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Signal transduction by Tga3, a novel G protein alpha subunit of Trichoderma atroviride.

Trichoderma species are used commercially as biocontrol agents against a number of phytopathogenic fungi due to their mycoparasitic characterisitics. The mycoparasitic response is induced when Trichoderma specifically recognizes the presence of the host fungus and transduces the host-derived signals to their respective regulatory targets. We made deletion mutants of the tga3 gene of Trichoderma atroviride, which encodes a novel G protein alpha subunit that belongs to subgroup III of fungal Galpha proteins. Deltatga3 mutants had changes in vegetative growth, conidiation, and conidial germination and reduced intracellular cyclic AMP levels. These mutants were avirulent in direct confrontation assays with Rhizoctonia solani or Botrytis cinerea, and mycoparasitism-related infection structures were not formed. When induced with colloidal chitin or N-acetylglucosamine in liquid culture, the mutants had reduced extracellular chitinase activity even though the chitinase-encoding genes ech42 and nag1 were transcribed at a significantly higher rate than they were in the wild type. Addition of exogenous cyclic AMP did not suppress the altered phenotype or restore mycoparasitic overgrowth, although it did restore the ability to produce the infection structures. Thus, T. atroviride Tga3 has a general role in vegetative growth and can alter mycoparasitism-related characteristics, such as infection structure formation and chitinase gene expression.

Acetylglucosaminidase↗

Fungi Associated with Softening of Bisulfite-Brined Cherries.

Softening of sound, calcium bisulfite-brined cherries was induced fairly quickly by brining them with cherries rotted by Aspergillus niger, Cytospora leucostoma, and Penicillium expansum, but not with cherries rotted by a variety of other microorganisms, including Alternaria sp., Aspergillus oryzae, Aureobasidium pullulans, Botrytis cinerea, Cladosporium sp., Mucor racemosus, Rhizopus stolonifer, and Sclerotinia fructicola. Rapid softening was correlated with the presence of a bisulfite-stable polygalacturonase, as demonstrated by a cup-plate test. A survey of naturally rotted cherries suggests the involvement of a bark-canker fungus, C. leucostoma, in softening of commercially brined cherries in the Pacific Northwest.

Journal Article↗

Sorbic Hydroxamic Acid, an Antifungal Agent Effective over a Wide pH Range.

Sorbic hydroxamic acid was prepared from sorbic acid by esterification and treatment with hydroxylamine (mp 133 to 135 C, pK(a) 8.8). Its ultraviolet spectrum in acid solution had a single absorption maximum at 262 mmu; in alkaline solution the maximal absorption shifted to 255 mmu and significant absorption appeared at 280 to 300 mmu. At concentrations of 0.1% (w/v), sorbic hydroxamic acid prevented the growth of Aspergillus niger, Penicillium notatum, Botrytis cinerea, Cladosporium herbarum, and a Rhizopus species in grape juice over the pH range 3.6 to 9.2, although sorbic acid was not effective at pH 5.7 and above.

Journal Article↗

Vitamin K5 as a fungistatic agent.

The effectiveness of vitamin K(5) in controlling the growth of different molds at varying pH levels in a culture medium, in tomato juice, and in several berry purees was studied. The molds studied were Aspergillus, Botrytis, Hormodendrum, Mucor, and Penicillium. The results showed that vitamin K(5) was effective as a fungistatic agent at concentrations ranging from 0.006 to 0.02%.

Antifungal Agents↗

Fungistatic effects of controlled atmospheres.

The fungistatic effects of controlled atmospheres composed of increased CO(2) and decreased O(2) was studied in a manner such that the condition of stored fruit was not a factor in the growth of the fungi. Varying concentrations of O(2) and CO(2) were used. The fungi used were Botrytis alli, Rhizopus nigricans, and Penicillium expansum. The results showed that controlled atmospheres, within the limits of concentrations usable for fruit storage, are effective fungistatic agents.

Antifungal Agents↗

Influence of phosphate compounds on certain fungi and their preservative effects on fresh cherry fruit (Prunus cerasus, L.).

