Title: Maldi-Tof Mass Spectrometry Applied to Identifying Species of Insect-Pathogenic Fungi from the Metarhizium Anisopliae Complex Authors
|Lopes, R -|
|Faria, M -|
|Souza, D -|
|Bloch, JR., C -|
Submitted to: Mycologia
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: February 5, 2014
Publication Date: July 1, 2014
Citation: Lopes, R.B., Faria, M., Souza, D.A., Bloch, Jr., C., Humber, R.A. 2014. MALDI-TOF mass spectrometry applied to identifying species of insect-pathogenic fungi from the Metarhizium anisopliae complex. Mycologia. 106(4):865-878. Interpretive Summary: Current standards for identification of increasing numbers of organisms depend upon obtaining and comparing gene sequences with related organisms. This manuscript presents an alternative technique for rapid and inexpensive molecularly based identifications for an important group of insect-pathogenic fungal species using a specialized form of mass spectroscopy (MALDI-TOF) rather than DNA extraction and gene sequencing. The results of MALDI-TOF are demonstrated to be both robust and extremely accurate when compared directly to sequence-based identifications of the same fungal isolates. The principal advantage of MALDI-TOF for those facilities that have access to the necessary hardware is in the ease of obtaining accurate identifications much faster and less expensively than by depending on gene-based methods. This study provides direct comparisons of the time and costs for identifications of fungi by both MALDI-TOF and standard gene sequence-based approaches that underscore the possible benefits of using MALDI-TOF for preliminary identifications. While mass spectrometric approach is not recommended to replace gene-based identifications, MALDI-TOF is proven to have enormous value for the initial, rapid, high-confidence identifications of large numbers of cultures while also flagging unusual cultures requiring further gene-based study as potential new or unusual representatives of their genus. These sorts of applications are particularly valuable for large culture collections needing to increase the accuracy of identifications for existing holdings or to support field studies on fungal biodiversity, population biology, establishment and dispersal of introduced fungal biocontrol agents, or other similar applications involving isolations and identifications of large amounts of biological material from field sites.
Technical Abstract: Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) has proven to be a powerful tool for taxonomic resolution of microorganisms. In this proof-of-concept study, we assessed the effectiveness of this technique to track the current gene sequence-based phylogenetic classification of species in the Metarhizium anisopliae complex. Initially, the phylogenetic analysis of 51 strains by sequencing of the 5 prime end of the TEF1-a gene region revealed seven species within M. anisopliae sensu lato and two varieties outside this complex. Preliminary assays showed that profiles from mycelial fragments or conidia produced on nutrient-poor medium may yield too much background noise and, subsequently, all spectrometric analyses were performed with acid-hydrolyzed conidia from 10 to 12-day-old PDA cultures. Two reference MALDI-TOF reference libraries were built: The first included protein spectral profiles from nine taxonomically distinct, molecularly identified isolates sharing high genetic homology to the ex-type or ex-epitype isolates of these taxa in Metarhizium. The second reference library added one isolate each for M. anisopliae sensu stricto and M. robertsii. The larger reference library (including 11 taxa) allowed nearly perfect MALDI-TOF matching of DNA-based species identification for 40 additional isolates molecularly recognized as M. anisopliae sensu stricto (n=19), M. robertsii (n=6), M. majus (n=3), M. lepidiotae (n=1), M. acridum (n=3), M. flavoviride var. pemphigi (1), plus seven unidentified strains (six of them phylogenetically close to M. anisopliae sensu stricto, and one outside the PARB clade). Due to the increasing frequency of phylogenetically (genomically) based taxonomic revisions of fungi, this approach is especially useful for culture collections, since once the protein profiles of Metarhizium isolates are obtained, taxonomic updating of MALDI-TOF library data is easily accomplished by comparing stored profiles with those of newly proposed taxa.