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PubMed1999

Occurrence of a lipase in spores of Alternaria brassicicola with a crucial role in the infection of cauliflower leaves.

Alternaria brassicicola is a pathogen that penetrates directly through the host cuticle thanks to several serine esterases, according to our findings. Among these, an 80-kDa lipase (E.C 3.1.1. 3) was detected by SDS-PAGE and immunoblotting in the water washings of ungerminated spores. The purified lipase cross-reacted with Botrytis cinerea anti-lipase antibodies, which were reported to inhibit the in vitro lipase activity. Anti-lipase antibodies were added to a conidial suspension of A. brassicicola prior to inoculation. As a result, blackspot lesions were reduced by 90% on intact cauliflower leaves, but not on leaves from which surface wax had been removed. Spore surface-bound lipase is thought to interact closely with epicuticular leaf waxes for adhesion and/or penetration of the fungal propagules during the early stages of host-parasite interactions.

Alternaria↗
PubMed2000

ITS1 region of the nuclear ribosomal DNA of the mycoparasite Pythium periplocum, its taxonomy, and its comparison with related species.

Pythium periplocum Drechsler was isolated from some soil samples taken in the botanical garden of Tenerife, Canary Islands. This fungus has been found to be an aggressive mycoparasite of Botrytis cinerea. It is unique amongst the members of the genus Pythium because of the character combination of inflated filamentous type of sporangia and ornamented oogonia. The taxonomic description of this fungus and its comparison with related species, together with the polymerase chain reaction of the internal transcribed spacer of its nuclear ribosomal DNA, are discussed in this article.

Base Sequence↗
PubMed2001

Antifungal activity of a novel endochitinase gene (chit36) from Trichoderma harzianum Rifai TM.

A novel 36-kDa endochitinase named chit36 has been isolated and characterized from Trichoderma harzianum Rifai TM. Partial amino acid sequences from the purified protein were used to clone the fungal cDNA, based on polymerase chain reaction with degenerate primers. The complete open reading frame encodes a 344-amino acid protein which shows 84% similarity to a putative chitinase from Streptomyces coelicolor. Chit36 was overexpressed under the pki1 constitutive promoter from Trichoderma reesei via biolistic transformation of T. harzianum TM. Stable transformants showed expression and endochitinase activity of chit36 in glucose-rich medium. Culture filtrates containing secreted CHIT36 as the sole chitinolytic enzyme completely inhibited the germination of Botrytis cinerea conidia. Growth of Fusarium oxysporum f. sp. melonis and Sclerotium rolfsii were significantly inhibited on agar plates on which the Trichoderma transformants had previously been grown.

Amino Acid Sequence↗
PubMed2005

Expression of an alpha-1,3-glucanase during mycoparasitic interaction of Trichoderma asperellum.

Trichoderma species have been investigated as biological control agents for over 70 years owing to their ability to antagonize plant pathogenic fungi. Mycoparasitism, one of the main mechanisms involved in the antagonistic activity of Trichoderma strains, depends on the secretion of complex mixtures of hydrolytic enzymes able to degrade the host cell wall. The antifungal activity of an alpha-1,3-glucanase (EC 3.2.1.59, enzymes able to degrade alpha-1,3-glucans and also named mutanases) has been described in T. harzianum and its role in mycoparasitic processes has been suggested. In this study, we report on the purification, characterization and cloning of an exo-alpha-1,3-glucanase, namely AGN13.2, from the antagonistic fungus T. asperellum T32. Expression at the transcription level in confrontation assays against the strawberry pathogen Botrytis cinerea strongly supports the role of AGN13.2 during the antagonistic action of T. asperellum.

Amino Acid Sequence↗
PubMed1976

Bovine nasal granuloma in Victoria -- a search for the causative allergens.

Cows with nasal granuloma showed skin sensitivity to a range of environmental allergens while clinically normal control animals showed little sensitivity. The allergens producing the greatest number of positive reactions included the pollens of dock, capeweed, clover, paspalum, sorrel, rye grass, pepper tree and wattle, the spores of penicillium, cladosporium, botrytis and rye grass rust and extracts of sheep's wool and mite. It is likely that different allergens are important in different animals and in different regions and their importance in the aetiology of nasal granuloma probably depends on their presence in the inhaled air in high concentration for long periods.

Allergens↗
PubMed1995

Molecular basis of the cauliflower phenotype in Arabidopsis.

