Identification of a universally primed-PCR-derived sequence-characterized amplified region marker for an antagonistic strain of Clonostachys rosea and development of a strain-specific PCR detection assay.
We developed a PCR detection method that selectively recognizes a single biological control agent and demonstrated that universally primed PCR (UP-PCR) can identify strain-specific markers. Antagonistic strains of Clonostachys rosea (syn. Gliocladium roseum) were screened by UP-PCR, and a strain-specific marker was identified for strain GR5. No significant sequence homology was found between this marker and any other sequences in the databases. Southern blot analysis of the PCR product revealed that the marker represented a single-copy sequence specific for strain GR5. The marker was converted into a sequence-characterized amplified region (SCAR), and a specific PCR primer pair was designed. Eighty-two strains, isolated primarily from Danish soils, and 31 soil samples, originating from different localities, were tested, and this specificity was confirmed. Two strains responded to the SCAR primers under suboptimal PCR conditions, and the amplified sequences from these strains were similar, but not identical, to the GR5 marker. Soil assays in which total DNA was extracted from GR5-infested and noninoculated field soils showed that the SCAR primers could detect GR5 in a pool of mixed DNA and that no other soil microorganisms present contained sequences amplified by the primers. The assay developed will be useful for monitoring biological control agents released into natural field soil. (+info)
A novel lactonohydrolase responsible for the detoxification of zearalenone: enzyme purification and gene cloning.
Zearalenone (ZEN) is converted into a far less oestrogenic product by incubation with Clonostachys rosea IFO 7063. An alkaline hydrolase responsible for the detoxification was purified to homogeneity from the fungus by a combination of salt precipitation and column chromatography methods. The purified enzyme was homodimeric with a subunit molecular mass of 30 kDa and contained an intra-subunit disulphide bridge. On the basis of the internal peptide sequences of the purified protein, we cloned the entire coding region of the gene (designated as zhd101) by PCR techniques. The ZEN degradation activity was detected in heterologous hosts (Schizosaccharomyces pombe and Escherichia coli) carrying the cloned gene. Zhd101 could be a promising genetic resource for in planta detoxification of the mycotoxin in important crops. (+info)
Core structure in roselipins essential for eliciting inhibitory activity against diacylglycerol acyltransferase.
Fungal roselipins, discovered as inhibitors of diacylglycerol acyltransferase (DGAT), consist of three parts; highly methylated C20 fatty acid, mannose and arabinitol. Demannosyl and/or dearabinitoyl roselipins were prepared chemically or enzymatically. Demannnosyl roselipins conserved the DGAT inhibitory activity, but the others lost the activity, indicating that the arabinitoyl fatty acid core is essential for eliciting the activity. (+info)
Classification of the guava wilt fungus Myxosporium psidii, the palm pathogen Gliocladium vermoesenii and the persimmon wilt fungus Acremonium diospyri in Nalanthamala.
Psidium guajava wilt is known from South Africa, Malaysia and Taiwan. The fungus causing this disease, Myxosporium psidii, forms dry chains of conidia on surfaces of pseudoparenchymatous sporodochia, which develop in blisters on bark. Similar sporodochia are characteristic of Nalanthamala madreeya, the type species of Nalanthamala. Nalanthamala, therefore, is the appropriate anamorph genus for Myxosporium psidii, while Myxosporium is a nomen nudum (based on M. croceum). For M. psidii the combination Nalanthamala psidii is proposed. Nalanthamala psidii, the palm pathogen Gliocladium (Penicillium) vermoesenii, another undescribed anamorphic species from palm, two species of Rubrinectria and the persimmon pathogen Acremonium diospyri are monophyletic and belong to the Nectriaceae (Hypocreales) based on partial nuclear large subunit ribosomal DNA (LSU rDNA) analyses. Rubrinectria, therefore, is the teleomorph of Nalanthamala, in which the anamorphs are classified as N. vermoesenii, N. diospyri or Nalanthamala sp. Nalanthamala squamicola, the only other Nalanthamala species, has affinities with the Bionectriaceae and is excluded from this group. Rubrinectria/Nalanthamala species form dimorphic conidiophores and conidia in culture. Fusiform, cylindrical, or allantoid conidia arise in colorless liquid heads on acremonium-like conidiophores; ovoidal conidia with somewhat truncated ends arise in long, persistent, dry chains on penicillate conidiophores. No penicillate but irregularly branched conidiophores were observed in N. diospyri. Conidia of N. psidii that are held in chains are shorter than those of N. madreeya, of which no living material is available. Nalanthamala psidii and N. diospyri are pathogenic specifically to their hosts. They form pale yellow to pale orange or brownish orange colonies, respectively, and more or less white conidial masses. Most strains of Rubrinectria sp., Nalanthamala sp. and N. vermoesenii originate from palm hosts, form mostly greenish or olive-brown colonies and white-to-salmon conidial masses. They form a monophyletic clade to which Nalanthamala psidii and N. diospyri are related based on analyses of the internal transcribed spacer regions and 5.8S rDNA (ITS rDNA), LSU rDNA, and partial beta-tubulin gene. Few polymorphic sites in the ITS rDNA and beta-tubulin gene indicate that Nalanthamala psidii comprises two lineages, one of which has been detected only in South Africa. (+info)
Role of zearalenone lactonase in protection of Gliocladium roseum from fungitoxic effects of the mycotoxin zearalenone.
