马铃薯甲虫病原细菌分离及生防菌的筛选与鉴定

Isolation,Screening and Identification of Bacterial Agents for Biological Control of Colorado Potato Beetle (Leptinotarsa decemlineata)

【目的】分离筛选对马铃薯甲虫(Colorado potato beetle,CPB)具强致病性的细菌生防菌株并对其鉴定。【方法】从CPB虫体上分离病原细菌,利用几丁质酶和蛋白酶活性初步筛选致病菌株,然后通过室内和田间试验筛选出可导致CPB大量死亡的生防菌。根据形态学、生理生化特性和16S rDNA序列分析等进行鉴定,确定生防菌株的分类学地位。【结果】从新疆CPB分布区采集的CPB发病成虫虫体上分离纯化获得126个细菌菌株,其中36个菌株对昆虫体壁几丁质和蛋白质具有降解活性。在室内马铃薯叶片饲养的CPB各龄幼虫和成虫上测试,11个菌株对1—2龄幼虫表现出不同程度致病力,在接种后1—3 d始见幼虫死亡,处理后7 d幼虫的累积死亡率达到21.0%—77.9%,所有菌株对2龄以上CPB的致病力不明显或无致病力。在田间种植的马铃薯植株上测试这11个致病菌株,处理后观察,CPB008、CPB012和CPB016等3个菌株在10 d内对CPB的累积致死率达到41.0%—49.9%;另外还有3个菌株对CPB的致死率也在21.1%以上。这6个菌株均为革兰氏阳性菌,从形态学和生理生化特性鉴定它们均属于芽孢杆菌(Bacillus),通过PCR扩增分别获得它们的16S rDNA序列并登录到NCBI GenBank中,由此分析比对,菌株CPB008、CPB012、CPB016和CPB111为苏云金芽孢杆菌(B.thuringiensis);CPB072、CPB108为萎缩芽孢杆菌(B.atrophaeus)。【结论】筛选获得的3株苏云金芽孢杆菌(CPB008、CPB012、CPB016)对马铃薯甲虫致病性强,具有重要的应用潜力。

[Objective] The objective of this study is to isolate and screen bacterial biocontrol agents against Colorado potato beetle （CPB） （Leptinotarsa decemlineata）. [Method] Diseased CPB adult samples were collected from Xinjiang and bacterial isolates were obtained from the samples. These isolates were tested for CPB pathogenicity. Highly insecticidal isolates were screened through laboratory and field assays and identified morphologically and molecularly. [ Result ] A total of 126 bacterial isolates were obtained from diseased adults of CPBs collected from Xinjiang Autonomous Region of China and 36 isolates showed protein- and chitin-degrading activity. Among these isolates, 11 i^fected 1st instar and 2nd instar CPB larvae and resulted in lethal rates of 21.0%-77.9% within 7 days after inoculation when tested on detached potato leaves in the laboratory. The 11 infectious isolates were selected for field tests and 6 of them induced high CPB mortality rates. The CPB-lethal rates by the 3 isolates （CPB008, CPB012 and CPB016） reached 41.0%-49.9%. The other 3 isolates （CPB072, CPB108 and CPBlll） also had significantly high CPB lethal rates （21.1%-37.8%）. CPB008, CPB012, CPB016 and CPBlll were identified as Bacillus thuringiensis and CPB072, CPB108 were B. atrophaeus, respectively. [Conclusion] The screened 3 isolates （CPB008, CPB012 and CPB016） have strong CPB pathogenicity, and could be potential applications in biological control of this destructive pest in Xinjiang of China.