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Inhibition of phosphatidylcholine and chitin biosynthesis in Pyricularia oryzae, Botrytis fabae and Fusarium graminearum by edifenphos.

Colony growth of the fungi Pyricularia oryzae, Botrytis fabae and Fusarium graminearum was reduced by 50% (ED50) by edifenphos concentrations of 7, 25 and 190 microM respectively; the phosphatidylcholine (PC) content of biomass of P. oryzae, B. fabae, and F. graminearum harvested from fungicide-containing-cultures was reduced by 50% by 6, 95 and 350 microM-edifenphos respectively. By contrast, the activities of membrane-bound chitin synthase preparations isolated from the three fungi were approximately equally sensitive to edifenphos. A direct relationship was observed between PC contents of biomass grown in the presence of ediphenphos and in vivo rates of chitin synthesis (biomass incubated with [3H]GlcNAc in the absence of fungicide). Membrane-bound chitin synthase preparations from P. oryzae grown in medium containing 3 or 6 microM-edifenphos had, at the same fungicide concentration, a lower rate of in vivo chitin synthesis than preparations isolated from biomass grown in the absence of edifenphos. Membrane-bound chitin synthase preparations from P. oryzae grown in the presence and absence of 6 microM-edifenphos had the same Km values for the substrate (UDP-[14C]GlcNAc) but different Vmax values. The results suggest that chitin synthesis is inhibited directly by non-competitive inhibition of chitin synthase activity, and indirectly following inhibition of PC biosynthesis. P. oryzae is very sensitive to edifenphos because inhibition of PC biosynthesis occurs at very low fungicide concentrations, and therefore in this fungus inhibition of PC biosynthesis probably represents the primary mode of action of the fungicide.

Antifungal Agents↗

Light-induced synchronous conidiation in the fungus Botrytis cinerea.

Botrytis cinerea Pers. ex Fr. in stationary liquid cultures conidiated asynchronously in darkness after 4 days' growth. Synchronous conidiation was induced by irradiating dark-grown cultures with near-ultraviolet light for 12 h. The number of conidia increased very rapidly 10 h after the end of the photo-induction period, and conidiation was completed by the 14th hour. Filter paper cultures of the fungus also showed synchronous conidiation upon irradiation with near-ultraviolet light, but the rapid increase in the number of conidia took place 2 h earlier, conidiation being completed by the 12th hour. Cultures irradiated with blue light, however, produced sterile mycelia and showed complete suppression of conidiation.

Cell Division↗

Locus of blue and near ultraviolet reversible photoreaction in the stages of conidial development in Botrytis cinerea.

The effect of the blue and near ultraviolet reversible photoreaction on conidial development in Botrytis cinerea was studied by observing microscopically selected conidiophores. Conidiophore development was divided into six stages; when the developing conidiophores from stage 2 (i.e. a mature conidiophore) to stage 5 (i.e. a conidiophore with conidium initial) were exposed to blue light for a short time, conidiation was suppressed; the conidiophores already formed de-differentiated to 'sterile' conidiophores with sharply pointed tips. The suppression of conidial development by blue light could be reversed by subsequent exposure to near ultraviolet light, and conidia then developed normally. This mycochrome system functioned reciprocally within the range of identified conidiophore developmental stages and near ultraviolet light acted only at the same development stage as was inhibited by blue light.

Light↗

Characterization of chitin synthase from Botrytis cinerea.

Chitin synthase in a microsomal preparation from Botrytis cinerea had an apparent Km for UDP-N-acetylglucosamine of 2.0 mM while nikkomycin Z and polyoxin D inhibited enzyme activity competitively with apparent Ki values of approximately 0.1 microM and 6 microM respectively. The organophosphorus fungicide edifenphos was a non-competitive inhibitor (Ki(app) 54 microM). Preincubation of microsomes for 2 h at 25 degrees C resulted in a maximum twofold stimulation of chitin synthase activity while preincubation with trypsin (25 micrograms ml-1) or cytosol (350 micrograms cytosolic protein ml-1) for 10 min at 25 degrees C resulted in approximately fourfold and 20-fold increases in chitin synthase activity, respectively. A range of protease inhibitors reduced the degree of activation of microsomal chitin synthase by cytosol. Most potent were phenylmethanesulphonyl fluoride and chymostatin; these compounds completely inhibited activation of enzyme activity. Two fragments (approx. 600 bp; CHS1 and CHS2) were amplified from B. cinerea genomic DNA using degenerate PCR primers based on regions of complete amino acid homology between previously published chitin synthase gene sequences. When the DNA and predicted amino acid sequences of CHS1 were used to probe computer databases for related sequences, B. cinerea CHS1 was found to be most similar to CHS1 from Neurospora crassa.

