蝙蝠蛾幼虫肠道真菌复合体中的多菌种与中国被毛孢分生孢子联合侵染蝙蝠蛾幼虫提高侵染效能

Synergy of Fungal Complexes Isolated from the Intestines of Hepialus lagii Larvae in Increasing Infection Potency

摘要：应用真菌特异性引物PCR测序、克隆测序和SNP质谱基因分型法，鉴定2株蝙蝠蛾幼虫活体肠道的真菌分离株（CHl和CH2）中的多种菌物。应用这2株分离株与中国被毛孢分生孢子联合侵染蝙蝠蛾幼虫，观察其协同侵染活性。结果表明这2株分离株呈现中国被毛孢的体外培养生长和显微形态特征。通用引物ITS4／5和蝙蝠蛾拟青霉特异性引物Pp4／5扩增子测序检出蝙蝠蛾拟青霉和中国被毛孢ITS序列。扩增子克隆一测序和SNP基因分型法证实多种转换和颠换点突变基因型在2株分离株中共存。反复固体、液体培养证实蝙蝠蛾拟青霉和中国被毛孢ITS序列共存，并非真菌间DNA片段漂移。应用子囊孢子或中国被毛孢侵染4～5龄蝙蝠蛾幼虫，幼虫染菌率为1．37％～3．52％，幼虫变僵时间为35～50d。应用2株分离株与中国被毛孢分生孢子联合侵染，幼虫变僵时间为5—8d，染菌率达55．2％（P〈0．001），侵染效能提高15～39倍。蝙蝠蛾拟青霉和多种点突变基因型冬虫夏草菌（包括中国被毛孢）在蝙蝠蛾幼虫肠道以天然真菌复合体形式共存；多种菌物组成的侵染源联合侵染蝙蝠蛾幼虫大幅度提高侵染效能，呈现多菌协同侵染的共生关系。

In order to identify 2 wild-type fungal isolates, CH1 and CH2, from the intestines of liv- ing larvae of Hepialus lagii Yah, and to examine the inoculation synergy of multiple fungal sources on H. armoricanus larvae. The two wild-type fungal isolates were examined through PCR using fun- gus-specific primers for Paecilomyces hepiali and mutant genotypes of Ophiocordyceps sinensis, mo- lecular cloning and sequencing, and Single Nucleotide Polymorphism （SNP） mass spectrometry genotyping techniques. Inoculated H. armoricanus larvae of 4- 5 instars with combined fungal sources to explore the inoculation synergy. Both CH1 and CH2 isolates featured with the in vitro culture-growth and morphologic characteristics of Hirsutella sinensis, but contained both ITS se- quences of P. hepiali and H. sinensis. Amplicon cloning-sequencing and SNP genotyping demon- strated coexistence of several genotypes of O. sinensis with multiple transition and transversion point mutations in these 2 wild-type isolates. Repeated alternative solid-/liquid-phase cultures revealed co-existence of living P. hepiali and H. sinensis, rather than genetic drifts among fungi. The phe- nomenon of the fungal （species） complex was further confirmed through examining several pure H. sinensis strains. Inoculating larvae of H. armoricanus with the ascospores of natural Cordyceps sinen- sis or the conidia or mycelia of H. sinensis resulted in infection rates of 1.37%-3.52% and the lar- val death latency of 35-50 days. The combined use of CH1, CH2 and the conidia of H. sinensis as the inoculation source significantly increased the infection rate to 55.2% （15-to 39-fold increases in inoculation potency, P〈0. 001 ） and largely shortened the larval death latency to 5-8 days. P. hepiali and several mutants of O. sinensis （including H. sinensis） co-exist as a natural symbiotic fungal （species） complex in the intestines of H. lagii larvae, and are difficult to be purified during fungal purification processes. The combined use of the natural fungal （species） complex with the conidia of H. sinensis enhances synergy of multiple fungi opens sinensis. the inoculation potency on H. an avenue in studying the key armoricanus larvae. The inoculation step of the development of natural C.