Characterization of cuticle-degrading proteases produced by the entomopathogen Metarhizium anisopliae

Two chymoelastases and three trypsinlike proteases were separated from culture filtrates of the entomopathogen Metarhizium anisopliae. A chymoelastase (Pr1) (pI 10.3 Mr 25,000) and trypsin (Pr2) (pI 4.42, Mr 28,500) were purified to homogeneity by ammonium sulphate precipitation, isoelectric focusing, and affinity chromatography. Inhibition studies showed that both enzymes possessed essential serine and histidine residues in the active site. Pr1 shows greater activity than Pr2 or mammalian enzymes against locust cuticle and also possesses activity vs elastin. Pr1 shows a broad primary specificity toward amino acids with hydrophobic side groups in synthetic ester and amide substrates. The kinetic properties of Pr1 demonstrate a preference for extended peptide chains with the active site recognising at least five substrate residues. The S5 and S4 subsites show a preference for negatively charged succinyl and hydrophobic acetyl groups, respectively. The S3 and S2 subsites both discriminated in favor of alanine and against proline. Pr2 rapidly hydrolyzed casein and synthetic substrates containing arginine or lysine. It possessed little or no activity vs cuticle, elastin, or synthetic substrates for chymotrypsin and elastase. Specific active site inhibitors confirmed the similarities between Pr2 and trypsin.     

Termite pathogens: Transfer of the entomopathogen Metarhizium anisopliae between Reticulitermes sp. termites

Subterranean termites (Reticulitermes sp.) exposed to whole cultures of Metarhizium anisopliae for 4, 8, 12, or 48 hr transfer disease to previously healthy termites. Healthy termites concentrate grooming activity on diseased individuals and thereby become infected. Termites which have been killed by the fungus are avoided by healthy individuals and are less effective in spreading disease than are exposed living termites.     

Cuticle-degrading proteases from the entomopathogen Metarhizium flavoviride and their distribution in secreted and intracellular fractions

AIMS: The aim of this study was to analyse a native isolate of Metarhizium flavoviride (CG423) which is being developed as a myco-insecticide against grasshoppers in Brazil for the production of the cuticle-degrading subtilisin-like (Pr1), and trypsin-like (Pr2) proteases. METHODS AND RESULTS: The results show that Pr1 activity occurred only in medium supplemented with grasshopper cuticle (Schistocerca pallens). In contrast, Pr2 was detected in higher amounts on defined growth substrate than on cuticle-supplemented medium. Both activities were detected after 48 h of growth, suggesting that in S. pallens cuticle-containing medium these protease types are not co-ordinately expressed. Low levels of enzyme activity were detected when pre-grown mycelium was used to investigate the induction of Pr1 proteases. CONCLUSIONS: The analysis of Pr1 and Pr2 distribution in both secreted and intracellular fractions revealed high percentage of extracellular activity, which may suggest the occurrence of an efficient mechanism of protein secretion by this fungus, probably related to substrate degradation which provides nutrients for fungal growth.     

The entomopathogen Metarhizium anisopliae can modulate the secretion of lipolytic enzymes in response to different substrates including components of arthropod cuticle

The filamentous fungus Metarhizium anisopliae is a well-characterized, arthropod pathogen used in the biological control of arthropod pests. Studies on the regulation of enzymes related to host infection such as proteases and chitinases have been reported but little is known about regulation of lipolytic enzymes in this fungus. Here we present the effects of different carbon sources such as components of the arthropod cuticle on the secretion of lipolytic enzymes by M. anisopliae. Differences in the induction of lipolytic activity were observed between the several carbon sources tested. Higher activities of lipase or lipase/esterase were found in culture media containing the arthropod integument components chitin and cholesteryl stearate. Several bands of lipolytic activity were also detected in zymograms, thus suggesting an important set of lipolytic enzymes secreted by the fungus. These results show that the fungus can modulate the secretion of lipolytic activity in response to host integument components, thus reinforcing the potential role of these enzymes during M. anisopliae infection.     

Variability in esterases of Metarhizium anisopliae

The variability in esterases of the entomogenous fungus Metarhizium anisopliae was determined electrophoretically on 8.5% polyacrylamide gel. Ten isolates from diverse taxonomic groups of insects were analyzed. The electrophoretic analysis showed differences and similarities between these isolates and it was possible to distinguish six different patterns. The results obtained show a great polymorphism for the esterase system of M. anisopliae.     