Amino Acid Sequence↗

On Autotropism in Botrytis: Measurement Technique and Control by CO(2).

The mutual orientation of the germination of nearby pairs of Botrytis spores growing in a simple, dilute medium was studied. If the medium is equilibrated with room air, they show a very strong tendency to germinate both toward each other and in a cis arrangement, i.e., toward the same side of the line joining their centers. If the medium is equilibrated with air enriched with 0.3% or 3% CO(2), (i.e., 10 or 100 times the normal CO(2) concentration) then the cells show an equally strong tendency to germinate away from each other. The interaction shows little dependence upon pH.

Journal Article↗

Serine Transhydroxymethylase of Cauliflower (Brassica oleracea var. botrytis L.): Partial Purification and Properties.

Serine transhydroxymethylase (EC 2.1.2.1) has been purified 46-fold from cauliflower (Brassica oleracea var. botrytis L.). The enzyme was completely dependent on the presence of tetrahydrofolic acid for the conversion of serine to glycine. The addition of pyridoxal phosphate gave a large increase in the reaction rate. A double pH optimum was observed with maxima at 7.5 and 9.5. The enzyme is specific for l-serine. The d-isomer is neither a substrate nor an inhibitor. The Michaelis constants for l-serine, tetrahydrofolic acid, and pyridoxal phosphate were 300 mum, 760 mum, and 24 mum, respectively. The addition of K(+) also stimulated the reaction rate considerably. The effect was quite specific since all other metal ions tested either had very little: influence or were extremely inhibitory.

Journal Article↗

Action Spectrum for Photosporogenesis in Botrytis cinerea Pers. ex Fr.

An action spectrum for photoinduced conidium formation in the fungus Botrytis cinerea Pers. ex Fr. was determined by exposing colonies to monochromatic radiation obtained from a diffraction grating monochromator. Wavelengths longer than 355 nm were ineffective even after exposures of 8 hours at intensities of 4,068 to 8,276 erg per cm(2) per second. Colonies were exposed at 22.5 C from 226 to 355 nm at about 5 nm intervals. Three prominent peaks of effectiveness occurred at about 231, 268, and 283 nm, and there was also a minor peak at about 303 nm. The most effective wavelength for inducing sporulation was 231 nm; it was 25% more effective than the second most effective wavelength (283 nm).

Journal Article↗

The C-S Lyases of Higher Plants : Isolation and Properties of Homogeneous Cystine Lyase from Broccoli (Brassica oleracea var botrytis) Buds.

Cystine lyase degrades l-cystine by a beta-elimination to form cysteine persulfide, pyruvate, and ammonia. This enzyme is common in Brassica sp. and has been purified to homogeneity from extracts of broccoli (Brassica oleracea var botrytis) buds. Two isozymes were separated on DEAE-Fractogel columns and the first peak, cystine lyase I further purified to homogeneity. The purified enzyme had a narrow range of substrate specificity with l-cystine and S-alkyl-l-cysteine sulfoxides being the primary substrates. The K(m) for l-cystine was 1.9 millimolar and for S-ethyl-l-cysteine sulfoxide was 15.6 millimolar, suggesting that l-cystine would be preferred in vivo. Using gel filtration and sodium dodecyl sulfate-polyacrylamide gel electrophoresis the molecular weight of the holoenzyme was estimated as 152,000 composed of subunits of approximately 49,000. This strongly suggests the native enzyme is a trimer. The presence of carbohydrate in the native enzyme was detected at the level of 5.8% on a weight basis. Except for the ability to utilize l-cystine as a substrate there are many similarities between cystine lyase I and the alliin lyase of onion (Allium cepa).

Journal Article↗

Bioassay methods for the detection of antifungal activity by Pseudomonas antimicrobica against the grey mould pathogen Botrytis cinerea.

