关键词: 球孢白僵菌, 松褐天牛成虫, 菌株筛选鉴定, 生物防治

Abstract:

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 1×10⁶~3×10⁶ or 1×10⁷~3×10⁷·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, 10⁸·cm^-2) were selected. The virulence test results showed that the cumulative corrected mortality of B9 strain on the 12^th day reached 100%, 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.

Key words: Beauveria bassiana, Monochamus alternatus adult, screening and identification of strains, biocontrol