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

Author of the article:LIU Si-Yu；XUE Rui；XIAO Guan-Li；LI Zheng-Yue；CHEN Bin

Author's Workplace： State Key Laboratory for Conservation and Utilization of Bio-Resources in Yunnan, College of Plant Protection, Yunnan Agricultural University, Kunming 650201, China; 2. College of Agronomy and Biotechnology, Yunnan Agricultural University, Kunming 650201, China

Key Words：Metarhizium anisopliae (Metschn.) Sorokin; Maladera ovatula (Fairmaire); orthogonal design; culture condition; colony growth; sporulation

Abstract：[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-5.8S 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 6.68×10⁶ spore/cm². [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.