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PubMed1999

Pythium periplocum, an aggressive mycoparasite of Botrytis cinerea causing the gray mould disease of grape-vine.

Pythium periplocum Dreschler has been found to be an aggressive mycoparasite of Botrytis cinerea, the causal agent of the gray mould disease of the grape-vine. When grown together, the former enters the latter's mycelium, branches freely within, coagulates its cytoplasm and finally tears its hyphae apart, bringing about widespread destruction of the grape-vine pathogen. Extensive coiling around the host, as reported in the case of other mycoparasites belonging to the genus Pythium, has not been observed here. The infected mycelium of B. cinerea fails to infect the grape-vine and does not induce the characteristic gray mould symptoms. Since P. periplocum is not a grape-vine parasite, it could be useful for the biological control of B. cinerea. A brief account of this mycoparasitism is discussed in this article.

Botrytis↗

PubMed1999

Purification and characterization of a 40.8-kDa cutinase in ungerminated conidia of Botrytis cinerea Pers.: Fr.

Cytoplasmic soluble proteins from ungerminated conidia of Botrytis cinerea exhibited cutinase activity. A 40.8-kDa cutinase was purified to homogeneity from this crude conidial protein extract. This cutinase does not correspond either to constitutive or to induced lytic cutin enzymes already described by other authors. The possible role of this constitutive cutinase in the induction of other cutinolytic proteins in the early stages of infection of plants by B. cinerea is discussed.

Antibodies, Monoclonal↗

PubMed1999

A new double-stranded RNA mycovirus from Botrytis cinerea.

A simple double-stranded RNA mycovirus was detected in a wild-type Botrytis cinerea 55k strain. The virus was located in the fungus cytoplasm as free particles of approximately 28 nm in diameter. The mycovirus possesses a single double-stranded genome segment of 1.8 kilobase pairs (kbp) encapsidated within an isometric protein coat whose main structural component is a polypeptide of 68 kDa. Cells infected with this virus showed an important degree of cellular degeneration.

Botrytis↗

PubMed2000

Pythium contiguanum nomen novum (syn. Pythium dreschleri Paul), its antagonism to Botrytis cinerea, ITS1 region of its nuclear ribosomal DNA, and its comparison with related species.

Pythium drechsleri Paul was described as a new species from soil samples taken in a salt-marsh of Arzew, Algeria [Paul, B. (1988) Une nouvelle espèce de Pythium isolée d'une saline de l'ouest Algérien. Cryptogam. Mycol. 9, 325-333]. The name of the fungus, P. drechsleri, is a nomen invalidum, as it is a later homonym of P. drechsleri Rajgopalan and Ramakrishnan [Rajagopalan, S. and Ramakrishnan, K. (1971) Phycomycetes in agricultural soils with special reference to the Pythiaceae. Madras Univ. J. Sect. B 37,38, 100-117]. A new name, Pythium contiguanum is now being given to P. drechsleri Paul. This species is characterised by its contiguous inflated type of sporangia, smooth-walled oogonia and mostly monoclinous antheridia. Although the fungus is not known to be a pathogen, it has a very well developed appressorial system comprised of both sickle shaped and coiled appressoria. Morphological features, its antagonism towards the grape-vine pathogen Botrytis cinerea, together with the sequences of the ITS1 region of its nuclear ribosomal DNA and its comparison with related species are given here.

Antibiosis↗

PubMed2000

Enzymes of Botrytis cinerea capable of breaking down hydrogen peroxide.

The amounts of intra- and extracellular guaiacol peroxidase, ascorbic peroxidase, glutathione peroxidase, superoxide dismutase, laccase, and catalase present in Botrytis cinerea, cultured in three different media: Kovac synthetic medium, Sabouraud fluid medium, and a medium containing malt extract, were determined. The activity of two enzymes, ascorbic peroxidase and glutathione peroxidase, has not been previously described in B. cinerea. The detected amount of the enzymes showed considerable variability in the three different culture media. The presence of an array of enzymes capable of metabolizing hydrogen peroxide, whose levels are determined by the conditions under which the fungus grows, shows that B. cinerea is well equipped to contend with the occurrence of host-produced active oxygen species.

Botrytis↗

PubMed2001

The possible function of the glucan sheath of Botrytis cinerea: effects on the distribution of enzyme activities.

