4株绿僵菌的鉴定及对松褐天牛成虫致病效果比较

Identification of Four Entomopathogenic Metarhizium Strains and Their Virulence against Monochamus alternatus Adults

【目的】为筛选对松褐天牛毒力更高的微生物资源,开展生物防治通过培养性状、分生孢子形态及系统发育树鉴定4个绿僵菌菌株,比较测定各自对松褐天牛的毒力。【方法】对4株绿僵菌的培养性状和分生孢子形态特征进行描述,并对4株绿僵菌的EF-1α基因进行克隆和测序,构建系统进化发育树,综合鉴定4株绿僵菌的分类地位。测定了不同孢子浓度及接种方法(浸虫、浸枝和无纺布)对松褐天牛成虫的致病效果,构建4个菌株对松褐天牛的毒力方程。【结果】1)菌株WTKH和WP08鉴定为平沙绿僵菌;菌株LV2鉴定为金龟子绿僵菌;菌株qc1401鉴定为瘿绵蚜绿僵菌。2)平沙绿僵菌WP08菌株和金龟子绿僵菌LV2菌株对松褐天牛校正死亡率较高,瘿绵蚜绿僵菌qc1401菌株次之,平沙绿僵菌WTKH菌株最低。4个菌株对松褐天牛的致死速度依次为WP08>LV2>qc1401>WTKH,LT50分别为5.52、10.38、16.93和20.80天。3)以平沙绿僵菌WP08为供试菌株,在1.0×10^8、1.0×10^7、1.0×10^6个·m L^-1共3个浓度下,孢子浓度越高则松褐天牛校正死亡率越高、半数死亡时间越短,孢子浓度为1.0×10^8、1.0×10^7时,试验期内校正死亡率均达到100%;半数死亡时间LT50分别为11.38、16.44和24.22天。4)以平沙绿僵菌WP08为供试菌株,3种接种方法40天内松褐天牛死亡率均为100%,LT50浸虫法为5.52天,浸枝法为11.38天,无纺布法为17.21天。【结论】4株绿僵菌均可侵染松褐天牛致其死亡,其中,平沙绿僵菌WP08的致死率和致死速度较快,为防治松褐天牛的优良菌株。

【Objective】Monochamus alternates,causing great economic losses every year,is not only an important borer pest in pinewood,but also the main vector of pine nematode disease.To screen for more effective biocontrol agents against M.alternates,four Metarhizium strains were identified by molecular and morphological characteristics,and their virulence against M.alternates adults was analyzed.【Method】In this study,cultural characteristics and conidia morphology of four Metarhizium strains were observed respectively.The EF-[STBX]1[STBZ]αgene of each strain was cloned and sequenced,and a phylogenetic tree was constructed to comprehensively identify the taxonomic status of 4 strains to species level.The virulence of four Metarhizium strains against M.alternatus adult was tested with different conidia concentrations and inoculation methods(dipping,twig dipping,and nonwoven cloth),and the pathogenesis was evaluated.【Result】1)WP08 and WTKH were clusted into the clade of M.pingshaense,and LV2 was into M.anisopliae,and qc1401 was into M.pemphogum.2)Among the four strains,WP08 and LV2 caused the highest corrected death rate(100%)in the 40-day experimental period,and the corrected death rate of qc1401 and WTKH was 91.9%and 87.9%,respectively.Lethal speed of each strain was in the order of WP08>LV2>qc1401>WTKH with the LT 50 of 5.52,10.38,16.93 and 20.80 d,respectively.3)Among the conidium concentration series of 1.0×10^8,1.0×10^7,1.0×10^6 conidia·mL^-1 of WP08,the higher the conidia concentration,the higher the corrected death rate and the shorter the lethal time of M.alternatus adults.The corrected death rate was as high as 100%at conidium concentration of 1.0×10^8 and 1.0×10^7 conidia·mL^-1 of WP08,and was 83.9%at conidium concentration of 1.0×10^6 conidia·mL^-1,and the LT 50 was 11.38,16.44 and 24.22 d at the three concentration,separately.4)For strain of WP08,all the three inoculation method could cause a death rate of 100%in the experimental period of 40 days,and the LT 50 of insect dipping,twig dipping,and nonwoven cloth were 5.52,11.38,and 17.21 d,respectively.【Conclusion】Four Metarhizium strains are identified as M.pingshaense,M.anisopliae,and M.pemphogum,and all the strains can infect M.alternatus adults and cause their death.M.pingshaense WP08 strain exhibits higher death rate and faster lethal speed than the other three stains,and shows great potential as biocontrol agent for M.alternatus.