Genetic studies demonstrate that two Arabidopsis genes, CAULIFLOWER and APETALA1, encode partially redundant activities involved in the formation of floral meristems, the first step in the development of flowers. Isolation of the CAULIFLOWER gene from Arabidopsis reveals that it is closely related in sequence to APETALA1. Like APETALA1, CAULIFLOWER is expressed in young flower primordia and encodes a MADS-domain, indicating that it may function as a transcription factor. Analysis of the cultivated garden variety of cauliflower (Brassica oleracea var. botrytis) reveals that its CAULIFLOWER gene homolog is not functional, suggesting a molecular basis for one of the oldest recognized flower abnormalities.

Alleles↗
PubMed1978

Detection of mutagens produced by fungi with the Salmonella typhimurium assay.

Forty-one fungal isolates (one isolate per species) representing common plant pathogens and food crop contaminants were grown on sterile, polished rice and assayed for mutagenic activity in the Salmonella typhimurium-microsome system. Initially, single doses of aqueous and chloroform extracts of the moldy rice were assayed against the TA100 tester strain by incorporating extracts into the growth medium and by applying small quantities on disks placed on the agar surface. Suspected activity was examined further by analysis of several doses in the plate incorporation assay. Extracts of two aflatoxin-producing isolates (Aspergillus flavus and A. parasiticus) showed pronounced mutagenic activity, as did extracts of five other isolates (A. heterothallicus, A. nidulans, A. terricola, Alternaria tenuis, and Fusarium moniliforme) which did not contain detectable aflatoxins. Seven additional isolates (Botrytis cineria, Ceratocystis fimbriata, Cladosporium herbarum, Fusarium solani f. sp. pisi, Penicillium oxalicum, Thermomyces lanuginosus, and Verticilium albo-atrum) revealed activity which was possibly mutagenic; i.e., mutagenic responses were not observed in both the disk and incorporation assays, and clear dose-related activity was not observed in the incorporation assay. Extracts of the remaining fungi were not mutagenic in the bacterial assay.

Food Microbiology↗
PubMed1984

Comparative studies of extracellular fungal laccases.

Various basidiomycetes, ascomycetes, and deuteromycetes, grown in a sugar-rich liquid medium, were compared for laccase-producing ability and for the inducing effect of 2,5-xylidine on laccase production. Clear stimulation of the extracellular enzyme formation by xylidine was obtained in the cultures of Fomes annosus, Pholiota mutabilis, Pleurotus ostreatus, and Trametes versicolor, whereas Rhizoctonia praticola and Botrytis cinerea were not affected by the xylidine, and in the case of Podospora anserina a decrease in laccase activity was observed. The laccases were purified, and electrophoresis on polyacrylamide gels indicated a particular pattern for each laccase. The bands of the induced forms appeared only with basidiomycetes. The optimal pH of R. praticola laccase was in the neutral region, whereas the optima of all the other exolaccases were significantly lower (between pH 3.0 and 5.7). All laccases oxidized the methoxyphenolic acids under investigation, but there existed quantitative differences in oxidation efficiencies which depended on pH and on the nature (noninduced or induced) of the enzyme. The sensitivity of all enzymes to inhibitors did not differ considerably.

Journal Article↗
PubMed1990

Characterization and reclassification of yeasts used for biological control of postharvest diseases of fruits and vegetables.

In previous studies workers have shown that three yeast strains (strains US-7, 82, and 101) have biological control activity against various postharvest fungal pathogens of fruits and vegetables, including Penicillium rots of apples and citrus and Botrytis rot of apples. In these reports the researchers have described these strains as Debaryomyces hansenii (anamorph, Candida famata) or Candida sp. strains. In this study we performed additional physiological, DNA reassociation, and mannan characterization tests that clearly established a new taxonomic classification for these strains, Candida guilliermondii. We also propose amendment of the physiological test profile in the taxonomic description of C. guilliermondii.

Journal Article↗
PubMed1994

Parallel formation and synergism of hydrolytic enzymes and peptaibol antibiotics, molecular mechanisms involved in the antagonistic action of Trichoderma harzianum against phytopathogenic fungi.

