香蕉根系免疫反应和活性氧产生,并延缓低浓度R31在根表定殖,最终影响其对枯萎病的防效。

The purpose of this paper is to study the effects of the inoculation concentrations of Bacillus subtilis strain R31 on banana roots reactive oxygen generation,its own roots colonization and their biological control on Fusarium wilt.The Mn-SOD activities and H_2O_2 concentrations of banana（Musa spp.AAA Cavendish cv.Baxi）seedlings roots were investigated followed by the inoculation with 1.0×10~6,1.0×10~7 or 1.0×10~8 cfu/mL R31,respectively.The influence of different concentrations of H_2O_2 on R31 pellicle formation in MSgg medium,and the colonization of different concentrations R31-gfp on Baxi seedlings roots were also studied.Then,the biological control effects of different concentrations of R31 on Fusarium wilt were tested in fields.Results showed that significantly changed Mn-SOD activities and improved H_2O_2 concentrations of Baxi seedlings roots were observed after inoculated by 1.0×10~6 or 1.0×10~7 cfu/mL of R31,respectively.R31 inoculation at 1.0×10~8 cfu/mL could also significantly influence the Mn-SOD activities,which kept at 4-5 U/g at all tested time.However,there is no significant H_2O_2 burst of Baxi roots after 1.0×10~8 cfu/mL R31 inoculation.The concentration of H_2O_2 higher than80 or 120μmol/mL could significantly inhibit the pellicle formation of R31 in MSgg medium with 1.0×10~5 or 1.0×10~6 cfu/mL cell density at 24 h,but significantly improve the pellicle formation with the same treatment at72 h.In the treatments with1.0×10~7 and 1.0×108 cfu/mL R31,40 to 240μmol/mL H_2O_2 concentrations had no influence on the pellicle formation of R31 at 24 h,but significantly improved the pellicle formation at 72 h.Green fluorescence was observed in the cuticle of Baxi seedlings roots inoculated by R31-gfp at 1.0×10~6 cfu/mL after 5 days,and those inoculated by R31 on 1.0×10~7 or 1.0×10~8 cfu/mL after 3 days.From December 2014 to May 2015,the Fusarium wilt control efficiency were 35.41%and 72.96%,respectively,after the Baxi seedlings were irrigated by 2000 mL 2.0×10~6 and 2.0×10~7 cfu/mL R31 spores wettable powder suspension broth and water for 4 times in the 3 experiment areas set in Xuwen county of Zhanjiang City.It was showed that low inoculation concentrations of B.subtilis strain R31 could activate the Baxi roots immune system and caused reactive oxygen generation,then delay its own colonization on roots cuticle,which further influenced the biological control efficiency of fusarium wilt.