The fungal species Metarhizium pingshaense, M. anisopliae, M. robertsii, and M. brunneum, 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 amplifications
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 nontarget
effects of applied biocontrol strains.