Studies were conducted to ascertain the retarding effects of four phosphate compounds (sodium hexametaphosphate, sodium tripolyphosphate, sodium tetraphosphate, and tetrasodium pyrophosphate) on molding of fresh cherries (Prunus cerasus, L.). In vitro studies on their antimycotic effects against the most common fungal spoilers, Penicillium expansum, Rhizopus nigricans, and Botrytis sp., were also carried out. Sodium tetraphosphate appeared to be the most effective compound in preserving cherries and also had the greatest antimycotic effects in the in vitro studies. A 10% concentration, when applied as a dip, inhibited fungal growth on fresh cherries for up to 30 days of storage at 1.1 C (34 F) and a relative humidity of 94%, whereas untreated controls showed fungal growth at 14 days. Following in order of effectiveness were sodium hexametaphosphate, sodium tripolyphosphate, and tetrasodium pyrophosphate.

Antifungal Agents↗

Whole-genome analysis of two-component signal transduction genes in fungal pathogens.

Two-component phosphorelay systems are minimally comprised of a histidine kinase (HK) component, which autophosphorylates in response to an environmental stimulus, and a response regulator (RR) component, which transmits the signal, resulting in an output such as activation of transcription, or of a mitogen-activated protein kinase cascade. The genomes of the yeasts Saccharomyces cerevisiae, Schizosaccharomyces pombe, and Candida albicans encode one, three, and three HKs, respectively. In contrast, the genome sequences of the filamentous ascomycetes Neurospora crassa, Cochliobolus heterostrophus (Bipolaris maydis), Gibberella moniliformis (Fusarium verticillioides), and Botryotinia fuckeliana (Botrytis cinerea) encode an extensive family of two-component signaling proteins. The putative HKs fall into 11 classes. Most of these classes are represented in each filamentous ascomycete species examined. A few of these classes are significantly more prevalent in the fungal pathogens than in the saprobe N. crassa, suggesting that these groups contain paralogs required for virulence. Despite the larger numbers of HKs in filamentous ascomycetes than in yeasts, all of the ascomycetes contain virtually the same downstream histidine phosphotransfer proteins and RR proteins, suggesting extensive cross talk or redundancy among HKs.

Amino Acid Sequence↗

Serological cross-reactivity among Sporothrix schenckii, Ceratocystis, Europhium, and Graphium species.

Ethanol-precipitable culture filtrate antigens of 100 strains of 75 species of the Sporothrix-Ceratocystis-Europhium-Graphium complex and 1 species of Botrytis were examined for neutral sugar components and for serological cross-reactivity with S. schenckii rabbit antiserum and human sporotrichosis sera by capillary precipitin and double immunodiffusion assay. Results revealed that cross-reactive species (60 of 77, ca. 80%) produced exoconidial forms and rhamnose- and mannose-containing polysaccharides and included Ceratocystis, the three known Europhium, and several Graphium-form species. Endoconidial-form Ceratocystis species did not cross-react.

Animals↗

Cell wall synthesis is a major target of mycoparasitic antagonism by Trichoderma harzianum.

We have investigated the molecular basis for the reported synergism between peptaibols and cell wall hydrolytic enzymes in the antagonism of phytopathogenic fungi by Trichoderma harzianum. beta-Glucan synthase activity on isolated plasma membranes of Botrytis cinerea was inhibited in vitro by the peptaibols trichorzianin TA and TB, and this inhibition was reversed by the addition of phosphatidylcholine. beta-Glucan synthesis in vivo, assayed by the incorporation of [2-(3)H]glucose into cell wall material, was inhibited by the presence of peptaibols, and this inhibition was synergistic with exogenously added T. harzianum beta-1,3-glucanase. This synergism is therefore explained by an inhibition of the membrane-bound beta-1,3-glucan synthase of the host by the peptaibols, which inhibit the resynthesis of cell wall beta-glucans, sustain the disruptive action of beta-glucanases, and all together enhance the fungicidal activity. Therefore, we have identified cell wall turnover as a major target of mycoparasitic antagonism.