The effect of a beta-1,3-glucanase (Glucanex) on cultures of Botrytis cinerea was examined. The enzyme released reducing sugars from the mycelium and from the glucan secreted into the culture medium. The morphology of the mycelium was changed in the presence of Glucanex. The measured activity of guaiacol peroxidase, laccase, and catalase was increased when the mycelium was treated with Glucanex. Culture of the mycelium in the presence of Glucanex resulted in an increase in catalase activity. We suggest that the glucan plays a role in protecting the fungus from host response and may assist in the initial stages of host infection.

Botrytis↗

PubMed2001

Expression of exo-polygalacturonases in Botrytis cinerea.

The pathogenic fungus, Botrytis cinerea, causing gray mold disease in a variety of plant species, secretes at least four polygalacturonases (PGs), cell wall degrading enzymes. Among them, we prepared polyclonal antibody against purified 66-kDa exo-PG in rabbit. Immunoblot analysis revealed that the antibody recognized two exo-PGs, 66 kDa and 70 kDa in molecular mass, secreted from B. cinerea cultured in the medium containing citrus pectin as a carbon source. By immunohistochemical analysis, the expression of exo-PGs was identified in cucumber leaves inoculated with spores of B. cinerea. The exo-PGs were observed 9 h after inoculation, and the amount of exo-PGs increased with time in the leaves. The exo-PGs were induced by polygalacturonic acid as well as its monomer, galacturonic acid, in vitro. The expression of 66-kDa exo-PG (exo-PG I) increased with time of culture, while 70-kDa exo-PG (exo-PG II) was transiently expressed soon after the start of culture. Therefore, exo-PGs might play an important role in pathogenesis at an early stage of infection as well as in tissue maceration of host plant.

Botrytis↗

PubMed2001

Characterisation of the yeast Pichia membranifaciens and its possible use in the biological control of Botrytis cinerea, causing the grey mould disease of grapevine.

Pichia membranifaciens strain FY-101, isolated from grape skins, was found to be antagonistic to Botrytis cinerea, the causal organism of the grey mould disease of the grapevine. When grown together on solid as well as liquid media, the yeast brings about the inhibition of this parasitic fungus, coagulation and leakage of its cytoplasm, and suppression of its ability to produce the characteristic grey mould symptoms on the grapevine plantlets. In vitro experiments confirm that this yeast can be used as a biological control organism against B. cinerea. An account of the molecular characterisation of P. membranifaciens (complete sequence of the ITS region of its ribosomal DNA, GenBank accession No. AF 270935), as well as the interaction between B. cinerea and the yeast, are given here.

Antibiosis↗

PubMed2001

Effects of long-term storage at different temperatures on conidia of Botrytis cinerea Pers.: Fr.

Survival of Botrytis cinerea conidia was studied after storage without pretreatments at different temperatures (-80 degrees C, -20 degrees C, 4 degrees C and 21 degrees C). Germination tests performed during 3 years showed that viability at 21 degrees C was completely lost after 1 month. Conidia stored for 30 months at -80 degrees C, -20 degrees C and 4 degrees C were able to germinate, respectively, at 79%, 8% and 0.2%. Changes in adenylate level, energy charge and respiration (O(2) consumption) made on each set of conidia were correlated to the germination rate. The 30-month-old stored conidia showed differences in pathogenicity tests on apples. While the pathogenic aggressiveness of conidia stored at -80 degrees C was almost the same as for fresh conidia, it decreased with increasing temperature of storage. An ultrastructural study made on conidia stored for 30 months at -80 degrees C has shown the emergence of a new wall layer in a retraction zone of the cytoplasm by comparison to fresh conidia. However, the integrity of the cytoplasmic content was maintained. The effects of low temperature storage, maintenance of cell integrity and pathogenicity of conidia of B. cinerea are discussed.

Adenine Nucleotides↗

PubMed2002

ITS region of Pythium canariense sp. nov., its morphology and its interaction with Botrytis cinerea.

A new species Pythium canariense (CI-07), isolated from soil samples taken in the San Nicolas region of the Canary islands (Gran Canaria, Spain), is being described here. This species is characterised by its spherical to pyriform, intercalary to catenulate sporangia, smooth-walled terminal oogonia supplied with monoclinous and diclinous antheridia which at times are branched and wrap around the female gametangia. The fungus has an antagonistic effect on Botrytis cinerea, the grey mould fungus. Morphological features are being given here together with the sequence of the complete internal transcribed spacer region of the nuclear ribosomal DNA of the fungus, its comparison with related species, and some aspects of its antagonistic behaviour with B. cinerea.

