Insect cuticle-degrading enzymes from the entomogenous fungusBeauveria bassiana

Beauveria bassiana is a promising agent for biological control of agricultural insect pests. Its broad host range includes both the Greater Wax Moth (Galleria mellonella) and the cabbage looper (Trichoplusia ni). Five strains ofB. bassiana from the Agricultural Research Service Culture Collection were obtained and grown on defined media and a medium containing purified cuticle from eitherG. mellonella orT. ni. The production of cuticle-degrading enzymes (such as proteases, chitinases, and esterase) was examined. Natural strain variability in enzyme levels was significant, and there was no evidence of coordinated expression. Further, enzyme expression differed considerably as a function of cuticle source. The results suggest that it may be possible to tailor biological control agents against specific targets. Natural strain variability could be used to rationally develop improved mycoinsecticides.     

Ambient pH Is a Major Determinant in the Expression of Cuticle-Degrading Enzymes and Hydrophobin by Metarhizium anisopliae

Secretion of proteolytic and chitinolytic enzymes is a hallmark of infection processes of Metarhizium anisopliae in response to host (insect) cuticular signals. The regulation of these enzymes (subtilisin-like proteases [Pr1a and Pr1b], trypsin-like proteases [Pr2], metalloproteases, aspartyl proteases, aminopeptidase, and chitinases) and a hydrophobin was investigated by Northern analysis and/or enzyme assay. The production of each enzyme showed a differential expression pattern in response to ambient pH; enzymes were synthesized only at pHs at which they function effectively, irrespective of whether the medium contained an inductive cuticle substrate. Three aspartyl proteases (pH optimum, 3), and chitinase (pH optimum, 5) showed maximal accumulation at acidic pHs. The highest level of aminopeptidase (pH optimum, 7) was detected at pH 7. The highest levels of five metalloproteases (pH optima, ca. 7) were detected over the pH range 6 to 8. Two trypsins and several subtilisin-like Pr1 isoforms with pH optima of ca. 8 were produced only under alkaline conditions. Northern analysis of RNA species corresponding to seven cDNA sequences encoding proteases and chitinase confirmed that the ambient pH played a major role in gene expression of secreted proteins. Hydrophobin was expressed almost equally at pHs 5 and 8 but was not expressed at pH 3. During fungal penetration, the pH of infected cuticle rises from about 6.3 to 7.7. Consistent with pH regulation of enzyme production, serine and metalloproteases were produced in situ during infection, but no production of aspartyl proteases was found. We propose that the alkalinity of infected cuticle represents a physiological signal that triggers the production of virulence factors.     

Molecular epidemiology ofCladosporium carrionii

The effects of cuticle from larvae ofTrichoplusia ni andHelicoverpa (=Heliothis)zea on expression of proteases and chitinases by germinating conidia ofNomuraea rileyi in submerged cultures were studied. Increasing the concentration ofT. ni orH. zea cuticle resulted in a 13- and 15-fold increase in protease activity, respectively. Endochitinase and N-acetylglucosaminidase activity on theT. ni andH. zea substrates increased as cuticular concentrations increased to 2.5%, then stabilized or decreased thereafter. The simultaneous expression of both proteases and chitinases suggests that they are controlled by a multiple-regulatory system.This article reports the results of research only. Mention of a proprietary product in this paper does not constitute a recommendation for use by US Department of Agriculture.     

Changes in the Activity and Pattern of Hemolymph Esterases in the Larvae of Greater Wax Moth Galleria mellonellaL. (Lepidoptera, Pyralidae) during Mycosis

The extended pattern of multiple esterase forms has been revealed in the hemolymph of wax moth Galleria mellonellalarvae infected by the fungi Metarhizium anisopliae, Beauveria bassianaor Paecilomyces fumoso-roseus. The total esterase activity of the hemolymph also increases during mycosis. Mechanical damage of the cuticle, treatment with deltamethrin, and chilling of the caterpillars induced similar changes in the hemolymph pattern of esterase activity. Presumably, the changed spectrum and activity of the hemolymph esterases during mycosis is due to the damaged cuticle and epidermis cells.     