Amino Acid Sequence↗

Identification of two different 14-alpha sterol demethylase-related genes (cyp51A and cyp51B) in Aspergillus fumigatus and other Aspergillus species.

Two cyp51-related genes (cyp51A and cyp51B) encoding 14-alpha sterol demethylase-like enzymes were identified in the opportunistic human pathogen Aspergillus fumigatus. PCR amplification using degenerate oligonucleotides based on conserved areas of cytochrome P450 demethylases of other filamentous fungi and yeasts allowed the cloning and sequencing of two different homologue genes in A. fumigatus. Southern analysis confirmed that both genes hybridized to distinct genomic loci and that both are represented as single copies in the genome. Comparison of the deduced Cyp51A and Cyp51B proteins with the CYP51 proteins from Penicillium italicum, Aspergillus nidulans, Erysiphe graminis, Uncinula necator, Botrytis cinerea, Ustilago maydis, Cryptococcus neoformans, Candida albicans, Saccharomyces cerevisiae, Candida tropicalis, and Candida glabrata showed that the percentages of identity of the amino acid sequences (range, 40 to 70%) were high enough to consider Cyp51A and Cyp51B to be members of the fungal CYP51 family. Fragments from both genes were also cloned from other Aspergillus spp. (A. flavus, A. nidulans, and A. terreus). Phylogenetic analysis showed that, at least in the most pathogenic species of Aspergillus, there are two fungal CYP51 proteins. This is the first report of the existence of two homologue genes coding for 14-alpha sterol demethylase in the fungal kingdom. This finding could provide insights into the azole resistance mechanisms operating in fungi. The primers used here may be useful molecular tools for facilitating the cloning of novel 14-alpha sterol demethylase genes in other filamentous fungi.

Amino Acid Sequence↗

Mycoparasitism of sclerotial fungi by Teratosperma oligocladum.

Sclerotia of Sclerotinia minor were parasitized by Teratosperma oligocladum, a recently described dematiaceous hyphomycete. The mycoparasite was cultured on living sclerotia placed on water agar and on sclerotia in moist sand. It grew poorly on several common laboratory media but growth in vitro was enhanced by supplements of soil extract and, especially, by aqueous extracts of sclerotia. Sclerotia of S. minor, S. sclerotiorum, S. trifoliorum, Sclerotium cepivorum, and Botrytis cinerea were parasitized in vitro, but sclerotia of Sclerotium rolfsii and Macrophomina phaseolina were not. Macroconidia of T. oligocladum germinated on membrane filters placed on soil containing sclerotia of S. minor but not on soil without sclerotia. Sclerotia of three Sclerotinia spp. were infected within 2 weeks in soil infested with the mycoparasite. Teratosperma oligocladum parasitized and destroyed all of the sclerotia of S. minor buried in a natural soil by 10 weeks. Parasitism was equally good at 20 and 25 degrees C, but occurred more slowly at 15 degrees C. No parasitic activity occurred at 30 degrees C. The morphology, cultural characteristics, and mycoparasitic habit of T. oligocladum indicated that it was similar in many respects to the mycoparasite, Sporidesmium sclerotivorum, and that it is a potentially useful agent fo the biological control for sclerotial plant pathogens.

Ascomycota↗

Phaseococcin, an antifungal protein with antiproliferative and anti-HIV-1 reverse transcriptase activities from small scarlet runner beans.

From the seeds of small scarlet runner beans (Phaseolus coccineus 'Minor'), an antifungal protein with an N-terminal sequence homologous to those of defensins was isolated. The antifungal protein bound to Affi-gel blue gel and Mono S but it did not bind to DEAE-cellulose. It was further purified by gel filtration on a Superdex peptide column. It exhibited a molecular mass of 5422 Da as determined by mass spectrometry. The protein, designated as phaseococcin, suppressed mycelial growth in a number of fungi including Botrytis cinerea, Coprinus comatus, Fusarium oxysporum, Mycosphaerella arachidicola, Physalospora piricola, and Rhizoctonia solani. It also inhibited proliferation in several Bacillus species and the leukemia cell lines HL60 and L1210 and curtailed the activity of HIV-1 reverse transcriptase. It did not affect proliferation of mouse splenocytes and neither did it inhibit protein synthesis in a cell-free rabbit reticulocyte lysate system.

