防治松褐天牛成虫的白僵菌菌株室内筛选和鉴定

Screening and Identification of Beauveria bassiana Strains for Biocontrol of Monochamus alternatus Adults(Coleoptera:Cerambycidae)

【目的】筛选对松褐天牛成虫具有高致病力的白僵菌菌株,以期用于防治松褐天牛成虫。【方法】收集被病原真菌感染的松褐天牛幼虫,25℃条件下PDA培养基培养分离纯化病原真菌。使用25℃、相对湿度60%、光周期16 L∶8D条件下羽化5~10^天的健壮松褐天牛成虫,生物测定病原真菌致死效果。将PDA培养基上培养获得的病原真菌孢子,分别溶于0.05%的吐温80溶液中,配制成终浓度分别为1×10^6~3×10^6或者1×10^6~3×10^7个·mL^-1的孢子悬浮液,0.05%的吐温80溶液作为空白对照。采用涂枝法和喷洒法处理松褐天牛成虫,以累计校正死亡率为指标评价菌株的致病力。折线图由R包“ggplot2”绘制。采用ITS1和ITS4扩增真菌保守序列,经连接转化后测序。采用软件muscle比对序列,MEGA软件中的最大似然法构建系统发育树。【结果】从感染的23头松褐天牛幼虫中共分离出23株白僵菌菌株,通过喷洒法初步筛选出18天累计校正死亡率在30%以上的6个菌株。选取B7和B92个产孢量大(14天,10^8·cm^-2)的菌株,涂枝法进行毒力测试结果表明,B9菌株在12天的累计校正死亡率即可达到10^0%,而B7菌株在12天时的累计校正死亡率约为50%±10^%,B9菌株的致死速率显著高于B7菌株。ITS扩增及测序结果表明与球孢白僵菌的同源性为99%。系统发育树结果也表明B9菌株与球孢白僵菌聚为同一分支上。结合形态、NCBI Blast比对结果和系统发育树共同确定B9菌株为球孢白僵菌。【结论】本研究得到对松褐天牛成虫高毒力的球孢白僵菌菌株,对于防治松褐天牛具有良好的应用前景,但实际应用价值需要进一步的田间试验进行确认。

【Objective】In this study,Beauveria bassiana strains with high pathogenicity against Monochamus alternatus adults were screened in order to control M.alternatus effectively.【Method】The Monochamus alternatus larvae infected by the pathogenic fungus were collected,and the pathogenic fungus was isolated and purified in PDA medium at 25℃.The robust Monochamus alternatus adults emerged in 5-10^ d(at 25℃,relative humidity 60%,photoperiod 16L∶8D)were used as bioassay object of pathogenic fungus lethal effect.The spores of pathogenic fungi cultured on PDA medium were collected and dissolved in 0.05%Tween 80 solution to prepare a spore suspension with a final concentration of 1×10^6~3×10^6 or 1×10^7~3×10^7·mL^-1,respectively,and 0.05%Tween 80 solution was used as a blank control.Two methods(applying and spraying spore suspension on branches)were used to treat the adult Monochamus alternatus,and the virulence of the strains was evaluated based on the cumulative corrected mortality.The line graph was drawn by R package"ggplot2".ITS1 and ITS4 were used to amplify the conserved sequences of fungi,and sequenced after connection and transformation.The phylogenetic tree was constructed by using the software muscle to compare sequences and the maximum likelihood method in MEGA software.【Result】A total of 23 Beauveria bassiana strains were isolated from the 23 infected Monochamus alternatus larvae,and 6 strains with a cumulative corrected mortality rate of more than 30%over 18 days were preliminarily screened by spraying method.Two strains,B7 and B9,with high spore production(14 d,10^8·cm^-2)were selected.The virulence test results showed that the cumulative corrected mortality of B9 strain on the 12th day reached 10^0%,while the cumulative corrected mortality rate of B7 strain in that time was about 50%±10^%.Thus the lethal rate of B9 strain was significantly higher than that of B7 strain.The results of ITS amplification and sequencing showed that the homology with Beauveria bassiana was 99%.The phylogenetic results also showed that B9 strain and B.bassiana clustered on the same branch.Combined with the morphology,NCBI Blast comparison results and phylogenetic tree,the B9 strain was determined to be Beauveria bassiana.【Conclusion】In this study,the strains of Beauveria bassiana with high virulence to adults of M.alternatus were successfully screened.It is expected that the use of entomopathogenic fungi to control M.alternatus adults has a good application prospect,but the practical application value needs to confirm in further field trials.