The production of toxic protease by the entomopathogenous fungus Metarhizium anisopliae in submerged culture

The production of a toxic complex of proteolytic enzymes by Metarhizium anisopliae was evaluated with 29 nitrogen sources in modified Czapek-Dox medium in submerged cultures. The proteolytic complex is more constitutive than that of Beauveria bassiana and its production is influenced by the quality of complex natural media. The highest activity was attained with Galleria mellonella proteins. The proteolytic complex manifests proteolytic activity of two pH optima, 5.5 and 8.0. The ratio of these two activities differs markedly with the nitrogen source used, but the major proteolytic activity occurs at pH 5.5.     

Cuticular and non-cuticular substrate influence on expression of cuticle-degrading enzymes from conidia of an entomopathogenic fungus,Nomuraea rileyi

Larval cuticle fromTrichoplusia ni, Helicoverpa (=Heliothis)zea, andHeliothis virescens and a cellulose substrate were used to quantify release of proteolytic, chitinolytic, and lipolytic enzymes by germinating conidia of the entomopathogenic fungus,Nomuraea rileyi. There was no significant difference in conidial viability incubated withT. ni, H. zea or cellulose substrates. Conidial viability onH. virescens cuticle, however, was significantly lower (ca. 19–25%) than the other three substrates. The presence of cuticle substrates, especially cuticle ofT. ni, stimulated germination. The nature of the substrate influenced both the time and quantity of the enzymes expressed. Specific proteases (aminopeptidase, chymoelastase, trypsin) generally were expressed earlier and/or in greater quantities on cuticular than on the cellulose substrate. Although both chitinolytic enzymes (endochitinase, N-acetylglucosaminidase) were detected on all three cuticular substrates, their activity was substantially lower than that of the proteolytic enzymes. Lipase activity was only minimally present. Early concurrent release of both proteases and chitinases suggested that both may be important in the penetration of the larval integument by germinating conidia ofN. rileyi. Expression of proteases and chitinases, especially aminopeptidase and endochitinase was probably a specific response to cuticle, because little or no activity was expressed on the non-host, cellulose substrate.This article reports the results of research only. Mention of a proprietary product in this paper does not constitute a recommendation for use by the US Department of Agriculture.     

Regulation of Extracellular Chitinases and Proteases in the Entomopathogen and Acaricide <Emphasis Type="Italic">Metarhizium anisopliae</Emphasis>

Metarhizium anisopliae infects insects and ticks via a combination of specialized structures and cuticle degradation. Hydrolytic enzymes are accepted as key factors for the penetration step. The search for pathogenicity determinants has demonstrated that the process is multifactorial. Host specificity is an important factor to be addressed. The study of the enzymes produced during infection is important to discover those with a role in the process. To address some of the enzymes that take part during the infection of the tick, Boophilus microplus, we have analyzed the secretion of proteases and chitinases in single and combined carbon/nitrogen sources as compared with such complex substrates as chitin and B. microplus cuticles. Two chitinases, endo- and N-acetylglucosaminidases, and two proteases, subtilisin and trypsin-like proteases, were analyzed. Enzyme activities were detected in all carbon sources tested, but higher levels were found when combinations of carbon sources were used. A major 30-kDa protein apparently secreted during M. anisopliae growth on all carbon/nitrogen sources tested was demonstrated by SDS–PAGE. Received: 8 May 2002 / Accepted: 8 June 2002     

Metarhizium spp. isolates from madagascar: Morphology and effect of high temperature on growth and infectivity to the migratory locust,Locusta migratoria

Five strains ofMetarhizium anisopliae (Metsch.) Sorokin and one strain ofMetarhizium flavoviride Gams &; Rozsypal originally isolated in Madagascar were studied. Measurements of conidia and, for the first time, also of blastospores produced in a liquid medium were used for species and variety determination. Blastospores ofM. flavoviride were more homogenous in their size than those ofM. anisopliae. Growth at high temperatures between 25° and 40°C showed that 4 isolates ofM. anisopliae grew at 36°C andM. flavoviride grew at 38°C. Using alternating day/night temperatures (8/16 h) the three strains tested could also tolerate 40°/25°C. In bioassays, fiveMetarhizium spp. isolates were tested against third and fourth instar larvae ofLocusta migratoria (L.) at two alternating day/night temperatures of 30°/25°C and 36°/25°C. In the cooler regime, all strains caused a mortality of 50% within 5.9 to 8.5 days (median lethal time), while in the 36°/25°C treatment only the thermophilicM. flavoviride and oneM. anisopliae strain isolated from a soil sample gave comparable results with median lethal times of 6.8 and 7.3 days, respectively.     