Zearalenone is a mycotoxin with estrogenic effects on mammals that is produced by several species of Fusarium. We found that zearalenone and its derivatives inhibit the growth of filamentous fungi on solid media at concentrations of < or =10 microg/ml. The fungitoxic effect declined in the order zearalenone > alpha-zearalenol > beta-zearalenol. The mycoparasitic fungus Gliocladium roseum produces a zearalenone-specific lactonase which catalyzes the hydrolysis of zearalenone, followed by a spontaneous decarboxylation. The growth of G. roseum was not inhibited by zearalenone, and the lactonase may protect G. roseum from the toxic effects of this mycotoxin. We inactivated zes2, the gene encoding zearalenone lactonase in G. roseum, by inserting a hygromycin resistance cassette into the coding sequence of the gene by means of Agrobacterium tumefaciens-mediated genetic transformation. The zes2 disruption mutants could not hydrolyze the lactone bond of zearalenone and were more sensitive to zearalenone. These data are consistent with a hypothesis that resorcylic acid lactones exemplified by zearalenone act to reduce growth competition by preventing competing fungi from colonizing substrates occupied by zearalenone producers and suggest that they may play a role in fungal defense against mycoparasites. (+info)
Quantitative isolation of biocontrol agents Trichoderma spp., Gliocladium spp. and actinomycetes from soil with culture media.
Soil biodiversity plays a key role in the sustainability of agriculture systems and indicates the level of health of soil, especially when considering the richness of microorganisms that are involved in biological control of soilborne diseases. Cultural practices may produce changes in soil microflora, which can be quantified through the isolation of target microorganisms. Rhizosphere soil samples were taken from an assay with different crop rotations and tillage systems, and populations of Trichoderma spp., Gliocladium spp. and actinomycetes were quantified in order to select the general and selective culture media that better reflect the changes of these microbial populations in soil. The most efficient medium for the isolation of Trichoderma spp. and Gliocladium spp. was potato dextrose agar modified by the addition of chloramphenicol, streptomycin and rose bengal, and for actinomycetes was Kuster medium, with cycloheximide and sodium propionate. (+info)
Cloning and expression analysis of a chitinase gene Crchi1 from the mycoparasitic fungus Clonostachys rosea (syn. Gliocladium roseum).
Clonostachys rosea (syn. Gliocladium roseum) is a well-known biocontrol agent and widely distributed around the world. In this study, an endochitinase gene Crchi1 was isolated from the mycoparasitic fungus C. rosea using the DNA walking strategy. The Crchi1 ORF is 1,746 bp long and interrupted by three introns. The cloned gene Crchi1 encodes 426 amino acid residues and shares a high degree of similarity with other chitinases from entomopathogenic and mycoparasitic fungi. Several putative binding sites for transcriptional regulation of Crchi1 in response to carbon (5'-SYGGRG-3') and nitrogen (5'-GATA-3') were identified in the upstream of Crchi1. Expression of Crchi1 gene in different carbon sources was analyzed using real-time PCR (RT-PCR). We found that the Crchi1 expression was suppressed by glucose but strongly stimulated by chitin or solubilized components of the cell wall from Rhizoctonia solani. Phylogenetic analysis of chitinases from entomopathogenic and mycoparasitic fungi suggests that these chitinases have probably evolved from a common ancestor. (+info)
The production of myco-diesel hydrocarbons and their derivatives by the endophytic fungus Gliocladium roseum (NRRL 50072).