Donald W. Roberts

Adaptation of proteases and carbohydrases of saprophytic, phytopathogenic and entomopathogenic fungi to the requirements of their ecological niches

The abilities of isolates of saprophytes (Neurospora crassa, Aspergillus nidulans), an opportunistic human pathogen (Aspergillus fumigatus), an opportunistic insect pathogen (Aspergillus flavus), plant pathogens (Verticillium albo-atrum, Verticillium dahliae, Nectria haematococca), a mushroom pathogen (Verticillium fungicola) and entomopathogens (Verticillium lecanii, Beauveria bassiana, Metarhizium anisopliae) to utilize plant cell walls and insect cuticle components in different nutrient media were compared. The pathogens showed enzymic adaptation to the polymers present in the integuments of their particular hosts. Thus, the plant pathogens produced high levels of enzymes capable of degrading pectic polysaccharides, cellulose and xylan, as well as cutinase substrate, but secreted little or no chitinase and showed no proteolytic activity against elastin and mucin. The entomopathogens and V. fungicola degraded a broad spectrum of proteins (including elastin and mucin) but, except for chitinase, cellulase (V. lecanii and V. fungicola only) and cutinase (B. bassiana only), produced very low levels of polysaccharidases. The saprophytes (Neu. crassa and A. nidulans) and the opportunistic pathogens (A. fumigatus and A. flavus) produced the broadest spectrum of protein and polysaccharide degrading enzymes, indicative of their less specialized nutritional status. V. lecanii and V. albo-atrum were compared in more detail to identity factors that distinguish plant and insect pathogens. V. albo-atrum, but not V. lecanii, grew well on different plant cell wall components. The major class of proteases produced in different media by isolates of V. albo-atrum and V. dahliae were broad spectrum basic (pI > 10) trypsins which degrade Z-AA-AA-Arg-NA substrates (Z, benzoyl; AA, various amino acids; Na, nitroanilide), hide protein azure and insect (Manduca sexta) cuticles. Analogous peptidases were produced by isolates of V. lecanii and V. fungicola but they were specific for Z-Phe-Val-Arg-NA. V. albo-atrum and V. dahliae also produced low levels of neutral (pI ca 7) and basic (pI ca 9.5) subtilisin-like proteases active against a chymotrypsin substrate (Succinyl-Ala2-Pro-Phe-NA) and insect cuticle. In contrast, subtilisins comprised the major protease component secreted by V. lecanii and V. fungicola. Both V. lecanii and V. albo-atrum produced the highest levels of subtilisin and trypsin-like activities during growth on collagen or insect cuticle. Results are discussed in terms of the adaptation of fungi to the requirements of their ecological niches.

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.

Productionin vitro of appressoria by the entomopathogenic fungusMetarhizium anisopliae

Abstract Germination on complex media induced conidia of the entomopathogenMetarhizium anisopliae to produce infection structures (appressoria and penetration hyphae) when the germ tube contacted a hard surface. The morphology of the infection structures and their rate of formation are very similar to those observed for blowfly cuticle. Differentiation frequencies were greater (more than 70% as compared with less than 40%) on hydrophobic surfaces [Teflon, polyvinyl chloride, polystyrene, polypropylene, polyester (GelBond), aluminum foil] than on hydrophilic surfaces (agarose-coated polyester and cellophane). Differentiation frequencies were similar on both positively and negatively charged surfaces. Differentiationin vitro was stimulated by low levels of complex nitrogenous nutrients. Analysis of one- or multicomponent media suggested that amino acids and the lipid component of epicuticle act in combination with the hydrophobic cuticle surface to stimulate differentiation during pathogenesis. Thigmotropic and chemical stimuli for production of appressoria appear to be translated primarily during the second round of nuclear division because inhibitors of DNA and RNA synthesis do not prevent germination but block differentiation if applied before the second nuclear division. Inhibition of protein synthesis blocked both germination and differentiation.

