R.J.St. Leger

The Role of Cuticle-Degrading Enzymes in Fungal Pathogenesis in Insects

Insects are members of the Arthropoda. Among the characteristics of this phyllum is the presence of an external skeleton or cuticle. Because of its location, the cuticle serves a variety of functions in addition to the skeletal roles of support and muscle anchorage. The defensive capability of the cuticle is clear since only one group of entomopathogens, the fungi, have acquired the ability to invade insects actively via this route. The other major groups of disease-causing microorganisms, the viruses and bacteria, are restricted primarily to the alimentary canal, where the midgut provides an exposed mucosal surface.

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.

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.

Antifungal and insecticidal properties of the efrapeptins: Metabolites of the fungus Tolypocladium niveum

Abstract Efrapeptins, peptides isolated from the fungus Tolypocladium niveum, showed toxic activity, as a crude mixture of five closely related compounds, against twospotted spider mite, Colorado potato beetle,southern armyworm, and tobacco budworm. The insecticidal activity of pure efrapeptins was characterized using two of the compounds against Colorado potado beetle in a foliar spray assay. Efrapeptin F (LC50 = 8.4 ppm) was about twice as potent as efrapeptin D (LC50 = 18.9 ppm). ATPase inhibitory activity of the efrapeptins was demonstrated using mitochondrial preparations from two fungal species, Metarhizium anisopliae and T. niveum, and from an insect, house fly. Against house fly mitochondria, efrapeptin D was the most potent ATPase inhibitor of five efrapeptins tested.

A model to explain differentiation of appressoria by germlings of Metarhizium anisopliae

Abstract The roles of the cell wall and cytoskeleton in cell differentiation of Metarhizium anisopliae were examined by treating germlings with antagonists of wall formation or with drugs which bind to microtubules and filamentous actin. Evidence for the greater plasticity of the appressorial wall compared with nongrowing regions comes from differential staining with fluorescent brighteners (Uvitex and Calcofluor) and fluorescently labeled wheat germ agglutinin; agents which detect chitin. Sorbose, which interferes with primary wall formation, was a potent inhibitor of differentiation which indicates that formation of appressoria involves addition of materials to the wall which matches the wall extension. Treatment with the anti-microfilament agent, cytochalasin A, disrupted polar growth and produced “ballooning” of the hyphal tip, indicating that actin stabilizes the tip against turgor pressure and may be involved in the transition to nonpolar growth of appressoria. Flooding differentiated germlings with 0.5% acetic acid produced bursting of appressoria and extrusion of the cytoplasm, indicating that growing appressoria are also turgescent and that internal hydrostatic pressure is a component in their growth. The effects of anti-microtubule agents are consistent with the well-known role for microtubules in the transport of vesicles. Taken together, our studies indicate that the microtubule-microfilament network is involved in the mediation of extracellular signals but not in initial reception. Inhibition of differentiation by vanadate (an inhibitor of the plasma membrane ATPase) and gadilonium (a blocker of stretch-activated ion channels) implies that changes in membrane potential may play a role in signal reception. We propose a model for the sequence of events during differentiation in which an induction signal, perhaps involving a localized change in transmembrane potential produced by deformation of the plastic hyphal tip, disrupts the apical Ca2+ gradient required for maintenance of polar growth and the actin cytoskeleton, the consequent differential redistribution of Ca2+ in the cell enlargement zone initiates germ tube swelling and appressorium formation by turgor pressure against an expanded area of the cell wall produced by disruption of exocytosis at the original apex and randomly dispersed intussusception of new wall material over the entire cell surface. A possible role for the Spitzenkorper (vesicle generating apparatus) is discussed.

Second messenger involvement in differentiation of the entomopathogenic fungus Metarhizium anisopliae

Summary: The conidial germling of Metarhizium anisopliae produces an appressorium upon contact with a hard hydrophobic surface. We have conducted an investigation into how this entomopathogen mediates intracellularly the inductive signal to shift from polarized germ-tube growth to non-polarized appressorial growth. During sporulation, conidia accumulated 45Ca2+ but there was no evidence for a gradient of Ca2+ in the spore which could establish the initial polarity. Calmodulin, however, was localized at the poles of the conidia, near the site of germ-tube emergence. Exposing conidia to Ca2+ deprivation or calmodulin antagonists inhibited germination and polar growth. Disruption of Ca2+ gradients by ionophoresis did not prevent germination but caused the multiple emergence of branched germ-tubes from conidia. These findings indicate that Ca2+ plays a fundamental role in establishing the dominance of apical growth. Although an external source of Ca2+ is not required for appressorium formation, germlings producing appressoria took up 45Ca2+ when available. The 45Ca2+ accumulated in the cell wall, plasma membrane and organelles suggesting that these may function as Ca2+ stores for Ca2+-stimulated exocytosis of cell-wall materials. Mitochondria and vacuoles sequestered 45Ca2+ indicating that they play a role in maintaining low cytoplasmic concentrations of 45Ca2+ consistent with the reorganization of the cytoskeleton required for appressorial growth. Several Ca2+-binding proteins in appressoria may provide an energy-independent component of Ca2+ buffering in the cytoplasm. The results indicate that the apical Ca2+ gradient is disrupted during differentiation and subsequent differential Ca2+ redistribution in the cell enlargement zone coincides with germ-tube swelling. cAMP may also be involved by potentiating the effects of small changes in Ca2+ concentration and stimulating exocytosis of mucus components required for adhesion, which is a prerequisite for differentiation.

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.