一株小阔胫绒金龟幼虫绿僵菌的分离鉴定及培养条件的优化研究

Use of an orthogonal rotation combination design to identify optimal culture conditions for a Metarhizium sp. isolated from Maladera ovatula(Fairmaire) larvae

【目的】为了弄清从云南省昆明市寻甸县大河桥农场田间自然罹病的小阔胫绒金龟Maladera ovatula(Fairmaire)幼虫僵虫上分离纯化获得的1株绿僵菌Metarhizium sp.(编号MAXD170705)的种类及其培养条件,为蛴螬生防制剂开发提供依据。【方法】室内通过菌株的分离与纯化,并根据形态特征及ITS-5.8SDNA基因序列,对该菌株种类进行鉴定,同时采用正交旋转组合试验设计,测定该菌株在不同培养基上于不同温度、光照、pH值条件下的菌落生长速度及产孢特性。【结果】经形态学与分子鉴定,确定菌株MAXD170705为金龟子绿僵菌Metarhiziumanisopliae,该金龟子绿僵菌MAXD170705菌株在SMAY、PDA和SDAY 3种培养基上均能生长,但在SDAY培养基上孢子萌发率以及菌落生长、产孢时间与产孢量均优于其他2种培养基。在(25±1)℃、16 L︰8 D和pH=7.0条件下MAXD170705菌株生长最快,菌落直径日增量为0.38 cm/d;菌落直径最大,第8天达2.73 cm;产孢时间最早,从第3天开始产孢,并且产孢量最高,达6.68×10^6孢子/cm^2。【结论】营养成分对菌株发育过程中具有重要作用,只有丰富而且恰当的营养条件才有利于不同来源的绿僵菌菌丝生长以及产孢。除此之外,外界环境条件也是影响其生长发育的重要因素。综合以上结果,利用pH=7.0的SDAY培养基,于25℃、光照16L︰8D为金龟子绿僵菌MAXD170705的最适培养条件。

[Objectives] To determine the optimal culture condition for a strain of Metarhizium sp.(MAXD170705)isolated from muscardine-infected cadavers of Maladera ovatula(Fairmaire)larvae from Daheqiao farm,Xundian county,Kunming city,Yunnan province.[Methods]The fungal strain MAXD170705 was identified based on morphological and culturing characteristics and its ITS-5.8 S gene sequence.Optimal culture conditions for the growth and sporulation of this strain,including culture medium,temperature,light and pH,were determined based on an orthogonal rotational combined design.[Results]The MAXD170705 strain was Metarhizium anisopliae(Metschn.)Sorokin.This strain can be cultured on either SMAY,PDA or SDAY medium,but cumulative germination,growth rate,sporulation time and spore quantity were best on SDAY medium.Optimal culture conditions were a pH of 7.0,25℃and a photoperiod of 16 h light and 8 h darkness.Cumulative germination was 100.00%by Day 4,the colony growth rate was 0.38 cm/d and the diameter was 2.73 cm.The colony began to sporulate on the day 3 and sporulation was 6.68×10^6 spore/cm^2.[Conclusion]Nutrients played an important role in the development of this strain.Optimal mycelium growth and sporulation of Metarhizium sp.obtained from different sources was only achieved when nutrients were abundant and appropriate.In addition,external environmental conditions were important factors affecting colony growth and development.Of the media evaluated,the SDAY medium was the best for the growth and sporulation of the MAXD170705 strain of M.anisopliae.Optimal culture conditions were a pH of 7.0,25℃and a photoperiod of 16 L︰8 D.