Isolation of beauvericin as an insect toxin from Fusarium semitectum and Fusarium moniliforme var. subglutinans

Nonpolar methylene chloride-soluble extracts from the mycelia of Fusarium semitectum and Fusarium moniliforme var. subglutinans were toxic to Colorado potato beetles. The major toxic metabolite was isolated and found to be the cyclodepsipeptide, beauvericin. This is the first report of the isolation of beauvericin from the genus Fusarium.

Genetic differences in allozymes and in formation of infection structures among isolates of the entomopathogenic fungus Metarhizium anisopliae

Abstract In this study, we examined variation among isolates of the entomopathogenic fungus Metarhizium anisopliae in their ability to germinate and produce infection structures (appressoria) in different nutrient media. We also examined allozyme variation within and among 120 isolates of Metarhizium spp. Electromorph differences were noted and genotypic designations were assigned for eight loci encoding the following enzymes: glucose phosphate isomerase, glutamate dehydrogenase, glutamic pyruvic transaminase, glutathione reductase, malate dehydrogenase, phosphogluconate dehydrogenase, peptidase with glycyl leucine, and triosephosphate isomerase. Forty-eight distinct genotypic classes were found. Only the thirteen M. anisopliae var. majus isolates and an isolate of Metarhizium flavoviride possessed multibanded phenotypes characteristic of heterozygous genotypes. Three isolates were heterozygous at one locus, seven isolates at two loci, and four isolates at four loci. Based on the level of genetic similarities between the three recognized species ( M. anisopliae, Metarhizium album , and M. flavoviriae ) we report that isolates currently assigned as M. anisopliae are a strongly heterogenous array of multilocus genotypes that includes five varieties and two undescribed cryptic species. Genetic similarities provided evidence for geographical clustering. For example, Brazil and Colombia contain a common, widely dispersed genotypic class; eight additional classes are more locally distributed. Nutrient requirements for germination and appessorium formation were frequently host related, irrespective of genetic and geographical distance. Most of those lines of M. anisopliae var. anisopliae and var. majus isolated from scarabaeids germinated readily only in the presence of a crude protein/chitin product. Lines isolated from other coleopterans were much more adaptable and produced appressoria in yeast extract media. Unlike the majority of isolates from other hosts, lines isolated from Hemiptera usually germinated well in media containing glucose as the sole carbon source, and glucose allowed formation of appressoria. The role of selectable strain variations in the development of pathogen strategies is discussed.

Ultrastructural Localization of a Cuticle-degrading Protease Produced by the Entomopathogenic Fungus Metarhizium anisopliae during Penetration of Host (Manduca sexto) Cuticle

SUMMARY: Gold-labelled rabbit antiserum was used to demonstrate that a cuticle-degrading protease (Pr1) is produced by Metarhizium anisopliae during penetration of host (Manduca sexta) procuticle. The protease was secreted by infection structures (appressoria) on the cuticle surface and by the penetrant hyphae within the cuticle. Penetration of the procuticle was by a combination of enzymic degradation and mechanical pressure. Initially Pr1 was confined to the immediate vicinity of the fungal structures; however the enzyme diffused throughout the cuticle during later stages of pathogenesis. When hyphae were labelled during growth in culture under conditions conducive to rapid synthesis of Pr1, gold particles distributed over the fungal cell wall, indicating binding of Pr1 to hyphae.