Amino Acid Sequence↗

Biological control in greenhouse systems.

The controlled environment of greenhouses, the high value of the crops, and the limited number of registered fungicides offer a unique niche for the biological control of plant diseases. During the past ten years, over 80 biocontrol products have been marketed worldwide. A large percentage of these have been developed for greenhouse crops. Products to control soilborne pathogens such as Sclerotinia, Pythium, Rhizoctonia and Fusarium include Coniothyrium minitans, species of Gliocladium, Trichoderma, Streptomyces, and Bacillus, and nonpathogenic Fusarium. Products containing Trichoderma, Ampelomyces quisqualis, Bacillus, and Ulocladium are being developed to control the primary foliar diseases, Botrytis and powdery mildew. The development of Pseudomonas for the control of Pythium diseases in hydroponics and Pseudozyma flocculosa for the control of powdery mildew by two Canadian research programs is presented. In the future, biological control of diseases in greenhouses could predominate over chemical pesticides, in the same way that biological control of greenhouse insects predominates in the United Kingdom. The limitations in formulation, registration, and commercialization are discussed, along with suggested future research priorities.

Canada↗

The distribution and evolutionary history of the PRP8 intein.

BACKGROUND: We recently described a mini-intein in the PRP8 gene of a strain of the basidiomycete Cryptococcus neoformans, an important fungal pathogen of humans. This was the second described intein in the nuclear genome of any eukaryote; the first nuclear encoded intein was found in the VMA gene of several saccharomycete yeasts. The evolution of eukaryote inteins is not well understood. In this report we describe additional PRP8 inteins (bringing the total of these to over 20). We compare and contrast the phylogenetic distribution and evolutionary history of the PRP8 intein and the saccharomycete VMA intein, in order to derive a broader understanding of eukaryote intein evolution. It has been suggested that eukaryote inteins undergo horizontal transfer and the present analysis explores this proposal. RESULTS: In total, 22 PRP8 inteins have been detected in species from three different orders of euascomycetes, including Aspergillus nidulans and Aspergillus fumigatus (Eurotiales), Paracoccidiodes brasiliensis, Uncinocarpus reesii and Histoplasma capsulatum (Onygales) and Botrytis cinerea (Helotiales). These inteins are all at the same site in the PRP8 sequence as the original Cryptococcus neoformans intein. Some of the PRP8 inteins contain apparently intact homing endonuclease domains and are thus potentially mobile, while some lack the region corresponding to the homing endonuclease and are thus mini-inteins. In contrast, no mini-inteins have been reported in the VMA gene of yeast. There are several examples of pairs of closely related species where one species carries the PRP8 intein while the intein is absent from the other species. Bio-informatic and phylogenetic analyses suggest that many of the ascomycete PRP8 homing endonucleases are active. This contrasts with the VMA homing endonucleases, most of which are inactive. CONCLUSION: PRP8 inteins are widespread in the euascomycetes (Pezizomycota) and apparently their homing endonucleases are active. There is no evidence for horizontal transfer within the euascomycetes. This suggests that the intein is of ancient origin and has been vertically transmitted amongst the euascomycetes. It is possible that horizontal transfer has occurred between the euascomycetes and members of the basidiomycete genus Cryptococcus.

Amino Acid Sequence↗

Monoclonal antibody TeM 106 reacts with a tonoplast intrinsic protein of 106 kDa from Brassica oleracea L.

