关键词: 绿僵菌, 蓝光照射, 产孢, fluG, 基因表达

Abstract: 【Objective】 The objective of this study is to determine the effect of blu-rays on Metarhizium anisopliae conidiation and related regulating gene fluG expression, and to provide a theoretical basis and technical parameters for the scale up conidia production of M. anisopliae. 【Method】 The testing strain, M. anisopliae M202 being highly virulent to white grubs, was inoculated and cultured on PDAY plates. The fungal development was observed every 12 h by microscope to confirm the developmental process of the sprout, early hyphae, exuberant hyphae, initial conidiating, exuberant conidiating and stable conidiating. The strain culture developed at 24, 48, and 72 h stages in dark were exposed to blu-rays for 0.75, 1.5, 2.25, 3, 3.75, 4.5, 5.25 and 6 h (equal to 6 804, 13 608, 20 412, 27 216, 34 020, 40 824, 47 628 and 54 432 J?m^-2), respectively. Then all of them were incubated until 10th day to finish conidiation. Each of treatments was measured conidiation by punch sampling and microsurgical counting. The conidial yield was calculated and used to assess the sensitivity of the fungal development stages to blu-rays as well as the impact of blu-rays levels on conidiation. In the meanwhile, the fluG was cloned and the real-time PCR reaction system was built. The strain culture developed at 48 h stages in dark were exposed to blu-rays at 8 gradient illuminances in 6 804-54 432 J?m^-2, respectively. Then the cultures were frozen immediately in liquid nitrogen. For the each treated culture, RNA was extracted and reverse-transcribed into cDNA, and then the expression of fluG was measured by real-time PCR. So it could assess the influence of blu-rays on gene fluG for regulating conidiation. 【Result】 Development of M. anisopliae M202 strain could be divided into several stages, including germination before 24 h, hyphae fast growing during 24-72 h with pre-conidiation at around 48 h, initial conidiation at around 60 h, rising conidiation in 72-96 h, late conidiation in 96-120 h, and conidiation mature after 120 h. After 7-10 d, the abundance of conidiation tended to constant. The results of blu-rays treatments showed that all of the 8 gradient illuminances did not cause significant difference in young mycelia of 24 h age compared with the control by no blu-rays. When older mycelia of 48 h age, growing rapidly at pre-conidiation, were exposed to the blu-rays, the conidiation could increase significantly. The optimal illuminances were in 20 412- 40 824 J?m^-2, and maximum conidiation by 34 020 J?m^-2 illuminance got up to 1.50 folds compared with the control. The developmental stage of 72 h age, forming conidiation structure and increment, was most sensitive to blu-rays. The low illuminance of 6 804 J?m^-2 could significantly improve conidiation, and a wide range would be valid. For fluG, the blu-rays could significantly improve expression in the mycelia of 48 h age. With the increase of illuminance, the fluG expression was positively correlated with illuminance in less than 20 412 J?m^-2 but negatively correlated when more than 20 412 J?m^-2. The maximum expression was 2.41 folds compared with the control. Grey relationship analysis indicated that the correlation of conidiation and fluG expression was r=0.74 level when the association factor was 0.5. 【Conclusion】 Blu-rays could promote M. anisopliae conidiation and fluG expression. Sensitivity of M. anisopliae developmental stages to blu-rays was significantly different. The 48-72 h aged cultures treated with blu-rays would yield maximum conidiation at 34 020 J?m^-2 illuminance. The high correlation between conidiation and fluG expression indicated that fluG involved in the regulation of conidiation in M. anisopliae development.

Key words: Metarhizium spp., blu-rays, conidiation, fluG, gene expression