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PubMed1997

Transformation of Botrytis cinerea with the nitrate reductase gene (niaD) shows a high frequency of homologous recombination.

The nitrate reductase (niaD) gene was isolated from the phytopathogenic ascomycete Botrytis cinerea using a probe obtained by a polymerase chain reaction (PCR) with degenerate oligonucleotides corresponding to domains conserved among three fungal nitrate reductases. The B. cinerea niaD gene encodes a predicted protein of 907 amino acids and contains no intron. Nitrate reductase-deficient mutants of B. cinerea have been isolated. One of them was transformed with the niaD genes of Fusarium oxysporum f.sp. melonis and B. cinerea. The transformation was always ectopic when the donor DNA originated from F. oxysporum, but there was 80% gene replacement when the donor DNA originated from B. cinerea.

Cloning, Molecular↗
PubMed1997

Flipper, a mobile Fot1-like transposable element in Botrytis cinerea.

A transposable element, Flipper, was isolated from the phytopathogenic fungus Botrytis cinerea. The element was identified as an insertion sequence within the coding region of the nitrate reductase gene. The Flipper sequence is 1842 bp long with perfect inverted terminal repeats (ITRs) of 48 bp and an open reading frame (ORF) of 533 amino acids, potentially encoding for a transposase; the element is flanked by the dinucleotide TA. The encoded protein is very similar to the putative transposases of three elements from other phytopathogenic fungi, Fot1 from Fusarium oxysporum, and Pot2 and MGR586 from Magnaporthe grisea. The number of Flipper elements in strains of B. cinerea varied from 0 to 20 copies per genome. Analysis of the descendants of one cross showed that the segregation ratio of Flipper elements was 2:2 and that the copies were not linked.

Amino Acid Sequence↗
PubMed1975

Polygalacturonase of Botrytis cinerea E-200 Pers.

A polygalacturonase (poly(1,4-alpha-D-galacturonide)glycanohydrolase, EC 3.2.1.15) was purified from the culture fluid of Botrytis cinerea. The polygalacturonase preparation, homogeneous on the basis of disc-gel electrophoresis also showed pectinesterase activity. Some properties of the purified polygalacturonase were studied. It had a molecular weight about 69 000. It was inactivated by p-chloromercuribenzoate, tetranitromethane and urea. A 50% loss in viscosity of sodium polypectate solution occurred when 4.6% of the glycosidic bonds were hydrolyzed. The only end product of sodium polypectate and oligogalacturonides hydrolysis was monogalacturonic acid.?

Chloromercuribenzoates↗
PubMed1995

Structural properties of native and sonicated cinerean, a beta- (1-->3) (1-->6)-D-glucan produced by Botrytis cinerea.

Cinerean, the extracellular beta-(1-->3) (1-->6)-D-glucan of the fungus Botrytis cinerea was studied. Electron micrographs of the native polysaccharide revealed quasi-endless fibrils with an estimated diameter of ca. 1.5 nm. A particle mass of 10(9)-10(10) daltons was determined from dilute solutions by low-angle laser light scattering. Sonication of increasing duration led to fragmentation of the native polymer with an approximately exponential decrease of mass in the range of average molecular masses between 250,000 and 50,000 daltons. Shadowed by platinum, cinerean fibril fragments with a weight-average molecular mass of 172,000 +/- 3000 daltons could be characterized from electron micrographs as a distribution of rods of most probable length of 45 nm and an average length of 72 nm. Small-angle X-ray scattering confirmed the fibrillar structure of the native cinerean and the rodlike structure of sonicated cinerean. A rod diameter of 1.9 +/- 0.2 nm and a mass per unit length of 2250 +/- 490 daltons/nm were found. The latter is in agreement with the value of 1830 daltons/nm calculated from the length distribution determined from the electron micrographs. These data-especially the mass per unit length-suggest a quaternary structure for the polysaccharide. Such a structure would explain the rigidity of the rods which, in turn, is responsible for the characteristic phase separation behaviour in aqueous solutions observed by nephelometry and viscometry.

Carbohydrate Sequence↗
PubMed1997

Stability and modulated expression of a hygromycin resistance gene integrated in Botrytis cinerea transformants.

When transformation of Botrytis cinerea occurred in mononucleated protoplasts the hygromycin resistance phenotype was stable and integrated plasmid DNA although rearranged was transmitted through meiosis. We observed that transformants were often heterokaryotic and using serial conidial transfer, we showed failure of expression of the entire copies of integrated plasmids in some conidial isolates. A non-Mendelian segregation of the hygromycin resistance phenotype was observed in most crosses between these transformants and sensitive strains. However, a 1:1 segregation ratio of plasmid DNA hybridisation was observed. Mechanisms of gene silencing in B. cinerea, in both the asexual and the sexual cycle, are discussed.