Antibiosis↗

PubMed2002

Presence of a vanadium-dependent haloperoxidase in Botrytis cinerea.

The presence of a haloperoxidase in the mycelium of Botrytis cinerea, extractable with buffer, is demonstrated. A low level of extracellular enzyme activity was also detected. The haloperoxidase from the fungus is a vanadium-dependent glycoprotein, with a pH optimum of about 5.5. Native gel electrophoresis indicates that it is a high molecular mass protein. It appears to react with antibodies against haloperoxidase from Caldariomyces fumago. Enzyme activity is increased 3.5-fold and 15-fold by culture of the fungus in the presence of NaCl or vanadium, respectively. Activity is partly reduced by removal of vanadium and activity can be restored by the addition of vanadium to the enzyme. The possible function of this haloperoxidase is discussed.

Botrytis↗

PubMed2006

Identification of an abscisic acid gene cluster in the grey mold Botrytis cinerea.

Like several other phytopathogenic fungi, the ascomycete Botrytis cinerea is known to produce the plant hormone abscisic acid (ABA) in axenic culture. Recently, bcaba1, the first fungal gene involved in ABA biosynthesis, was identified. Neighborhood analysis of bcaba1 revealed three further candidate genes of this pathway: a putative P450 monooxygenase-encoding gene (bcaba2), an open reading frame without significant similarities (bcaba3), and a gene probably coding for a short-chain dehydrogenase/reductase (bcaba4). Targeted inactivation of the genes proved the involvement of BcABA2 and BcABA3 in ABA biosynthesis and suggested a contribution of BcABA4. The close linkage of at least three ABA biosynthetic genes is strong evidence for the presence of an abscisic acid gene cluster in B. cinerea.

Abscisic Acid↗

PubMed2000

A new method to monitor airborne inoculum of the fungal plant pathogens Mycosphaerella brassicicola and Botrytis cinerea.

We describe a new microtiter immunospore trapping device (MTIST device) that uses a suction system to directly trap air particulates by impaction in microtiter wells. This device can be used for rapid detection and immunoquantification of ascospores of Mycosphaerella brassicicola and conidia of Botrytis cinerea by an enzyme-linked immunosorbent assay (ELISA) under controlled environmental conditions. For ascospores of M. brassicicola correlation coefficients (r(2)) of 0.943 and 0.9514 were observed for the number of MTIST device-impacted ascospores per microtiter well and the absorbance values determined by ELISA, respectively. These values were not affected when a mixed fungal spore population was used. There was a relationship between the number of MTIST device-trapped ascospores of M. brassicicola per liter of air sampled and the amount of disease expressed on exposed plants of Brassica oleracea (Brussels sprouts). Similarly, when the MTIST device was used to trap conidia of B. cinerea, a correlation coefficient of 0.8797 was obtained for the absorbance values generated by the ELISA and the observed number of conidia per microtiter well. The relative collection efficiency of the MTIST device in controlled plant growth chambers with limited airflow was 1.7 times greater than the relative collection efficiency of a Burkard 7-day volumetric spore trap for collection of M. brassicicola ascospores. The MTIST device can be used to rapidly differentiate, determine, and accurately quantify target organisms in a microflora. The MTIST device is a portable, robust, inexpensive system that can be used to perform multiple tests in a single sampling period, and it should be useful for monitoring airborne particulates and microorganisms in a range of environments.

Air Microbiology↗

PubMed2002

Yeast diversity and persistence in botrytis-affected wine fermentations.