不同磷源对金龟子绿僵菌CQMa102生物量及生成胞外磷酸酶的影响(英文)

金龟子绿僵菌Metarhizium anisopliae是一种广泛分布于世界各地土壤中的重要的昆虫病原真菌。已有研究表明,胞外磷酸酶在绿僵菌侵染并致死寄主过程中发挥了重要作用。利用摇瓶培养方法探究了无机磷(KH2PO4)、简单有机磷(植酸钠、磷酸苯二钠)和蛋白有机磷(酪蛋白)为单独磷源条件下,绿僵菌生物量、产胞外酸性磷酸酶以及酪氨酸蛋白磷酸酶、丝/苏氨酸蛋白磷酸酶活性的变化。实验结果显示:加入酪蛋白的培养基,最有利于绿僵菌生长、胞外蛋白的分泌和产酸性磷酸酶;其次为加入KH2PO4和磷酸苯二钠的培养基;加入植酸钠的培养基不利于绿僵菌的生长代谢。然而,加入磷酸苯二钠的培养基,最有利于酪氨酸蛋白磷酸酶、丝/苏氨酸蛋白磷酸酶的分离纯化。     

Regulation of production of a trypsin-like protease by the insect pathogenic fungus Metarhizium anisopliae

Abstract The entomopathogenic fungus metarhizium anisopliae produces several cuticle-degrading proteases which may play a role in pathogenesis. The regulation of one of these, a trypsin-like protease PR2, has been investigated using depressed mycelia. Three insoluble protein sources, insect cuticle, elastin and collagen, as well as two soluble proteins, BSA and gelatin, induced PR2. The polymeric carbon sources cellulose and xylan resulted in depressed basal levels but not induced production of PR2. An approximately 15-fold increase in PR2 activity per mg dry weight of mycelium was observed when the fungus was grown in the presence of bovine serum albumin (BSA), as compared with conditions of depression alone. This indicates that PR2 is induced by BSA, and probably by other proteins. Basal levels of PR2 were detected after 8 h when mycelium was starved for both carbon and nitrogen but only after 16 h when starved for either nitrogen or carbon. In the presence of a protein source, nitrogen strongly repressed PR2 whereas carbon had little effect. There was no effect of sulphur on PR2 production.     

Efficacy of Metarhizium anisopliae microsclerotial granules

The entomopathogenic fungus Metarhizium anisopliae has very recently been shown to produce microsclerotia (MS) – compact, heavily melanised, hyphal aggregates – in liquid media. Soil incorporation bioassays of dried MS preparations of three isolates of M. anisopliae were conducted using third instar Tetanops myopaeformis (sugarbeet root maggot) in clay and/or clay loam field soils as a model system to demonstrate efficacy. At rates as low as 23 mg MS granules/100 g dry soil, the biocontrol efficacy of MS granules of M. anisopliae Strain F52 produced in liquid media with a high carbon concentration (36 g/L) and high C:N ratios (30:1, 50:1) were superior to MS preparations produced in low carbon (8 g carbon/L) media and a high carbon medium with a 10:1 C:N ratio. Bioassays using MS formulations of M. anisopliae strains MA1200 and TM109 produced in high carbon and high C:N ratio media were superior in efficacy to the other MS production media tested. MS preparations of M. anisopliae F52 showed superior efficacy against the sugarbeet root maggot in comparison with more conventional, conidia-covered nutritive (corn grit) granules in a clay and clay soil. The MS granules were also highly efficacious against the sugarbeet root maggot at soil moisture levels as low as 0.983 A _w (?2.33 MPa). Granular preparations incorporating Metarhizium MS can serve as a viable formulation for the use of this fungus against soil insects.     

Compatability of Metarhizium anisopliae var. anisopliae with chemical pesticides

The effects of various insecticides on the mycelial growth, sporulation and conidial germination of Metarhizium anisopliae var. anisopliae isolate E₉ were studied in the laboratory. Chlorpyrifos was the most toxic organophosphate to mycelial growth and sporulation at all concentrations. Temephos, malathion and leptophos were highly toxic to sporulation while malathion was the most inhibitory to germination. The carbamates, carbofuran, methomyl and oxamyl were moderately toxic to mycelial growth and sporulation while oxamyl had an adverse effect on germination. The pyrethroids (pyrethrin, permethrin and resmethrin) and the insect growth regulators (diflubenzuron and methoprene) were not inhibitory to the various developmental stages of isolate E₉. The chlorinated hydrocarbons (chlordane, lindane and toxaphene) were more deleterious than all other insecticide groups tested. Among the fungicides, benomyl and maneb produced the greatest inhibition.     