Antagonism against the grey mould pathogen Botrytis cinerea by Pseudomonas antimicrobica was demonstrated in vitro and in vivo. Cell-free filtrates showed activity against B. cinerea growing on Potato Dextrose Agar (PDA) in a media-dependent manner with the most distinct antagonism being produced in Czapek Dox Broth (CDB). Cell-free filtrates of CDB-grown cultures also significantly reduced conidial germination of B. cinerea. An assay based on the inhibition of conidial germination was compared with two assays measuring the antagonism of mycelial growth on PDA. The conidial germination bioassay was more sensitive in the detection of this antifungal activity than the Petri dish bioassay while a bioassay using Microdetection plates did not detect antagonism due to the small loading capacity of the latter. The conidial germination bioassay was modified for detection of antibiosis on the surface of strawberry leaves. Significant reductions in percentage conidial germination were recorded on the surface of leaves of both micropropagated and glasshouse grown strawberry plants when the antifungal compounds of Ps. antimicrobica were applied to the leaf tissue with the conidia. In addition, antifungal compounds were also detectable when conidia were applied to leaf tissue which had previously been sprayed with cells of Ps. antimicrobica. These tests indicate that Ps. antimicrobica would be a suitable biocontrol agent for the control of B. cinerea.

Antifungal Agents↗

Onychomycosis due to Ulocladium botrytis.

A case of disto-lateral onychomycosis of the third toe of the right foot of a 45-year-old man is reported. The agent of infection was Ulocladium botrytis, a mycete not hitherto reported to infect humans. The patient underwent mycological examination consisting of direct microscope observation and culture of pathological material. Diagnosis was based on the macro- and microscopic characteristics of the colonies. Fragments of the latter were also examined by scanning electron microscope. Clinical and mycological recovery was achieved after 3 months of topical therapy with ciclopiroxolamine.

Ascomycota↗

An autonomously replicating plasmid transforms Botrytis cinerea to phleomycin resistance.

A transformation system has been developed for the pathogen fungus Botrytis cinerea, based on the utilization of the wide host plasmid pUT737 that contains the Sh ble gene, conferring resistance to phleomycin. Transformed protoplasts were regenerated at 10-25 micrograms ml(-1) of phleomycin, at a frequency of 25-40 transformants per microgram of DNA, and they were resistant up to 50 micrograms ml(-1). Southern hybridization using undigested and digested total DNA showed the presence of circular autonomously replicating plasmid pUT737 in the transformants. Reisolated plasmid from transformed fungus transformed E. coli and rescued plasmid was identified as PUT737. Transformants were grown for four generations under non-selective conditions and replicative plasmids were still detected. Plasmids present in all transformants at this stage had been modified from native pUT737 and showed the same size and configuration indicating that selection through stabilizing plasmid forms has happened.

Antifungal Agents↗

A double-stranded RNA mycovirus in Botrytis cinerea.

In wild-type Botrytis cinerea CVg25 strain we have detected the presence of extrachromosomal genetic elements corresponding to double-stranded RNA molecules. These genetic elements have been designated L, M1 and M2 with molecular sizes of 8.3, 2.0 and 1.4 kb, respectively. The visualization by electron microscopy of mycelium ultrathin sections from B. cinerea CVg25 showed the presence of isometric virus-like particles of about 40 nm in diameter. Linear sucrose gradient centrifugation of mycelium-free extracts was done to determine if the double-stranded RNAs were associated with virus-like particles. The gradient profile obtained at 260 and 280 nm revealed a major peak that was analyzed by both agarose-gel electrophoresis and electron microscopy. It was observed that only the L-double-stranded RNA molecule copurified with isometric virus-like particles. These virus-like particles had a similar morphology and size as those detected by electron microscopy in the mycelium sections. These results suggest that only the L-double-stranded RNA would be encapsidated.

Extrachromosomal Inheritance↗

Telomeric DNA of Botrytis cinerea: a useful tool for strain identification.

Telomeric DNA was isolated from the phytopathogenic fungus Botrytis cinerea by PCR using only the oligonucleotide primer (CCCTAA)4. As with other filamentous fungi, B. cinerea has a short TTAGGG telomeric repeat. Telomere-linked restriction fragment length polymorphism (RFLP) was found in strains of B. cinerea isolated from different host plants collected from different regions at different periods. Almost every strain had a specific RFLP pattern, including those collected from the same plant one month apart. Thus, this marker appears to be an excellent tool to show the great polymorphism of B. cinerea strains by fingerprinting. The Southern blots of some strains of B. cinerea showed one band which was much more intense than the others, suggesting that the majority of telomere-associated sequences have the same sequence.