Chitinase, beta-1,3-glucanase, and protease activities were formed when Trichoderma harzianum mycelia, grown on glucose as the sole carbon source, were transferred to fresh medium containing cell walls of Botrytis cinerea. Chitobiohydrolase, endochitinase, and beta-1,3-glucanase activities were immunologically detected in culture supernatants by Western blotting (immunoblotting), and the first two were quantified by enzyme-linked immunosorbent assay. Under the same conditions, exogenously added [U-14C]valine was incorporated in acetone-soluble compounds with an apparent M(r) of < 2,000. These compounds comigrated with the peptaibols trichorzianines A1 and B1 in thin-layer chromatography and released [U-14C]valine after incubation in 6N HCl. Incorporation of radioactive valine into this material was stimulated by the exogenous supply of alpha-aminoisobutyric acid, a rare amino acid which is a major constituent of peptaibols. The obtained culture supernatants inhibited spore germination as well as hyphal elongation of B. cinerea. Culture supernatants from mycelia placed in fresh medium without cell walls of B. cinerea did not show hydrolase activities, incorporation of [U-14C]valine into peptaibol-like compounds, and inhibition of fungal growth. Purified trichorzianines A1 and B1 as well as purified chitobiohydrolase, endochitinase, or beta-1,3-glucanase inhibited spore germination and hyphal elongation, but at concentrations higher than those observed in the culture supernatants. However, when the enzymes and the peptaibols were tested together, an antifungal synergistic interaction was observed and the 50% effective dose values obtained were in the range of those determined in the culture supernatants. Therefore, the parallel formation and synergism of hydrolytic enzymes and antibiotics may have an important role in the antagonistic action of T. harzianum against fungal phytopathogens.

Amino Acid Sequence↗
PubMed1994

Chlorine Dioxide for Reduction of Postharvest Pathogen Inoculum during Handling of Tree Fruits.

Alternatives to hypochlorous acid and fungicides are needed for treatment of fruit and fruit-handling facilities. Chlorine dioxide was evaluated and found effective against common postharvest decay fungi and against filamentous fungi occurring on fruit packinghouse surfaces. In vitro tests with conidial or sporangiospore suspensions of Botrytis cinerea, Penicillium expansum, Mucor piriformis, and Cryptosporiopsis perennans demonstrated >99% spore mortality within 1 min when the fungi were exposed to aqueous chlorine dioxide at 3 or 5 mug . ml. Longer exposure times were necessary to achieve similar spore mortalities with 1 mug . ml. Of the fungi tested, B. cinerea and P. expansum were the least sensitive to ClO(2). In comparison with the number recovered from untreated control areas, the number of filamentous fungi recovered was significantly lower in swipe tests from hard surfaces such as belts and pads in a commercial apple and pear packinghouse after treatment of surfaces with a 14.0- to 18.0-mug . ml ClO(2) foam formulation. Chlorine dioxide has desirable properties as a sanitizing agent for postharvest decay management when residues of postharvest fungicides are not desired or allowed.

Journal Article↗
PubMed1998

A novel ATP-binding cassette transporter involved in multidrug resistance in the phytopathogenic fungus Penicillium digitatum.

Demethylation inhibitor (DMI)-resistant strains of the plant pathogenic fungus Penicillium digitatum were shown to be simultaneously resistant to cycloheximide, 4-nitroquinoline-N-oxide (4NQO), and acriflavine. A PMR1 (Penicillium multidrug resistance) gene encoding an ATP-binding cassette (ABC) transporter (P-glycoprotein) was cloned from a genomic DNA library of a DMI-resistant strain (LC2) of Penicillium digitatum by heterologous hybridization with a DNA fragment containing an ABC-encoding region from Botrytis cinerea. Sequence analysis revealed significant amino acid homology to the primary structures of PMR1 (protein encoded by the PMR1 gene) and ABC transporters of Saccharomyces cerevisiae (PDR5 and SNQ2), Schizosaccharomyces pombe (HBA2), Candida albicans (CDR1), and Aspergillus nidulans (AtrA and AtrB). Disruption of the PMR1 gene of P. digitatum DMI-resistant strain LC2 demonstrated that PMR1 was an important determinant of resistance to DMIs. The effective concentrations inhibiting radial growth by 50% (EC50s) and the MICs of fenarimol and bitertanol for the PMR1 disruptants (Deltapmr1 mutants) were equivalent to those for DMI-sensitive strains. Northern blot analysis indicated that severalfold more PMR1 transcript accumulated in the DMI-resistant strains compared with those in DMI-sensitive strains in the absence of fungicide. In both DMI-resistant and -sensitive strains, transcription of PMR1 was strongly enhanced within 10 min after treatment with the DMI fungicide triflumizole. These results suggested that the toxicant efflux system comprised of PMR1 participates directly in the DMI resistance of the fungus.

4-Nitroquinoline-1-oxide↗
PubMed1999

Expression of two major chitinase genes of Trichoderma atroviride (T. harzianum P1) is triggered by different regulatory signals.