Genetic diversity of Metarhizium anisopliae var. anisopliae in southwestern British Columbia

The abundance and genetic diversity of the entomopathogenic fungus, Metarhizium anisopliae var. anisopliae, in southwestern British Columbia (BC) and southern Alberta was examined. The fungus was found to be widespread in soil throughout southwestern BC, and was recovered from 56% of 85 sample sites. In contrast to southwestern BC, no M. anisopliae isolates were recovered in southern Alberta. An automated fluorescent amplified fragment length polymorphism (AFLP) method was used to examine genetic diversity. In excess of 200 isolates were characterized. The method identified 211 polymorphic amplicons, ranging in size from ≈92 to 400 base pairs, and it was found to be reproducible with a resolution limit of 86.2% similarity. The AFLP method distinguished Metarhizium from other entomopathogenic fungal genera, and demonstrated considerable genetic diversity (25 genotypes) among the reference strains of M. anisopliae isolates examined (i.e. recovered from various substrates and geographical locations). Although 13 genotypes of M. anisopliae var. anisopliae were recovered from southwestern BC soils, the vast majority of isolates (91%) belonged to one of two closely-related genotypes. Furthermore, these two genotypes predominated in urban, agricultural and forest soils. The reasons for the limited diversity of M. anisopliae var. anisopliae in southwestern BC are uncertain. However, findings of this study are consistent with island biogeography theory, and have significant implications for the development of this fungus for microbial control of pest insects.     

Protoplast fusion and genetic recombination in Metarhizium anisopliae

Using two strains of Metarhizium anisopliae var. minor designated E6 and RJ from different origins, inter-strain crosses were readily performed by orthodox methods from mutants of strain E6 through the parasexual cycle. However, in most cases crosses between mutants of strain RJ or between RJ and E6 mutant strains were not achieved. Protoplast fusion was carried out in an attempt to cross such strains. Protoplasts were obtained after treatment of mycelium with lytic enzymes using 0.7 m KCl as osmotic stabilizer. Regeneration frequency varied from 1.2 to 3.0%. Poly (tethylene glycol) was used for the fusion of protoplasts and fusion frequencies varied from 0.9 to 44.1 in 10⁵ protoplasts according to the cross. Sectors which emerge from fusion products were analysed and recombinants were obtained even from crosses between mutant strains which could not be crossed by hyphal fusion. In this way protoplast fusion proved to be a valuable tool for further studies of genetics and breeding of Metarhizium anisopliae.     

Swainsonine production in fed-batch fermentations of Metarhizium anisopliae

Summary Extended and enhanced production of swainsonine was achieved from fed-batch fermentations of the fungus Metarhizium anisopliae. A complex medium based on oatmeal extract was intermittently fed with D-glucose and/or lysine. Swainsonine titres were improved eleven-fold and the duration of production extended was from 240 to 550 hours compared with a batch culture under the same conditions.     

Biochemical characterization and ultrastructural localization of two extracellular trypsins produced by Metarhizium anisopliae in infected insect cuticles.

Proteinase 2 (Pr2) is a fungal (Metarhizium anisopliae) serine proteinase which has a tryptic specificity for basic residues and which may be involved in entomopathogenicity. Analytical and preparative isoelectric focusing methods were used to separate two trypsin components, produced during growth on cockroach cuticle, with isoelectric points of 4.4 (molecular mass, 30 kDa) and 4.9 (27 kDa). The catalytic properties of the proteases were analyzed by their kinetic constants and by a combination of two-dimensional gelatin-sodium dodecyl sulfate-polyacrylamide gel electrophoresis and enzyme overlay membranes. Both Pr2 isoforms preferentially cleave at the carboxyl sides of positively charged amino acids, preferring arginine; the pI 4.4 Pr2 isoform also possessed significant activity against lysine. Compared with the pathogen's subtilisin-like enzyme (Pr1), the pI 4.4 Pr2 isoform shows low activity against insoluble proteins in a host (Manduca sexta) cuticle. However, it degrades most cuticle proteins when they are solubilized, with high-molecular-weight basic proteins being preferentially hydrolyzed. Polyclonal antibodies raised against each Pr2 isoform were isotype specific. This allowed us to use ultrastructural immunocytochemistry to independently visualize each isoform during penetration of the host (M. sexta) cuticle. Both isoforms were secreted by infection structures (appressoria) on the cuticle surface and by the penetrant hyphae within the cuticle. The extracellular sheath, which is commonly observed around fungal cells, often contained Pr2 molecules. Intracellular labelling was sparse.     