Base Sequence↗

Bioconversion of alpha-Damascone by Botrytis cinerea.

Bioconversion of alpha-damascone (compound 1) was studied with four strains of Botrytis cinerea in grape must (pH 3.2). As biotransformation products of compound 1, 3-oxo-alpha-damascone, cis- and trans-3-hydroxy-alpha-damascone, gamma-damascenone, 3-oxo-8, 9-dihydro-alpha-damascone, and cis- and trans-3-hydroxy-8,9-dihydro-alpha-damascone were identified. In addition, acid-catalyzed chemical transformation of compound 1 to the diastereomers of 9-hydroxy-8,9-dihydro-alpha-damascone was observed. Identifications were performed by capillary gas chromatography (HRGC) and coupled HRGC techniques, i.e., on-line HRGC-mass spectrometry and HRGC-Fourier transform infrared spectroscopy, after extractive sample preparation.

Journal Article↗

Degradation of Extracellular beta-(1,3)(1,6)-d-Glucan by Botrytis cinerea.

During growth on glucose, Botrytis cinerea produced extracellular beta-(1,3)(1,6)-d-glucan (cinerean), which formed an adhering capsule and slime. After glucose was exhausted from the medium, cinereanase activity increased from <0.4 to 30 U/liter, effecting a striking loss in the viscosity of the culture. Cinerean was cleaved into glucose and gentiobiose. Gentiobiose was then hydrolyzed to glucose. While cinereanase activity was strongest in the culture supernatant, gentiobiase activity was located mainly in the cell wall fraction. The addition of extra glucose or cycloheximide prevented the cinerean degradation caused by an effect on cinereanase formation. Cinerean degradation was accompanied by microconidiation and sclerotium formation. B. cinerea was found to grow on cinerean with the latter as its single carbon and energy source. In this case, cinerean degradation occurred during hyphal growth, and no microconidiation or sclerotium formation was observed. Growth experiments with various carbon sources indicated that cinerean had a positive effect on the formation of cinerean-degrading enzymes.

Journal Article↗

Adhesion of Nongerminated Botrytis cinerea Conidia to Several Substrata.

Conidia of the plant pathogenic fungus Botrytis cinerea adhered to tomato cuticle and to certain other substrata immediately upon hydration. This immediate adhesion occurred with both living and nonliving conidia. Adhesion was not consistently influenced by several lectins, sugars, or salts or by protease treatment, but it was strongly inhibited by ionic or nonionic detergents. With glass and oxidized polyethylene, substrata whose surface hydrophobicities could be conveniently varied, there was a direct relationship between water contact angle and percent adhesion. Immediate adhesion did not involve specific conidial attachment structures, although the surfaces of attached conidia were altered by contact with a substratum. Freshly harvested conidia were very hydrophobic, with more than 97% partitioning into the organic layer when subjected to a phase distribution test. Percent adhesion of germinated conidia was larger than that of nongerminated conidia. Evidence suggests that immediate adhesion of conidia of B. cinerea depends, at least in part, on hydrophobic interactions between the conidia and substratum.

Journal Article↗

Boty, a long-terminal-repeat retroelement in the phytopathogenic fungus Botrytis cinerea.

The phytopathogenic fungus Botrytis cinerea can infect an extremely wide range of host plants (tomato, grapevine, strawberry, and flax) without apparent specialization. While studying genetic diversity in this fungus, we found an element which is present in multiple copies and dispersed throughout the genome of some of its isolates. DNA sequence analysis revealed that the element contained direct, long-terminal repeats (LTRs) of 596 bp whose features were characteristic of retroviral and retrotransposon LTRs. Within the element, we identified an open reading frame with sequences homologous to the reverse transcriptase and RNase H domains of retroelement pol genes. We concluded that the element we had identified was a retroelement and named it Boty. By comparing its open reading frame with sequences from other retroelements, we found that Boty is related to the gypsy family of retrotransposons. Boty was present in numerous strains isolated from grapes and tomatoes but not in isolates from lentils. We propose that Boty-containing and Boty-deficient groups represent two lineages in the population of B. cinerea.

Amino Acid Sequence↗