Michael J. Bidochka

Differentiation of species and strains of entomopathogenic fungi by random amplification of polymorphic DNA (RAPD).

Polymerase chain reaction (PCR)-based technology, involving random amplification of polymorphic DNA (RAPD), was used to assess the genomic variability between 24 isolates of deuteromycetous fungi (Metarhizium anisopliae, Metarhizium flavoviride, unidentified strains of Metarhizium and Beauveria bassiana) which were found to infect grasshoppers or locusts. M. flavoviride showed little intraspecific variability in PCR-amplified fragments when compared to M. anisopliae. The high level of variability in PCR-amplified fragments contained within M. anisopliae was similar to the total variability between B. bassiana, M. anisopliae and M. flavoviride, and suggests that M. anisopliae may include a number of cryptic species. Four polymorphic RAPD fragments were used to probe the genomic DNA of the various species and strains. On the basis of these probes the fungi can be grouped into M. flavoviride, M. anisopliae, or B. bassiana. According to PCR-amplified fragments, previously-unidentified Metarhizium strains were characterized as M. flavoviride. There was little evidence that these fungi, all isolated from, or virulent towards, grasshoppers or locusts, showed host-selection in PCR-amplified fragments. Nor was geographical origin a criterion for commonalty based on PCR-amplified fragments. PCR-fragment-pattern polymorphisms and the construction of probes from one or more of these fragments may provide a useful and rapid tool for identifying species and strains of entomopathogenic fungi.

The rodlet layer from aerial and submerged conidia of the entomopathogenic fungus Beauveria bassiana contains hydrophobin

The entomopathogenic fungus Beauveria bassiana produces aerial conidia, submerged conidia or blastospores depending on the culture conditions. Rodlets are observed on aerial and submerged conidia but not on blastospores. Cell wall rodlets were removed by sonication and the proteins dissolved with formic acid. A single major 9·7 kDa protein was found in rodlets of aerial and submerged conidia and showed N terminal sequence similarities to the hydrophobin class of fungal proteins. Hydrophobin could not be recovered from blastospores. Oxidation of the hydrophobin with performic acid produced a protein of higher molecular weight (14·0 kDa). The rodlet layer could not be removed from intact conidia by boiling in sodium dodecyl sulphate (SDS) but could be removed with formic acid. Formic acid treated conidia retained the ability to bind to insect cuticles and retained hydrophobicity as indicated by a phase exclusion assay.

Protein synthesis in Metarhizium anisopliae growing on host cuticle

In vitro protein synthesis using poly(A + )RNA and a two-step gel system for proteins were used in this study on the entomopathogenic fungus Metarhizium anisopliae to provide an estimate of the magnitude of differential protein synthesis and secretion that may be involved in adapting to growth on insect cuticle. Shortly after being transferred to a media containing cockroach cuticle, mRNAs for certain proteins are repressed while a broad array of mRNAs for other proteins is induced. Concurrent with this, a least 42 proteins were secreted into the media in a process which was sensitive to actinomycin D. The majority of these proteins were acidic (pI range 4·2–5·6) and co-migrated with Con-A/peroxidase stained bands indicating that they might be glycoconjugates. Microsequencing of those polypeptides accumulated in large amounts revealed two NH 2 -terminal amino acid sequences from acidic proteins that were highly homologous to those of animal trypsins. The trypsin nature of the two proteins was confirmed using a combination of gelatin-SDS-polyacrylamide gel electrophoresis and enzyme overlay membranes. The NH 2 -terminal sequence of the major basic protein identified it as a know subtilisin-like proteinase (Pr1). A second basic sequence was identified as a carboxypeptidase. No other homologies were found.

Cloned DNA probes distinguish endemic and exotic Entomophaga grylli fungal pathotype infections in grasshopper life stages

Entomophaga grylli is a fungal pathogen of grasshoppers and at least three pathotypes are recognized world‐wide. Pathotypes 1 and 2 are endemic to North America while the Australian pathotype 3 had been released into two field sites in North Dakota between 1989 and 1991. Grasshoppers were collected over the summer at the field sites in 1992 and assessed for pathotype infection by cloned DNA probe analysis. The three most predominant grasshopper species that were infected (Melanoplus sanguinipes, M. bivittatus and Camnula pellucida) were assessed for pathotype infection with respect to their life stages (nymphal instars and adult males and females). Pathotype 1 predominantly infected grasshoppers in the subfamilies Oedipodinae and Gomphocerinae and pathotype 2 predominantly infected grasshoppers in the subfamily Melanoplinae. Early‐instar M. sanguinipes and M. bivittatus had higher pathotype 2 infection frequencies, while late‐instar and adult C. pellucida had higher pathotype 1 infection frequencies. Cross‐infection by the pathotypes did occur in up to 3% of the individuals, on a per species basis, and primarily in later instar and adult grasshoppers. Pathotype 3 infections occurred in later instar and adults of the three grasshopper species. Infection of grasshoppers by E. grylli pathotypes is discussed with reference to the fungal life cycles.