ORIGINAL ARTICLE Comparison of the germination rates of Metarhizium spp. conidia from Japan at high and low temperatures O. Nishi, K. Iiyama, C. Yasunaga-Aoki and S. Shimizu Laboratory of Insect Pathology and Microbial Control, Institute of Biological Control, Kyushu University, Higashi-ku, Fukuoka city, Japan Significance and Impact of the Study: The relationships between the growth characteristics and phylo- genetic placements have not been adequately investigated in species of the entomopathogenic fungus Metarhizium. This study determined the relationships between the germination rates in hot and cold conditions and the phylogenetic placements of 89 Japanese soil isolates of Metarhizium spp. Fourteen isolates each of M. brunneum and M. flavoviride var. pemphigi, identified by molecular phylogenetic analysis, showed relatively high germination rates at lower temperatures and low germination rates at higher temperatures compared with isolates, which were identified as six other species. This study detected a strong relationship between the phylogenetic placements of Japanese Metarhizium spp. isolates and their in vitro germination characteristics. Keywords elongation factor 1-alpha, entomopathogenic fungi, Metarhizium, phylogenetic analysis, physiological characteristics, thermal condition. Correspondence Oumi Nishi, Kyushu University Faculty of Agriculture Build. VI (Room 224), 6-10-1, Hakozaki, Higashi-ku, Fukuoka 812-8581, Japan. E-mail: ag105154@agr.kyushu-u.ac.jp 2013/0514: received 16 March 2013, revised 25 July 2013 and accepted 22 August 2013 doi:10.1111/lam.12150 Abstract The knowledge of the growth characteristics of entomopathogenic fungi at different temperatures is very important for understanding their ecology and field efficacy as biological control agents. However, the relationships between their DNA-based phylogenetic tree classifications and growth characteristics at different temperatures have not been adequately investigated. In this study, we compared the phylogenetic relationships between Metarhizium anisopliae sensu lato and M. flavoviride isolates obtained from soils in various environments in Japan and the germination rates of their conidia on agar medium in hot and cold conditions. The results showed that the 89 Japanese isolates belonged to the clade of eight species, according to the molecular phylogenetic analysis. The germination rates of isolates belonging to the M. brunneum and M. flavoviride var. pemphigi clades were higher at lower temperatures (9Á7– 11Á0°C) and lower at higher temperatures (34Á3–35Á2°C) compared with the other six species. The isolates of these two species originated from different geographical regions in Japan, despite their uniform germination characteristics. This study detected the clear interspecific differences in the in vitro germination characteristics of the Japanese isolates of Metarhizium spp. at two different temperature regimes. Introduction Metarhizium is one of the most commercially important entomopathogenic fungi, and some isolates of this genus have been used as biological control agents to facilitate the pest management for locusts, termites, spittlebugs and white grubs (Zimmermann 1993). M. anisopliae sensu lato (s. l.), the most common group in this genus, is now known to be a complex of cryptic species, which can be distinguished by their DNA sequences (Bischoff et al. 2006, 2009). M. anisopliae s. l. has been detected in soils from various environments and more than 200 species of insects (Zimmermann 1993). Metarhizium anisopliae s. l. has variable growth charac- teristics and levels of virulence against insects. For exam- ple, isolates of M. anisopliae s. l. from Tasmanian soil, which were later classified as M. frigidum (Bischoff et al. 2006), could grow at 4°C and were pathogenic to scar- abaeid larvae at 10°C and 15°C (Rath et al. 1995). Some varieties of M. flavoviride were also active in cold condi- Letters in Applied Microbiology 57, 554--560 © 2013 The Society for Applied Microbiology 554 Letters in Applied Microbiology ISSN 0266-8254