Regulation of the expression of the two major chitinase genes, ech42 (encoding the CHIT42 endochitinase) and nag1 (encoding the CHIT73 N-acetyl-beta-D-glucosaminidase), of the chitinolytic system of the mycoparasitic biocontrol fungus Trichoderma atroviride (= Trichoderma harzianum P1) was investigated by using a reporter system based on the Aspergillus niger glucose oxidase. Strains harboring fusions of the ech42 or nag1 5' upstream noncoding sequences with the A. niger goxA gene displayed a glucose oxidase activity pattern that was consistent under various conditions with expression of the native ech42 and nag1 genes, as assayed by Northern analysis. The expression product of goxA in the mutants was completely secreted into the medium, detectable on Western blots, and quantifiable by enzyme-linked immunosorbent assay. nag1 gene expression was triggered during growth on fungal (Botrytis cinerea) cell walls and on the chitin degradation product N-acetylglucosamine. N-Acetylglucosamine, di-N-acetylchitobiose, or tri-N-acetylchitotriose also induced nag1 gene expression when added to mycelia pregrown on different carbon sources. ech42 expression was also observed during growth on fungal cell walls but, in contrast, was not triggered by addition of chitooligomers to pregrown mycelia. Significant ech42 expression was observed after prolonged carbon starvation, independent of the use of glucose or glycerol as a carbon source, suggesting that relief of carbon catabolite repression was not involved in induction during starvation. In addition, ech42 gene transcription was triggered by physiological stress, such as low temperature, high osmotic pressure, or the addition of ethanol. Four copies of a putative stress response element (CCCCT) were found in the ech42 promoter.

Aspergillus niger↗
PubMed1999

Comparison of fungal laccases and redox mediators in oxidation of a nonphenolic lignin model compound.

Several fungal laccases have been compared for the oxidation of a nonphenolic lignin dimer, 1-(3, 4-dimethoxyphenyl)-2-(2-methoxyphenoxy)propan-1,3-diol (I), and a phenolic lignin model compound, phenol red, in the presence of the redox mediators 1-hydroxybenzotriazole (1-HBT) or violuric acid. The oxidation rates of dimer I by the laccases were in the following order: Trametes villosa laccase (TvL) > Pycnoporus cinnabarinus laccase (PcL) > Botrytis cinerea laccase (BcL) > Myceliophthora thermophila laccase (MtL) in the presence of either 1-HBT or violuric acid. The order is the same if the laccases are used at the same molar concentration or added to the same activity (with ABTS [2, 2'-azinobis (3-ethylbenzothiazoline-6-sulfonic acid)] as a substrate). During the oxidation of dimer I, both 1-HBT and violuric acid were to some extent consumed. Their consumption rates also follow the above order of laccases, i.e., TvL > PcL > BcL > MtL. Violuric acid allowed TvL and PcL to oxidize dimer I much faster than 1-HBT, while BcL and violuric acid oxidized dimer I more slowly than BcL and 1-HBT. The oxidation rate of dimer I is dependent upon both kcat and the stability of the laccase. Both 1-HBT and violuric acid inactivated the laccases, violuric acid to a greater extent than 1-HBT. The presence of dimer I or phenol red in the reaction mixture slowed down this inactivation. The inactivation is mainly due to the reaction of the redox mediator free radical with the laccases. We did not find any relationship between the carbohydrate content of the laccases and their inactivation. When the redox potential of the laccases is in the range of 750 to 800 mV, i.e., above that of the redox mediator, it does not affect kcat and the oxidation rate of dimer I.

Barbiturates↗
PubMed2000

Molecular characterization and identification of biocontrol isolates of Trichoderma spp.

The most common biological control agents (BCAs) of the genus Trichoderma have been reported to be strains of Trichoderma virens, T. harzianum, and T. viride. Since Trichoderma BCAs use different mechanisms of biocontrol, it is very important to explore the synergistic effects expressed by different genotypes for their practical use in agriculture. Characterization of 16 biocontrol strains, previously identified as "Trichoderma harzianum" Rifai and one biocontrol strain recognized as T. viride, was carried out using several molecular techniques. A certain degree of polymorphism was detected in hybridizations using a probe of mitochondrial DNA. Sequencing of internal transcribed spacers 1 and 2 (ITS1 and ITS2) revealed three different ITS lengths and four different sequence types. Phylogenetic analysis based on ITS1 sequences, including type strains of different species, clustered the 17 biocontrol strains into four groups: T. harzianum-T. inhamatum complex, T. longibrachiatum, T. asperellum, and T. atroviride-T. koningii complex. ITS2 sequences were also useful for locating the biocontrol strains in T. atroviride within the complex T. atroviride-T. koningii. None of the biocontrol strains studied corresponded to biotypes Th2 or Th4 of T. harzianum, which cause mushroom green mold. Correlation between different genotypes and potential biocontrol activity was studied under dual culturing of 17 BCAs in the presence of the phytopathogenic fungi Phoma betae, Rosellinia necatrix, Botrytis cinerea, and Fusarium oxysporum f. sp. dianthi in three different media.