Anti-Bacterial Agents↗
PubMed1997

A structure-specific endonuclease from cauliflower (Brassica oleracea var. botrytis) inflorescence.

A protein with structure-specific endonuclease activity has been purified to near homogeneity from cauliflower ( Brassica oleracea var. botrytis) inflorescence through five successive column chromatographies. The protein is a single polypeptide with a molecular mass of 40 kDa. Using three different branched DNA structures (flap, pseudo-Y and stem-loop) we found that the enzyme, a cauliflower structure-specific endonuclease, cleaved the single-stranded tail in the 5'-flap and 5'-pseudo-Y structures, whereas it could not incise the 3'-flap and 3'-pseudo-Y structures. The incision points occur around the single strand-duplex junction in these DNA substrates and the enzyme leaves 5'-PO4 and 3'-OH termini on DNA. The protein also endonucleolytically cleaves on the 3'-side of the single-stranded region at the junction of unpaired and duplex DNA in the stem-loop structure. The structure-specific endonuclease activity is stimulated by Mg2+ and by Mn2+, but not by Ca2+. Like mammalian FEN-1, the protein has weak 5'-->3' double-stranded DNA-specific exonuclease activity. These results indicate that the cauliflower protein is a plant structure-specific endonuclease like mammalian FEN-1 or may be the plant alternative.

Brassica↗
PubMed1997

RFLP markers show genetic recombination in Botryotinia fuckeliana (Botrytis cinerea) and transposable elements reveal two sympatric species.

Molecular markers revealed that Botryotinia fuckeliana (the teleomorph of Botrytis cinerea), a haploid, filamentous, heterothallic ascomycete, contained a large amount of intrapopulation genetic variation. The markers were used to determine the mode of reproduction and the population structure of this fungus. We did not detect any differentiation between isolates from different organs, collection dates, varieties of grape, or locations in the Champagne region of France, but two unexpected sympatric populations were identified. One group of isolates (transposa) contained the transposable elements Boty and Flipper; the other (vacuma) did not. These groups differed from one another for all the other markers. RFLP markers showed that there was genetic recombination in both groups of isolates. We conclude that there are two sympatric populations of B. fuckeliana in Champagne. One species (transposa) seems to be local and well adapted, while the other one (vacuma) is presumably a heterogeneous migrant population.

Alleles↗
PubMed1997

Cloning and expression of the cutinase A gene of Botrytis cinerea.

Cutinase of Botrytis cinerea has been suggested to play an important role in penetration of host tissues. A protein fraction with cutin hydrolyzing activity was purified from culture filtrates of B. cinerea induced for cutinase activity. An 18-kDa protein in this fraction was identified as cutinase and the corresponding gene cutA was cloned. The gene is present in a single copy in the genome of B. cinerea strain SAS56 and its predicted amino acid sequence shows significant homology (31 to 35% identity) to other fungal cutinases. RNA blot analysis showed that cutA mRNA is induced in vitro by the cutin monomer 16-hydroxyhexadecanoic acid and repressed by glucose. The expression of cutA during infection of tomato leaves is low during early phases of infection, but high when the fungus has colonized the leaf and starts to sporulate.

Amino Acid Sequence↗
PubMed1997

Cutinase A of Botrytis cinerea is expressed, but not essential, during penetration of gerbera and tomato.

The plant pathogen Botrytis cinerea can infect undamaged plant tissue directly by penetration of the cuticle. This penetration has been suggested to be enzyme-mediated, and an important role for cutinase in the infection process has been proposed. In this study the expression of the cutinase encoding gene cutA of B. cinerea was analyzed using a cutA promoter-GUS reporter gene fusion. Transformants containing the fusion construct were examined for GUS expression on gerbera flowers and tomato fruits. High GUS activity was detected from the onset of conidial germination and during penetration into epidermal cells, indicating that cutA is expressed during the early stages of infection. To determine the biological relevance of cutinase A for successful penetration, cutinase A-deficient mutants were constructed by gene disruption. Pathogenicity of two transformants lacking a functional cutA gene was studied on gerbera flowers and tomato fruits. Their ability to penetrate and cause symptoms was unaltered compared to the wild-type strain. These results exclude an important role for cutinase A during direct penetration of host tissue by B. cinerea.

Base Sequence↗
PubMed1993

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↗
PubMed1976

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↗
PubMed1977

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↗
PubMed1994

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↗
PubMed1996

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↗
PubMed1996

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↗
PubMed1997

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↗
PubMed1997

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↗