关键词: 松材线虫, 生防真菌, 伊氏线虫真菌, 转录组学

Abstract:

Objective: Pine wilt disease has been intensifying in China in recent years, and its pathogenic factor is pine wood nematode (Bursaphelenchus xylophilus), and it is a globally recognized quarantine pest. Esteya vermicola (EV) is an important biocontrol fungus infesting the pine wood nematode. The lunate spores produced by EV can infect and kill the pine wood nematode, thus EV has a good application prospect in the biological control of the pine wood nematodes. How to increase the growth rate, sporulation ability, and the virulence of EV to the pine wood nematode through cultivation, and then apply it to the field biological control is an important problem that needs to be solved urgently. Method: In this study, EV was cultured in carbon and nitrogen medium respectively, and the growth rate and spore production were determined. The mycelia were harvested and RNA was extracted for library construction and differential analysis. The corrected P-value (P_adj) and the absolute value of the logarithm of the base 2 fold change (|log₂FoldChange|) were used to judge the significance of the differential expression. Result: EV fungus grew fast under nitrogen culture conditions, and the total number of expressed genes and specifically expressed genes of EV fungi under nitrogen culture conditions were higher than those under carbon culture conditions. A total of 7 138 genes were significantly differently expressed in the nitrogen group compared to the carbon group, of which 3 571 genes were significantly up-regulated, and 3 567 genes were significantly down-regulated. There were 474 genes specifically expressed in the nitrogen group and 295 genes specifically expressed in the carbon group, and the number of genes specifically expressed in the nitrogen group involved nematicidal genes was greater than that in the carbon group. After cluster analysis, the variation trend of differential gene expression levels was divided into 4 clusters, among which there were 165 genes expressed much higher in the nitrogen group than in the carbon group; and 65 genes expressed much higher in the carbon group than in the nitrogen group. The expression of subtilisin and toxin synthesis-related genes involved in the activity of lethal nematodes and in the process of infecting nematodes were significantly up-regulated in the nitrogen group. The expression of subtilisin in the nitrogen group was 2.29-363.52 times higher relative to that in the carbon group. Conclusion: Carbon and nitrogen media have significant effects on the transcription of EV biocontrol fungi. The total number of genes and specifically expressed genes of EV fungi under nitrogen culture conditions are higher than those under carbon culture conditions. Subtilisin and toxin synthesis-related genes with nematicidal effects are significantly up-regulated under nitrogen culture. The research results will provide an important theoretical basis for the cultivation, gene function research and strain improvement of highly virulent EV strains, and then lay a foundation for improving the control efficacy of EV biocontrol fungi.

Key words: pine wood nematode, biological control, Esteya vermicola, transcriptomics