Purification and characterization of a novel thermostable extracellular protein tyrosine phosphatase from Metarhizium anisopliae strain CQMa102

An extracellular phosphatase was purified to homogeneity from the entomopathogenic fungus Metarhizium anisopliae with a 41.0% yield. The molecular mass and isoelectric point of the purified enzyme were about 82.5 kDa and 9.5 respectively. The optimum pH and temperature were about 5.5 and 75 degrees C when using O-phospho-L-tyrosine as substrate. The protein displayed high stability in a pH range 3.0-9.5 at 30 degrees C and was remarkably thermostable at 70 degrees C. The purified enzyme showed high activity on O-phospho-L-tyrosine and protein tyrosine phosphatase substrate monophosphate (a specific substrate of protein tyrosine phosphatase). Although one peptide of the phosphatase shared identity with one alkaline phosphatase of Neurospora crassa, its substrate specificity and inhibitor sensitivity indicate that the enzyme is a protein tyrosine phosphatase.     

The genome sequence of the biocontrol fungus Metarhizium anisopliae and comparative genomics of Metarhizium species

Background

Metarhizium anisopliae is an important fungal biocontrol agent of insect pests of agricultural crops. Genomics can aid the successful commercialization of biopesticides by identification of key genes differentiating closely related species, selection of virulent microbial isolates which are amenable to industrial scale production and formulation and through the reduction of phenotypic variability. The genome of Metarhizium isolate ARSEF23 was recently published as a model for M. anisopliae, however phylogenetic analysis has since re-classified this isolate as M. robertsii. We present a new annotated genome sequence of M. anisopliae (isolate Ma69) and whole genome comparison to M. robertsii (ARSEF23) and M. acridum (CQMa 102).

Results

Whole genome analysis of M. anisopliae indicates significant macrosynteny with M. robertsii but with some large genomic inversions. In comparison to M. acridum, the genome of M. anisopliae shares lower sequence homology. While alignments overall are co-linear, the genome of M. acridum is not contiguous enough to conclusively observe macrosynteny. Mating type gene analysis revealed both MAT1-1 and MAT1-2 genes present in M. anisopliae suggesting putative homothallism, despite having no known teleomorph, in contrast with the putatively heterothallic M. acridum isolate CQMa 102 (MAT1-2) and M. robertsii isolate ARSEF23 (altered MAT1-1). Repetitive DNA and RIP analysis revealed M. acridum to have twice the repetitive content of the other two species and M. anisopliae to be five times more RIP affected than M. robertsii. We also present an initial bioinformatic survey of candidate pathogenicity genes in M. anisopliae.

Conclusions

The annotated genome of M. anisopliae is an important resource for the identification of virulence genes specific to M. anisopliae and development of species- and strain- specific assays. New insight into the possibility of homothallism and RIP affectedness has important implications for the development of M. anisopliae as a biopesticide as it may indicate the potential for greater inherent diversity in this species than the other species. This could present opportunities to select isolates with unique combinations of pathogenicity factors, or it may point to instability in the species, a negative attribute in a biopesticide.

Electronic supplementary material

The online version of this article (doi:10.1186/1471-2164-15-660) contains supplementary material, which is available to authorized users.     

Molecular detection of the airborne entomopathogen fungus Metarhizium acridum using specific oligonucleotides

We describe a technique to detect the presence of airborne conidia from the fungus M. acridum (formerly Metarhizium anisopliae var. acridum) (Hypocreales: Clavicipitaceae) with great accuracy. Airborne conidia were collected using Hirst-type spore traps. DNA extractions were optimized to achieve the best possible recovery. DNA was examined using polymerase chain reaction (PCR) with specific oligonucleotides to enable the detection of a single conidium. Experiments using a mini-wind tunnel were conducted to validate the method. Subsequently, this technique was applied to an agricultural region of Mexico, where M. acridum was sprayed to control the grasshopper, Sphenarium purpurascens, population (Orthoptera: Pyrgomorphidae). M. acridum conidia were detected 2 days after spraying in San Mateo Coatepec (Puebla, site of grasshopper study).