A monoclonal antibody, designated TeM 106, that recognizes an intrinsic protein from the vacuole membrane (tonoplast) of cauliflower (Brassica oleracea L. var. botrytis) is described. Mice were immunized with a tonoplast fraction that had been purified from differentiating meristematic cells from the cauliflower head. Hybridomas were generated and screened by means of Enzyme Linked Immuno Sorbent Assays for differential reactivity to tonoplast over non-related proteins (bovine serum albumin). One out of 14 reactive murine clones was selected on the basis of its stability, secretory efficiency, and high affinity of the secreted antibodies. TeM 106 is an IgM which was shown by indirect immunofluorescence microscopy of frozen thin sections to bind specifically to the tonoplast of highly vacuolated cells as well as to the tonoplast of small vacuoles in meristematic cells. The molecular specificities of TeM 106 were preliminarily determined using electrophoretic transfer procedures (immunoblotting). TeM 106 reacted with a single protein band of 106,000 M(r) from the tonoplast of cauliflower. Using two-dimensional gel electrophoresis, it was shown that the epitope is borne by a single polypeptide. The antigen is a glycopeptide containing mannose and/or glucose residues in the oligosaccharide side chain but the epitope, resistant to the metaperiodate oxidation, is contained in the polypeptide backbone. Salt elution experiments indicated that the antigen, unlike several proteins from the tonoplast, is not eluted from the membrane by KCl treatments and is, therefore, tentatively considered as a tonoplast intrinsic protein, designated TIP 106.

Animals↗

Virus-induced silencing of FtsH gene in Nicotiana benthmiana causes a striking bleached leaf phenotype.

A recombinant Potato virus X (PVX) vector, pTXS.FtsH, harboring partial sequence of FtsH gene of Nicotiana benthamiana was constructed to silence the expression of endogenous FtsH homologous gene in N. benthamiana. Inoculation with in vitro runoff transcript of pTXS.FtsH to N. benthamiana plants allowed silencing of FtsH, causing striking bleaching of upper leaves reminiscent of var2 mutant phenotype of Arabidopsis thaliana. FtsH-silenced plants exhibited no resistance against Tobacco mosaic virus (TMV) and a phytopathogenic fungus Botrytis cinerea. Virus-induced gene silencing (VIGS) of N. benthamiana with PVX as demonstrated here would be an efficient and rapid method to study the function of other elements of photosystem II (PSII) in planta.

Base Sequence↗

Blasticidin S deaminase gene (BSD): a new selection marker gene for transformation of Arabidopsis thaliana and Nicotiana tabacum.

Arabidopsis thaliana and Nicotiana tabacum were transformed to blasticidin S (BS) resistance with BSD (the BS deaminase gene from Aspergillus terreus) using the Agrobacterium-mediated transformation method. Expression of BSD allowed direct selection of transformants by the fungicide, and both kinds of transgenic plants showed high level of resistance phenotype at 100 ppm of BS sprayed on the leaves. Using Botrytis cinerea, the causal fungus of gray mold disease, it was exemplified that application of BS could control the disease in transgenic tobacco with negligible phytotoxicity.

Aminohydrolases↗

S-methyl methanethiosulfonate, bio-antimutagen in homogenates of Cruciferae and Liliaceae vegetables.

The isolation of a new type of bio-antimutagen, S-methyl methanethiosulfonate (MMTS), from cauliflower (Brassica oleracea var. botrytis) and the distribution and formation of MMTS in Cruciferae and Liliaceae vegetables are described. For the separation and purification, cauliflower curds were homogenized, extracted with acetone, and then purified by organic solvent extraction and by various processes of chromatographic separation. The chemical structure of an active principle was identified as MMTS by GC-MS and 1H-NMR analyses. MMTS was widely found in vegetable homogenates of the Cruciferae and Liliaceae species, and is found from its precursor S-methyl-L-cysteinesulfoxide (SMCS), by wounding the vegetable tissues. Wounding may induce C-S lyase, which converts SMCS to MMTS. The amount of MMTS formed was affected by the pH value for C- lyase, but not by the SMCS content in a tissue homogenate.

Antimutagenic Agents↗