Species Discrimination within the Metarhizium PARB Clade: Ribosomal Intergenic Spacer (rIGS)-Based Diagnostic PCR and Single Marker Taxonomy.

(1) Background: The entomopathogenic fungus Metarhizium anisopliae sensu lato forms a species complex, comprising a tight cluster made up of four species, namely M. anisopliae sensu stricto, M. pinghaense, M. robertsii and M. brunneum. Unambiguous species delineation within this "PARB clade" that enables both the taxonomic assignment of new isolates and the identification of potentially new species is highly solicited. (2) Methods: Species-discriminating primer pairs targeting the ribosomal intergenic spacer (rIGS) sequence were designed and a diagnostic PCR protocol established. A partial rIGS sequence, referred to as rIGS-ID800, was introduced as a molecular taxonomic marker for PARB species delineation. (3) Results: PARB species from a validation strain set not implied in primer design were clearly discriminated using the diagnostic PCR protocol developed. Using rIGS-ID800 as a single sequence taxonomic marker gave rise to a higher resolution and statistically better supported delineation of PARB clade species. (4) Conclusions: Reliable species discrimination within the Metarhizium PARB clade is possible through both sequencing-independent diagnostic PCR and sequencing-dependent single marker comparison, both based on the rIGS marker.

Isolation, Identification, and Characterization of the Nematophagous Fungus Arthrobotrys oligospora from Kyrgyzstan.

PURPOSE: Predatory fungi have been the subject of fundamental studies and their potential as biological control agents against parasitic plant nematodes has been assessed. The aim of the present study was to isolate and identify predatory fungi, performing in vitro and in vivo screening to select highly active strains to control parasitic nematodes. METHODS: Different nutrient media were used to isolate predatory fungi and determine their morphological and cultural properties. Identification was performed by classical and molecular biology methods. In vitro and in vivo screening was conducted to select highly active strains. RESULTS: Twelve isolates of Arthrobotrys oligospora (Orbiliomycetes) found in nature were investigated for their predaceous efficacy against garlic stem nematodes (Ditylenchus dipsaci). The effect of temperature and pH on the growth rate and trap formation of representative isolates was determined and isolates were characterized by light microscopy and molecular markers. BLAST was used to sequence the rDNA internal transcribed spacer of A. oligospora isolate KTMU-7. The optimum growth of A. oligospora strains was achieved at 20-25 °C on 1-2% corn meal agar (CMA) within the pH range of 5.6-8.6. The factors responsible for the trap formation of these fungal strains were identified. In vitro and in vivo experiments were performed to evaluate the nematicidal activity of local predatory fungal isolates against soil nematodes. CONCLUSIONS: Preliminary studies proved A. oligospora to be a potentially effective biological control agent, immobilizing 85.7 ± 2.19% of garlic stem nematodes in soil from the rhizosphere of potato plants.