Prepenetration Events during Infection of Host Cuticle by Metarhizium anisopliae

Abstract The early involvement of cuticle degrading enzymes in infection was suggested by the presence of chymoelastase protease (Prl), esterase, and N-acetylglucosaminidase in ungerminated spores of Metarhizium anisopliae. Enzymes were released from spores by washing in water or dilute buffer solutions. This, together with the degradation of enzyme substrates by intact spores, indicates that enzyme activities are located at the surface of spores, and probably parallels the availability or release of enzymes under natural conditions. Levels of enzymes on conidia from infected Manduca sexta larvae were higher than those harvested from Sabauraud dextrose agar (SDA), indicating that environmental conditions in which spores develop can preadapt them for the pathogenic life style. Cell-bound enzymes were released from differentiating germlings in varying degrees by water, salt solutions, detergents, and mercaptoethanol. Release of Pr1 and esterase activities by salt solutions suggests that their binding to cell walls involves ionic bonds. By contrast, release of N-acetylglucosaminidase required the disruption of membranes. We suggest that binding of enzymes to fungal walls could, in part, explain localized action of cuticle degrading enzymes during host penetration. The esterase activity in conidia is represented by a single major activity (pI 7) and in infection structures by multiple isoenzymes, all characterizable as β-esterases. Esterase activites were greatest against short and intermediate length fatty acids with little activity above C10. The effects of surface topography on appressorium formation was studied using M. sexta cuticles and plastic replicas of the cuticles. Appressoria were only produced after extensive growth over the microfolds of the cuticle surface of early (1 day) fifth instar larvae. By contrast germination on the comparatively flat surface of 5-day fifth instar larvae allows appressorium formation close to the conidium. The results are discussed in the context of cuticle degradation during prepenetration fungal growth, and the effects of host morphology on pathogen behavior.

World-wide distribution of genetic variation among isolates of Beauveria spp.

Genetic variation among isolates of Beauveria spp. was defined by an analysis of electrophoretically demonstrable allelic variations of allozyme coding genes. One hundred and forty-six isolates from diverse geographical locations were assigned to forty-seven distinctive genotypic classes. Clustering analysis demonstrated that four morphological species ( B. brongniartii, B. vermiconia, B. caledonica and Tolypocladium cylindrosporum ) were genetically distinct from each other. However, with the exception of B. brongniartii , which clustered separately with B. amorpha , they were not clearly resolved from lines of B. bassiana . The level of genetic distance shown between cluster groups of B. bassiana indicated that it represents a species aggregate, components of which display overlapping genetic variability with isolates currently assigned to other species. All isolates were homozygous at each locus, consistent with haploidy, except for B. caledonica , which was heterozygous at one of the four loci. In spite of the maintenance of high diversity in B. bassiana , three geographically widespread genotypic classes were found to contain the majority of isolates. This suggests that, in many situations, B. bassiana exists with a clonal population structure. Various other aspects of the allozyme data (the magnitude of genetic distances between populations, gene diversity, and the pattern of distribution of genotypic classes) indicate that chromosomal recombination between different genotypes of B. bassiana spp. is rare or absent.

Studies on the prolonged storage of Metarhizium anisopliae conidia: effect of temperature and relative humidity on conidial viability and virulence against mosquitoes

Abstract Unformulated conidia of three strains of Metarhizium anisopliae were stored at four temperatures (viz., 37°, 26°, 19°, and 4°C) at each of six relative humidities (RH) (viz., 0, 12, 33, 53, 75, and 98%) and at −20°C at 0% RH. Conidial viabilities and virulence against two species of mosquitoes were determined at various intervals over a 2-year period. Conidia were found to survive longest when moderate temperature-high RH (26°C–97% RH or 19°C–97% RH) or low temperature-low RH (4°C–0% RH) were used, while intermediate RH values between 33 and 75%, depending upon the strain, were most lethal. As the temperature declined from 37° to 4°C at nearly all RH values the longevity of spores increased. Conidia remained highly virulent under favorable storage conditions for at least 18 months against Culex pipiens pipiens or 12 months against Anopheles stephensi larvae.

Synthesis of proteins including a cuticle-degrading protease during differentiation of the entomopathogenic fungusMetarhizium anisopliae

Abstract Studies of polypeptide synthesis thesis utilizing [ 35 S]methionine, sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and “Western blot” analyses revealed that a cuticle-degrading protease with a chymoelastase specificity is the major protein product when Metarhizium anisopliae produces infection structures on an artificial surface or in situ during penetration of host cuticle. Production of chymoelastase by infection structures is favored by levels of nutrients insufficient to induce catabolite repression. Addition of readily utilized nutrients, e.g., alanine, caused extensive growth on the host cuticle but repressed penetration and synthesis of chymoelastase. Thus, the pathogenic process involving infection-related morphogenesis and enzyme production occurs only when it is necessary for the pathogen to establish a nutritional relationship with the host. Studies with inhibitors (8-azaguanine and actinomycin D) suggest that control of enzyme production is at the level of transcription.