Ferulic Acid Esterase Activity from Schizophyllum commune

Schizophyllum commune produced an esterase which released ferulic acid from starch-free wheat bran and from a soluble ferulic acid-sugar ester that was isolated from wheat bran. The preferred growth substrate for the production of ferulic acid esterase was cellulose. Growth on xylan-containing substrates (oat spelt xylan and starch-free wheat bran) resulted in activity levels that were significantly lower than those observed in cultures grown on cellulose. Similar observations were made for endoglucanase, p-nitrophenyllactopyranosidase, xylanase, and acetyl xylan esterase. Of the enzymes studied, only arabinofuranosidase was produced at maximum levels during growth on xylan-containing materials. Ferulic acid esterase that had been partially purified by DEAE chromatography released significant amounts of ferulic acid from wheat bran only in the presence of a xylanase-rich fraction, indicating that the esterase may not be able to readily attack high-molecular-weight substrates. The esterase acted efficiently, without xylanase addition, on a soluble sugar-ferulic acid substrate.     

Development and encapsulation of the endoparasitoid,Microplitis croceipes (Hym.: Braconidae), in six candidate host species (Lep.)

Encapsulation and development of the endoparasitoid,Microplitis croceipes (Cresson), were studied in six atypical lepidopteran host species whose usual host isHelicoverpa zea (Boddie). The candidate hosts examined were: the fall armywormSpodoptera frugiperda (J. E. Smith); the beet armyworm,Spodoptera exigua (Hübner); the cabbage looper,Trichoplusia ni (Hübner); the greater wax moth,Galleria mellonella (L.); the Indian meal moth,Plodia interpunctella (Hübner); and the diamondback moth,Plutella xylostella (L.). BothS. exigua andT. ni were completely unsuitable forM. croceipes development due to the high rate of eggs that were encapsulated within three days after parasitism. Encapsulation inS. frugiperda included mainly parasitoid eggs and was first detected six days after parasitization at 25°C and two days at 30°C. Encapsulation inG. mellonella occurred only in the larval stage of the parasitoid. InP. interpunctella, parasitoid larvae reached the 3rd stadium, but none of them pupated. OnlyS. frugiperda andG. mellonella supported successful development ofM. croceipes from egg to adult. The percentage of parasitoids reaching the adult stage in these hosts was higher at 30°C than at 25°C (13% vs. 4% inS. frugiperda, and 21% vs. 3% inG. mellonella, respectively). However, these percentages were too low to substitute them as a more economical host for rearingM. croceipes. This biological information will be useful in additional laboratory studies directed toward reducing the rate of encapsulation (e.g., manipulation of host rearing temperature) to increase production ofM. croceipes on these hosts.     

Proteases from the fungus Metarhizium anisopliae toxic for Galleria mellonella larvae

The high molecular fraction of the extract from Metarhizium anisopliae grown on wheat bran contains proteolytic enzymes which are toxic for Galleria mellonella larvae. The complex of proteases was fractionated using precipitation with ammonium sulfate, gel filtration, and electrofocusing. Two components have been found: one with the optimum of activity on hemoglobin at pH 6.5, and the second with the optimum around pH 9. The prevailing protease acting at pH 6.5 was inhibited by phenylmethylsulfonyl fluoride and the inhibition was followed by decrease of toxicity. The molecular weights of the enzymes are 35 × 10³ and 71 × 10³.     

Production of lytic enzymes by<Emphasis Type="Italic">Trichoderma</Emphasis> spp. and their effect on the growth of phytopathogenic fungi

The production of β-1,3-glucanases and chitinases by three strains ofTrichoderma in submerged cultures was determined. The synthesis of enzymes was induced by cell wall biopolymers of phytopathogenic fungi (Botrytis cinerea, Fusarium culmorum andF. oxysporum). T. hamatum produced the highest β-1,3-glucanase activity; the most effective inducer of enzyme synthesis was the biomass ofF. oxysporum. All examined strains ofTrichoderma inhibited phytopathogen growth in biotic tests. The diffusion tests showed that the lytic enzymes take part in growth inhibition of phytopathogenic fungi.     

