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  • 1
    ISSN: 1574-6968
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Biology
    Notes: Abstract The major extracellular proteases from the nematophagous fungus Verticillium chlamydosporium and the entomophagous fungus Metarhizium anisopliae, VCP1 and Pr1, respectively, are closely related both functionally and serologically. Antibodies raised against either enzyme cross-reacted with both antigens, suggesting that they have common epitopes. The VCP1 and Prl antisera labelled bovine pancreatic elastase and proteinase K, respectively. Neither antiserum reacted with commercial chymotrypsin. An antiserum to a serine protease from the closely related V. suchlasporium also cross-reacted with VCP1 and Prl. In contrast, a polyclonal antibody to an isoform of Pr1 exclusive to M. anisopliae isolate ME1 failed to recognize Prl from M. anisopliae V245 or VCP1. The N-terminal amino acid sequence of VCP1 revealed similarities with subtilisin-like enzymes from other fungi, but the closest match was with Pr1. The pure enzymes, VCP1 and Prl, failed to hydrolyse mono-aminoacyl-naphthylamide substrates but demonstrated dipeptidyl peptidase activity against Gly-Pro-βNA and Leu-Ala-βNA, respectively. These results are discussed in the context of specificity of invertebrate mycopathogens.
    Type of Medium: Electronic Resource
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  • 2
    Electronic Resource
    Electronic Resource
    Oxford, UK : Blackwell Publishing Ltd
    FEMS microbiology letters 251 (2005), S. 0 
    ISSN: 1574-6968
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Biology
    Notes: Nutrition influenced growth, sporulation and virulence of the insect pathogenic fungus, Metarhizium anisopliae. Virulent conidia were produced on susceptible insect hosts, 1% yeast extract, 2% peptone, osmotic stress medium (OSM) and CN 10:1 medium. Several strain independent markers were identified that could be used to predict the virulence of M. anisopliae conidia. Virulent conidia typically had high levels of spore bound Pr1, an important cuticle degrading protease, and high germination rates. We also show for the first time that virulent conidia have an endogenous CN ratio below 5.2:1. Real Time PCR revealed that virulent conidia from insects contained significantly higher levels of transcripts of pr1 A and other pathogenicity-related genes than inoculum from artificial media. Of the artificial media studied, 1% yeast extract medium yielded the most virulent conidia, these had higher levels of transcripts of these pathogenicity-related genes than the least virulent conidia from the high conidia yielding CN 35:1 medium (= SDA), however, the levels were significantly lower than those in insect-derived conidia. Our study shows for the first time that the passaged inoculum is virulent irrespective of the original culture medium or insect host. Virulent conidia were consistently produced on OSM even though growth and sporulation were poor. We postulate that starvation conditions, whether in vivo or in vitro, results in de-repression of Pr1 and that elevated levels of this enzyme enhance fungal virulence.
    Type of Medium: Electronic Resource
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  • 3
    Electronic Resource
    Electronic Resource
    Oxford, UK : Blackwell Publishing Ltd
    FEMS microbiology letters 251 (2005), S. 0 
    ISSN: 1574-6968
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Biology
    Notes: Increasing sensitivity towards secondary metabolites from fungal biological control agents (BCAs) has prompted the toxicological risk assessment of metabolites produced by the insect pathogenic fungus Metarhizium anisopliae. Viability studies on one human and one insect cell line were used to compare the two approaches of testing individual metabolites (destruxins A, B and E) or the complete crude extract from liquid cultures. Furthermore, crude extract was separated into fractions, which did not contain the main destruxins A, B and E. Evaluation of the cytotoxic activity of these different compounds suggested that a wide range of metabolites with synergistic or adverse effects are present in the crude extract. The results indicate that identification and toxicological assessment of each individual metabolite produced by a BCA is not only time and cost-intensive, but also does not convey the whole picture. Testing of the crude extract offers an alternative approach and is recommended when assessing the risks of metabolites for registration purposes.
    Type of Medium: Electronic Resource
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  • 4
    Electronic Resource
    Electronic Resource
    Oxford, UK : Blackwell Publishing Ltd
    FEMS microbiology letters 250 (2005), S. 0 
    ISSN: 1574-6968
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Biology
    Notes: Metarhizium anisopliae strains V245 and V275 differed in their stability when grown on different nutrient media. V275 produced fewer sectors than V245 irrespective of the cultural conditions. Both strains produced more sectors on nutrient rich media. At least four distinct types of sectors were produced in vitro. Most sectors were sterile or sporulated poorly and produced significantly lower quantities of virulence determining enzymes like Pr1. Real-time PCR confirmed differential expression of the pathogenicity-related genes pr1 A, ste 1, try 1, and chy 1 encoding for the subtilisin Pr1A, esterase, trypsin and chymotrypsin, respectively. API-ZYM revealed that the enzyme profiles of sectors differed from those of the parent cultures and also from other sectors. Sectors of M. anisopliae also produced less destruxins than the parent cultures independent of the strain.
    Type of Medium: Electronic Resource
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  • 5
    Electronic Resource
    Electronic Resource
    Oxford, UK : Blackwell Publishing Ltd
    FEMS microbiology letters 226 (2003), S. 0 
    ISSN: 1574-6968
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Biology
    Notes: In a previous study, a spontaneous subtilisin pr1A and pr1B gene-deficient mutant of the entomopathogenic fungus Metarhizium anisopliae strain V275 has been identified [Wang, C.-S. et al. (2002) FEMS Microbiol. Lett. 213, 251–255]. The insecticidal metabolites of this mutant were studied further. High-performance liquid chromatography (HPLC) analysis indicated that the mutant isolate lost the ability to produce cyclic peptide toxins, destruxins, both in vitro and in vivo. Pulsed-field gel electrophoresis revealed that the mutant concurrently lost a 1.05 Mb (approximately) chromosome, demonstrating for the first time that a conditionally dispensable (CD) chromosome exists in the insect pathogenic fungus, M. anisopliae. Concurrence of losing the ability to produce destruxins and a CD chromosome in the mutant suggests that the toxin synthetase genes of M. anisopliae are located on this CD chromosome, as similarly described for plant pathogenic fungi. Semi-quantitative api ZYM analysis showed more biochemical disparities between the mutant and the wild-type strain.
    Type of Medium: Electronic Resource
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  • 6
    Electronic Resource
    Electronic Resource
    Oxford, UK : Blackwell Publishing Ltd
    FEMS microbiology letters 213 (2002), S. 0 
    ISSN: 1574-6968
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Biology
    Notes: In an initial attempt to characterise single-spore isolates from Metarhizium anisopliae wild-type strain V275, three genetically identical spontaneous pr1A- and pr1B-deficient mutants were detected. These mutants were identified by Nested-PCR amplification and verified by Southern hybridisation. Sequencing and comparison of the highly conserved sequences of 28S rDNA domains 9–11 confirmed that the mutant isolates were Metarhizium anisopliae, but each had both morphological and genetic differences from the wild-type parent. RAPD analyses indicated that the overall genetic similarity between wild-type strain and the mutant was less than 70%. Enzyme assays proved that the mutant isolates had significantly lower Pr1 and elastase activities. Bioassays showed reduced lethal activity of the mutants (20%) on Tenebrio molitor, but lethal activities were similar to those of the wild-type parent on the larvae of Galleria mellonella. This paper shows for the first time that a stable mutant strain lacking the most important pr1 genes is still able to infect its respective hosts. Our observations suggest that it is not uncommon for Metarhizium anisopliae to lose its virulence genes during maintenance on artificial medium.
    Type of Medium: Electronic Resource
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