不同培养基继代培养球孢白僵菌对西花蓟马毒力和产孢量的影响

Influence of Subculture on Virulence to Frankliniella occidentalis and Conidial Production of the Entomopathogenic Fungus Beauveria bassiana

【目的】通过开展连续继代培养对高毒菌株产孢和毒力的影响研究,获得对西花蓟马（Frankliniella occidentalis）毒力和产孢量均高且稳定的球孢白僵菌（Beauveria bassiana）菌株,进一步提出有效防止菌株退化、优良菌株保存和改良的方法与措施,为高效菌株的规模化生产提供参考。【方法】室内条件下,采用浸虫法,用浓度为1×10^7个孢子/mL球孢白僵菌悬浮液处理西花蓟马初羽化成虫,分别测定28株不同来源的菌株对西花蓟马的毒力;并将筛选出的4株高毒力菌株的分生孢子分别涂布于玉米琼脂（CMA）培养基与SDAY培养基上,比较不同菌株以及不同培养基之间产孢量的差异,筛选出高产孢菌株与产孢培养基,以筛选得到的白僵菌菌株为初始菌株F_0,把F_0代球孢白僵菌分别接种于CMA培养基、添加蝉蜕的CMA培养基和添加西花蓟马虫尸粉的CMA培养基,分别测定经过不同培养基连续5代继代培养得到的球孢白僵菌对西花蓟马成虫的毒力,并测定产孢量。使用模型模拟分析球孢白僵菌菌株经过不同培养基继代培养后培养代数与产孢量的关系,并对不同培养基培养得到的不同培养代数的白僵菌菌株的产孢量与其对西花蓟马的致死中时（LT50）进行回归分析,研究毒力与产孢量的相关关系。【结果】经过室内毒力测定,处理5 d后,菌株DZDC-9、GZGY-5、WLMQ1-8、SZ-26对西花蓟马成虫的校正累积死亡率均高于90%,LT50均在3 d内,毒力显著高于其他菌株;比较高毒力菌株在两种产孢培养基上的产孢量,发现4株球孢白僵菌的产孢量存在显著差异,菌株DZDC-9的产孢量显著高于其他3株,4株菌株在CMA培养基中的产孢量均明显高于SDAY培养基。随着白僵菌菌株在CMA培养基上连续5代的继代培养,菌株对西花蓟马成虫的致病力呈现下降趋势,从第3代开始致病力显著降低,而添加蝉蜕的CMA培养基培养得到的不同培养代数的分生孢子对西花蓟马致病力则没有显著差异,添加西花蓟马虫尸粉的CMA培养基培养得到的不同代数的分生孢子对西花蓟马致病力有一定的增强趋势;通过模型可以发现单纯以CMA培养基继代培养白僵菌的产孢量随培养代数呈现指数下降,而在培养基中添加蝉蜕或西花蓟马虫尸粉白僵菌产孢量则随培养代数呈现指数上升,蝉蜕或西花蓟马虫尸粉的添加对产孢量有一定的增强作用。白僵菌菌株产孢量与毒力存在一定正相关关系,产孢量相对较高的菌株对西花蓟马成虫的致病力相对较高,致死中时短。【结论】筛选得到一株对西花蓟马高效的白僵菌菌株DZDC-9,产孢培养基中加入蝉蜕和西花蓟马虫尸可以维持菌株生长特性,延缓毒力退化趋势;同一菌株的产孢量可以作为菌株毒力评价的一个指标。

【Objective】The objective of this study is to assess the capability of the Beauveria bassiana against Frankliniella occidentalis in the field, and explore the influence of subculture on virulence and conidial production of the B. bassiana, propose an effective method for preventing the retrogression during successive subculture, and provide a basis for the large-scale production technology of B. bassiana. 【Method】 The virulence of 28 fungal strains to adult F. occidentalis was evaluated by soaking method with B. bassiana concentration of 1×10^7 conidia/mL in the laboratory. The adults were dipped for 5 s in the conidial suspension. Conidial production of the 4 high virulent strains was measured on corn meal agar（CMA） medium and sabouraud dextrose agar yeast（SDAY） medium, screening high conidial production strain and spore production medium by comparing conidial production of different strains on different media. Subsequently, strain DZDC-9 was subcultured on three media（CMA medium, CMA medium with cicada exuviae, and CMA medium with F. occidentalis cadaver powder） to compare its virulence and conidial production over five subcultures. The relationship between conidial production and virulence was analyzed. 【Result】Among 28 strains of entomopathogenic fungi B. bassiana tested for virulence against F. occidentalis in laboratory bioassays, strains DZDC-9, GZGY-5, WLMQ1-8 and SZ-26 were found as the most potent. The corrected cumulative mortalities were over 90% after 5 days, and LT50〈3 d. Conidial production obtained from the CMA medium of each strain was higher than from SDAY medium, and strain DZDC-9 had the higher conidial production among the four strains. The results demonstrated that the virulence of strain DZDC-9 declined with successive subculture. However, when the strain was subcultured on the CMA medium with 0.5% cicada exuviae or 0.5% F. occidentalis cadaver powder as an additive, the virulence increased with successive subculture. Conidial production was not significantly different between the same generation subculture on CMA medium with cicada exuviae and CMA medium with F. occidentalis cadaver powder, but they were significantly greater than conidial production obtained from CMA medium. A positive correlation was found between sporulation and virulence of B. bassiana conidia under different media and culture conditions.【Conclusion】The potential of B. bassiana strain DZDC-9 for the control of F. occidentalis was studied, and proposed an effective method for stabilizing the virulence and sporulation of B. bassiana. Conidial production could be used as an indicator for virulence evaluation.