大豆高隆象致病球孢白僵菌菌株BEdy1的鉴定及毒力测定

Species Identification and Virulence Determination of Beauveria bassiana Strain BEdy1 from Ergania doriae yunnanus

【目的】大豆高隆象(Ergania doriae yunnanus)是我国南方大豆田间主要害虫之一。本研究旨在明确一株高隆象自然致病真菌的种类及其生物学特性,并测定该菌株对大豆高隆象成虫的毒力,为大豆高隆象的生物防治提供新材料。【方法】对采集的真菌进行分离、纯化培养,形态学观察;提取真菌DNA进而扩增rDNA-ITS,利用BLAST比对和系统进化树构建,鉴定田间致高隆象自然死亡的真菌种类;设置不同培养温度,通过十字交叉法和血球计数板方法测量计算真菌生长速率和产孢量;配置不同浓度的球孢白僵菌(Beauveria bassiana)BEdy1孢子悬浮液,采用浸虫处理法测定对大豆高隆象的毒力,与商品化球孢白僵菌产品LH毒力进行对比;大豆叶片喷施BEdy1孢子悬浮液,评价高隆象取食后的致死效果。【结果】致使高隆象田间自然发病死亡的真菌为球孢白僵菌,菌株命名为BEdy1。该菌株在22—28℃有较高的生长速率,26℃下生长速率最高,达4.34 mm·d^-1;在20—26℃有较高的产孢量,22℃下产孢量最大,为4.63×10^6孢子/mm^2。浓度为1.0×10^5、1.0×10^6、1.0×10^7和1.0×10^8孢子/mL的BEdy1孢子悬浮液处理高隆象成虫10 d,死亡率分别为49.33%、77.33%、88.67%和98.00%,对照死亡率仅为10%。1×10^8孢子/mL孢子悬浮液处理下,LT50为(6.79±0.13)d,僵虫率为74.67%,发病严重度最高。10 d的LC50和LC95分别为4.80×10^4和3.57×10^7孢子/mL。浓度为1×10^8孢子/mL的商品化球孢白僵菌产品LH处理高隆象成虫,10 d死亡率为58.00%,僵虫率为4.67%,极显著低于同浓度的BEdy1处理。大豆叶片喷施BEdy1孢子悬浮液后喂食高隆象,10 d死亡率为100%,僵虫率为63.33%,LT50为(5.27±0.35)d。【结论】适当环境条件下,球孢白僵菌菌株BEdy1生长速度快,产孢量高,对大豆高隆象成虫有很高的致死率,具有较大的开发应用潜力。

【Objective】Ergania doriae yunnanus has become one of the main pests in soybean fields in southern China.The objective of this study is to investigate the species and biological characteristics of an entomopathogenic fungus that naturally caused the death of E.d.yunnanus,determine the virulence of this fungal strain to E.d.yunnanus adults,and to provides a new way for biological control of E.d.yunnanus.【Method】The fungus from E.d.yunnanus was isolated and purified,and the fungal DNA was extracted for amplification of rDNA-ITS.Subsequently,BLAST alignment and phylogenetic tree construction were used to identify fungal species that caused the natural death of E.d.yunnanus.The growth rate and sporulation of the fungus were measured by cross method and hemocytometer method at different culture temperatures.The virulence of fungus BEdy1 to E.d.yunnanus was assessed on the lethality of the pests by treatment with different concentrations of spore suspension and estimated by comparison with the commercial fungal agent LH.Finally,the soybean leaves sprayed with BEdy1 spore suspension were used to evaluate the lethal effect after E.d.yunnanus feeding.【Result】The fungus caused the death of E.d.yunnanus under natural conditions was identified as Beauveria bassiana,named BEdy1.BEdy1 had a high growth rate at 22-28℃,and the highest growth rate was 4.34 mm·d^-1 at 26℃.There was a high sporulation at 20-26℃,and the highest sporulation was 4.63×10^6 spores/mm^2 at 22℃.The mortality at 10 th day of E.d.yunnanus adults treated with 1.0×10^5,1.0×10^6,1.0×10^7 and 1.0×10^8 spores/m L of B.bassiana BEdy1 spore suspension was 49.33%,77.33%,88.67%and 98.00%,respectively.The mortality of the control treatment was only 10%.The effect was best under the treatment of 1×10^8 spores/mL,with the LT50 of(6.79±0.13)d and the cadaver rate of 74.67%.The LC50 and LC95 at 10 th day were 4.80×10^4 and 3.57×10^7 spores/mL,respectively.Treatment with 1×10^8 spores/mL of a commercial B.bassiana agent LH,the mortality and cadaver rate at 10 th day were only 58.00%and 4.67%,which were significantly lower than those of the same concentration of BEdy1 treatment.After 10 days of E.d.yunnanus feeding on BEdy1 treated soybean leaves,the mortality and cadaver rate were 100%and 63.33%,respectively,and the LT50 was(5.27±0.35)d.【Conclusion】Under appropriate conditions,the B.bassiana strain BEdy1 has fast growth rate,high sporulation,and high lethality rate to E.d.yunnanus adults,which has great potential for development and application in the future.