Cuticle degrading proteases from insect moulting fluid and culture filtrates of entomopathogenic fungi

Insects degrade their own cuticle during moulting, a process which is catalysed by a complex mixture of enzymes. Entomopathogenic fungi infect the insect host by penetration of the cuticle, utilizing enzymatic and/or physical mechanisms. Protein is a major component of insect cuticle and a major recyclable resource for the insect and, therefore, represents a significant barrier to the invading fungus. To this end, both insects and entomopathogenic fungi produce a variety of cuticle degrading proteases. The aim of this paper is to review these proteases and to highlight their similarities, with particular reference to the tobacco hornworm, Manduca sexta, and the entomopathogenic fungus, Metarhizium anisopliae     

Insecticidal evaluation of <Emphasis Type="Italic">Beauveria bassiana</Emphasis> engineered to express a scorpion neurotoxin and a cuticle degrading protease

To improve the insecticidal efficacy of the entomopathogen Beauveria bassiana, the fungus was genetically modified with an insect-specific scorpion neurotoxin AAIT and an insect cuticle degrading protease PR1A from another insect pathogen (Metarhizium anisopliae). The wild-type and the transformants were bioassayed against the larvae of Masson’s pine caterpillar Dendrolimus punctatus and the wax moth Galleria mellonella. In comparison to the wild-type strain, engineered isolates took fewer spores to kill 50% of pine caterpillars, 15-fold less for the aaIT single transformant Bb13T and eightfold less for the double transformant Bb13TPR1A, respectively. The median lethal times for Bb13T and Bb13TPR1A were reduced by 40% and 36.7%, respectively against D. punctatus and 24.4% and 20.9%, respectively against G. mellonella. Our data showed that the cotransformation of these two genes produced no synergistic effects on virulence improvement. It is evident from this study that AAIT could be degraded by the protease PR1A when they are expressed together, emphasizing that protein interactions need to be evaluated when working with multiple genes, particularly if they include proteases.     

Degradation of insect cuticle by Paecilomyces farinosus proteases

The entomopathogenic fungus Paecilomyces farinosus showed proteolytic activity in both solid and semi-liquid culture with gelatin as sole N and C source. Semi-liquid cultures were used to characterise proteases. Zymography of crude culture filtrates showed several bands of gelatin degradation in electrophoresis gels. Gel filtration chromatography of these filtrates revealed two peaks of proteolytic activity. Ion-exchange absorption eliminated gelatin from culture filtrates while retaining activity and was used to semipurify P. farinosus proteases. Semipurified culture filtrates had basic pH (8.5 approx.) optimum for proteolytic activity. Treatment of these filtrates with effectors revealed that P. farinosus proteases are serine proteases containing sulphydryl groups. Isoelectrofocusing combined with zymography revealed the presence of several active basic isoforms. Larvae of the lepidopteran Galleria mellonella showed cuticle damage and protein release 1h after incubation with semipurified extracts of P. farinosus. These results indicate that proteolytic enzymes could be involved in insect host penetration by P. farinosus.     

Xylanolytic enzyme activities produced by mesophilic and thermophilic actinomycetes grown on graminaceous xylan and lignocellulose

Mesophilic and thermophilic strains of actinomycetes were grown on media containing graminaceous xylan or lignocellulose. Aliquots of the culture fluids were sampled and assayed for enzyme activities involved in the degradation of hemicellulose. Xylanase, acetyl esterase and α-arabinofuranosidase activities could be detected after different times of incubation; their production was also dependent on the growth medium. The highest levels of xylanase activity were found in cultures of strains of Streptomyces, Actinomadura sp. and Saccharomonospora viridis. Streptomyces cyaneus produced the highest amount of arabinofuranosidase whereas acetyl esterase activities were highest in S. cyaneus, S. viridis and Pseudonocardia thermophila .     

Extracellular enzyme production in <Emphasis Type="Italic">Metarhizium anisopliae</Emphasis> isolates

Extracellular enzymes produced by Metarhizium anisopliae are believed to play a key role in cuticle hydrolysis. The in-vitro production of cuticle-degrading enzymes, such as chitinase, proteinase, caseinase, lipase and amylase in fourteen isolates of M. anisopliae exhibited significant natural isolate variability. The isolates were also evaluated for chitinase and proteinase enzyme assays in order to quantify the enzyme production. The growth characteristics and colony morphology of the isolates showed variation and few isolates formed sectors and the colonies were either fluffy or powdery. Among the isolates studied, isolate UM2 was found to show good consistence with the results on enzyme measurements as well as the growth characteristics and colony morphology. Such characterization of isolate variability could rationally be used in the selection of isolates for the production of improved myco-pesticides in the integrated pest management programs.     

