A species-specific multiplexed PCR amplicon assay for distinguishing between Metarhizium anisopliae, M. brunneum, M. pingshaense and M. robertsii

Authors: MAYERHOFER, JOHANNA - Agroscope; LUTZ, ANDY - Agroscope; DENNERT, FRANCESCA - Eth Zurich; Rehner, Stephen; Kepler, Ryan; WIDMER, FRANCO - Agroscope; ENKERLI, JURG - Agroscope

Submitted to: Journal of Invertebrate Pathology
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 6/1/2019
Publication Date: 1/11/2019
Citation: Mayerhofer, J., Lutz, A., Dennert, F., Rehner, S.A., Kepler, R.M., Widmer, F., Enkerli, J. 2019. A species-specific multiplexed PCR amplicon assay for distinguishing between Metarhizium anisopliae, M. brunneum, M. pingshaense and M. robertsii. Journal of Invertebrate Pathology. 161(1):23-28.
DOI: https://doi.org/10.1016/j.jip.2019.01.002

Interpretive Summary: Some groups of fungi are natural enemies of insects and can be used as biological control agents for insects that are pests of important plant crops. Some of the most effective insect biological control species are found in the fungal genus Metarhizium, but they can be difficult to identify. The ability to rapidly identify these fungi and their naturally occurring relatives is an important component in the design, implementation and monitoring of insect biological control programs. In this study, a combination of new species specific DNA sequences was used to develop a single PCR-based test for the identification of four species of Metarhizium. This assay is significant because it provides a robust method for quick and inexpensive identification of Metarhizium isolates from any environmental source. This diagnostic assay will be used by insect pathologists and mycologists investigating and implementing insect pest management programs for field crop and forest pests.

Technical Abstract: The fungal species Metarhizium anisopliae, M. brunneum, M. pingshaense and M. robertsii, a monophyletic group informally referred to as the PARB species complex, are well known facultative entomopathogens, including many commercialized strains used for biological pest control. Accurate and expedient species identification of Metarhizium isolates represents an important first step when addressing ecological as well as application-related questions involving these fungi. To this end, a species-specific multiplexed polymerase chain reaction (PCR) assay was developed for identification and discrimination among Metarhizium PARB complex species, based on unique sequence signature differences within the nuclear ribosomal intergenic spacer (rIGS) and nuclear intergenic spacer regions MzFG546 and MzIGS2. Species-specificities of the four primer pairs were assessed following a three-step approach including: 1) in silico verification of sequence signatures by BLASTN searches against publically available genome and amplicon sequence data, 2) corroboration of assay specificity and robustness by performing test PCR amplification against a taxonomically curated reference strain collection of 68 Metarhizium strains representing 12 species, and 3) testing against a field collection of 19 unknown Metarhizium isolates from soil of a Swiss meadow. The specificity of these four primer pairs provide an efficient means to detect and discriminate PARB species in studies targeting ecological aspects of indigenous isolates, as well as efficacy, persistence and potential non-target effects of applied biocontrol strains.