DNA, Mitochondrial↗
PubMed2002

Differentiation of Phytophthora infestans sporangia from other airborne biological particles by flow cytometry.

The ability of two different flow cytometers, the Microcyte (Optoflow) and the PAS-III (Partec), to differentiate sporangia of the late-blight pathogen Phytophthora infestans from other potential airborne particles was compared. With the PAS-III, light scatter and intrinsic fluorescence parameters could be used to differentiate sporangia from conidia of Alternaria or Botrytis spp., rust urediniospores, and pollen of grasses and plantain. Differentiation between P. infestans sporangia and powdery mildew conidia was not possible by these two methods but, when combined with analytical rules evolved by genetic programming methods, could be achieved after staining with the fluorescent brightener Calcofluor white M2R. The potential application of these techniques to the prediction of late-blight epiphytotics in the field is discussed.

Air Microbiology↗
PubMed2002

Identification of novel hexapeptides bioactive against phytopathogenic fungi through screening of a synthetic peptide combinatorial library.

The purpose of the present study was to improve the antifungal activity against selected phytopathogenic fungi of the previously identified hexapeptide PAF19. We describe some properties of a set of novel synthetic hexapeptides whose D-amino acid sequences were obtained through screening of a synthetic peptide combinatorial library in a positional scanning format. As a result of the screening, 12 putative bioactive peptides were identified, synthesized, and assayed. The peptides PAF26 (Ac-rkkwfw-NH(2)), PAF32 (Ac-rkwhfw-NH(2)), and PAF34 (Ac-rkwlfw-NH(2)) showed stronger activity than PAF19 against isolates of Penicillium digitatum, Penicillium italicum, and Botrytis cinerea. PAF26 and PAF32, but not PAF34, were also active against Fusarium oxysporum. Penicillium expansum was less susceptible to all four PAF peptides, and only PAF34 showed weak activity against it. Assays were also conducted on nontarget organisms, and PAF26 and PAF32 showed much-reduced toxicity to Escherichia coli and Saccharomyces cerevisiae, demonstrating selectivity towards certain filamentous fungi. Thus, the data showed distinct activity profiles for peptides differentiated by just one or two residue substitutions. Our conclusion from this observation is that a specificity factor is involved in the activity of these short peptides. Furthermore, PAF26 and PAF32 displayed activities against P. digitatum, P. italicum, and B. cinerea similar to that of the hemolytic 26-amino acid melittin, but they did not show the high toxicity of melittin towards bacteria and yeasts. The four peptides acted additively, with no synergistic interactions among them, and PAF26 was shown to have improved activity over PAF19 in in vivo orange fruit decay experiments.

Antifungal Agents↗
PubMed2005

Production of an engineered killer peptide in Nicotiana benthamiana by using a potato virus X expression system.

The decapeptide killer peptide (KP) derived from the sequence of a single-chain, anti-idiotypic antibody acting as a functional internal image of a microbicidal, broad-spectrum yeast killer toxin (KT) was shown to exert a strong microbicidal activity against human pathogens. With the aim to exploit this peptide to confer resistance to plant pathogens, we assayed its antimicrobial activity against a broad spectrum of phytopathogenic bacteria and fungi. Synthetic KP exhibited antimicrobial activity in vitro towards Pseudomonas syringae, Erwinia carotovora, Botrytis cinerea, and Fusarium oxysporum. KP was also expressed in plants by using a Potato virus X (PVX)-derived vector as a fusion to the viral coat protein, yielding chimeric virus particles (CVPs) displaying the heterologous peptide. Purified CVPs showed enhanced antimicrobial activity against the above-mentioned plant pathogens and human pathogens such as Staphylococcus aureus and Candida albicans. Moreover, in vivo assays designed to challenge KP-expressing plants (as CVPs) with Pseudomonas syringae pv. tabaci showed enhanced resistance to bacterial attack. The results indicate that the PVX-based display system is a high-yield, rapid, and efficient method to produce and evaluate antimicrobial peptides in plants, representing a milestone for the large-scale production of high-added-value peptides through molecular farming. Moreover, KP is a promising molecule to be stably engineered in plants to confer broad-spectrum resistance to phytopathogens.

Anti-Bacterial Agents↗