Argentine Metarhizium spp. isolates: Molecular identification, blastospores production, and its pathogenicity against Blattella germanica adults

Abstract

Blattella germanica is the most prevalent synanthropic pest and negatively impacts human health, as do the synthetic chemical insecticides used to control them. In contrast, Metarhizium spp. is a well known fungi that can infect insects and other arthropods, causing death to their hosts. When cultivated in liquid medium it produces blastospores. This propagule has the potential to be used as a biological control agent for cockroaches. The aim of this study was to identify eleven native Metarhizium strains from Argentina through molecular taxonomy, evaluate blastospores and biomass production in submerged fermentation; and assess the pathogenicity of the blastospores against adult B. germanica cockroaches. For the molecular identification of the strains, diagnostic PCR amplification was performed using previously developed primers, followed by sequence alignment and phylogeny reconstruction using the amplification and sequencing of the markers EF1A, RPB1, RPB2, 5TEF, MzIGS3, and rIGS- ID800. In order to elucidate blastospores production, a conidia suspension was inoculated into Adamek liquid medium. Samples were taken at 48 h, 72 h, and 96 h to measure blastopore production, and at 96 h blastospores were harvested, dry biomass were weighted and the pH of the liquid medium was assessed. The strains showed blastospores production at 96 h or earlier, except for two strains, and statistically significant maximum blastospores production was reached at 48 h for most strains. A blastospores suspension of 1E+07 blastopore/mL was applied by spray to B. germanica adults, and mortality was assessed every two days for 20 days after application. Death cockroaches were placed in a humid chamber and periodically observed for Metarhizium growth. Blastospores significantly reduced the survivorship of B. germanica adults. Metarhizium spp. grew and sporulated from cockroach cadavers, with up to 91% of adults dead. Species-discriminating diagnostic PCR and phylogenetic reconstruction were successful to assign ten of the strains to different species within the Metarhizium PARB clade: four strains to Metarhizium brunneum and three strains each to Metarhizium hybridum and Metarhizium robertsii. One strain had previously been shown to belong to the distantly related species Metarhizium argentinense.This study, therefore, constitutes the first description of M. hybridum from Argentina and provides the first report of direct contact application of fungal blastospores as a pathogenic approach against cockroaches, reducing B. germanica adult survivorship.