Production and regulation of extracellular proteases from the entomopathogenic fungus Isaria fumosoroseus (Cordycipitaceae; Hypocreales) in the presence of diamondback moth,Plutella xylostella cuticle

Extracellular proteases of entomopathogenic fungi have been implicated as components of the insect infection process. To elucidate their role in the infection process, it is essential to characterize these enzymes. During the current studies synthesis and regulation of extracellular proteases (Pr1 and Pr2) by Isaria fumosoroseus (Cordycipitaceae; Hypocreales) isolate IF28.2 were investigated as a function of carbon source (with special reference to diamondback moth cuticle), temperature and pH. The highest level of Pr1 and Pr2 activity were found in the supernatants from 1% glucose plus 1% diamondback moth cuticle (18.83±1.25 and 12.44±1.36 U/mg per h for Pr1 and Pr2, respectively). Maximum Pr1 production by the depressed mycelia was observed from the supernatants having diamondback moth cuticle as a nutrient source, whereas depressed mycelia showed maximum Pr2 activity from the cultures having chitin as the basic nutrient. The optimum pH for Pr1 and Pr2 activity was 8 while 35°C was the best temperature for protease production.     

Production of microsclerotia of the fungal entomopathogen Metarhizium anisopliae and their potential for use as a biocontrol agent for soil-inhabiting insects

Microsclerotia (MS), overwintering structures produced by many plant pathogenic fungi, have not been described for Metarhizium anisopliae. Three strains of M. anisopliae – F52, TM109, and MA1200 – formed MS in shake flask cultures using media with varying carbon concentrations and carbon-to-nitrogen (C:N) ratios. Under the conditions of this study, all strains produced MS, compact hyphal aggregates that become pigmented with culture age, in addition to more typical blastospores and mycelia. While all strains formed desiccation tolerant MS, highest concentrations (2.7–2.9 × 10⁸ L⁻¹ liquid medium) were produced in rich media with C:N ratios of 30:1 and 50:1 by strain F52. All three strains of M. anisopliae produced similar biomass concentrations when media and growth time were compared. Strain MA1200 produced higher concentrations of blastospores than the other two strains of M. anisopliae with highest blastospore concentrations (1.6 and 4.2 × 10⁸ blastospores ml⁻¹ on days 4 and 8, respectively) in media with the highest carbon and nitrogen concentrations. Microsclerotial preparations of M. anisopliae containing diatomaceous earth survived air-drying (to <5 % moisture) with no significant loss in viability. Rehydration and incubation of air-dried MS granules on water agar plates resulted in hyphal germination and sporogenic germination to produce high concentrations of conidia. Bioassays using soil-incorporated, air-dried MS preparations resulted in significant infection and mortality in larvae of the sugar beet root maggot, Tetanops myopaeformis. This is the first report of the production of sclerotial bodies by M. anisopliae and provides a novel approach for the control of soil-dwelling insects with this entomopathogenic fungus.     

Exocellular proteases ofSerratia marcescens and their toxicity to larvae ofGalleria mellonella

Out of 18 strains ofSerratia marcescens producing exocellular proteases the strainSerratia marcescens CCEB 415 was selected according to preliminary experiments. It could be shown that the train exhibits proteolytic activity reaching up to 10 TU per 1 ml of the culture filtrate in a medium with gelatine and peptone. Two proteolytic enzymes could be demonstrated by means of specific inhibitors EDTA and diisopropyfluorophosphate: metaloprotease with optimum activity at pH 7.5 and serine protease with pH optimum of 10.9. The enzymes were purified on Sephadex and DEAE cellulose columns and by means of gel electrophoresis. However, it was not possible to separate them. The optimum temperature for activity of the mixture of the two enzymes was 50 ° C, molecular weight varied around 37000 (according to gel filtration); certain kinetic characteristics of their activity were determined. Excess subtrate (casein) inhibited activity of the enzyme mixture. Toxicity of proteases expressed as LD₅₀ units equals 78. 10⁻³ TU per larva ofGalleria mellonella.