Culture-dependent and -independent methods were used to examine the yeast diversity present in botrytis-affected ("botrytized") wine fermentations carried out at high ( approximately 30 degrees C) and ambient ( approximately 20 degrees C) temperatures. Fermentations at both temperatures possessed similar populations of Saccharomyces, Hanseniaspora, Pichia, Metschnikowia, Kluyveromyces, and Candida species. However, higher populations of non-Saccharomyces yeasts persisted in ambient-temperature fermentations, with Candida and, to a lesser extent, Kluyveromyces species remaining long after the fermentation was dominated by Saccharomyces. In general, denaturing gradient gel electrophoresis profiles of yeast ribosomal DNA or rRNA amplified from the fermentation samples correlated well with the plating data. The direct molecular methods also revealed a Hanseniaspora osmophila population not identified in the plating analysis. rRNA analysis also indicated a large population (>10(6) cells per ml) of a nonculturable Candida strain in the high-temperature fermentation. Monoculture analysis of the Candida isolate indicated an extreme fructophilic phenotype and correlated with an increased glucose/fructose ratio in fermentations containing higher populations of Candida. Analysis of wine fermentation microbial ecology by using both culture-dependent and -independent methods reveals the complexity of yeast interactions enriched during spontaneous fermentations.

Botrytis↗

PubMed2002

Bcmfs1, a novel major facilitator superfamily transporter from Botrytis cinerea, provides tolerance towards the natural toxic compounds camptothecin and cercosporin and towards fungicides.

Bcmfs1, a novel major facilitator superfamily gene from Botrytis cinerea, was cloned, and replacement and overexpression mutants were constructed to study its function. Replacement mutants showed increased sensitivity to the natural toxic compounds camptothecin and cercosporin, produced by the plant Camptotheca acuminata and the plant pathogenic fungus Cercospora kikuchii, respectively. Overexpression mutants displayed decreased sensitivity to these compounds and to structurally unrelated fungicides, such as sterol demethylation inhibitors (DMIs). A double-replacement mutant of Bcmfs1 and the ATP-binding cassette (ABC) transporter gene BcatrD was more sensitive to DMI fungicides than a single-replacement mutant of BcatrD, known to encode an important ABC transporter of DMIs. The sensitivity of the wild-type strain and mutants to DMI fungicides correlated with Bcmfs1 expression levels and with the initial accumulation of oxpoconazole by germlings of these isolates. The results indicate that Bcmfs1 is a major facilitator superfamily multidrug transporter involved in protection against natural toxins and fungicides and has a substrate specificity that overlaps with the ABC transporter BcatrD. Bcmfs1 may be involved in protection of B. cinerea against plant defense compounds during the pathogenic phase of growth on host plants and against fungitoxic antimicrobial metabolites during its saprophytic phase of growth.

ATP-Binding Cassette Transporters↗

PubMed2002

Ethylene production by Botrytis cinerea in vitro and in tomatoes.

A laser-based ethylene detector was used for on-line monitoring of ethylene released by the phytopathogenic fungus Botrytis cinerea in vitro and in tomato fruit. Ethylene data were combined with the results of a cytological analysis of germination of B. cinerea conidia and hyphal growth. We found that aminoethoxyvinylglycine and aminooxyacetic acid, which are competitive inhibitors of the 1-aminocyclopropane-1-carboxylic acid pathway, did not inhibit the ethylene emission by B. cinerea and that the fungus most likely produces ethylene via the 2-keto-4-methylthiobutyric acid pathway. B. cinerea is able to produce ethylene in vitro, and the emission of ethylene follows the pattern that is associated with hyphal growth rather than the germination of conidia. Ethylene production in vitro depended on the L-methionine concentration added to the plating medium. Higher values and higher emission rates were observed when the concentration of conidia was increased. Compared with the ethylene released by the fungus, the infection-related ethylene produced by two tomato cultivars (cultivars Money Maker and Daniela) followed a similar pattern, but the levels of emission were 100-fold higher. The time evolution of enhanced ethylene production by the infected tomatoes and the cytological observations indicate that ethylene emission by the tomato-fungus system is not triggered by the ethylene produced by B. cinerea, although it is strongly synchronized with the growth rate of the fungus inside the tomato.

Botrytis↗

PubMed2002

Novel fungitoxicity assays for inhibition of germination-associated adhesion of Botrytis cinerea and Puccinia recondita spores.

Botrytis cinerea and Puccinia recondita spores adhere strongly to polystyrene microtiter plates coincident with germination. We developed assays for inhibition of spore adhesion in 96-well microtiter plates by using sulforhodamine B staining to quantify the adherent spores. In both organisms, fungicides that inhibited germination strongly inhibited spore adhesion, with 50% effective concentrations (EC(50)s) comparable to those for inhibition of germination. In contrast, fungicides that acted after germination in B. cinerea inhibited spore adhesion to microtiter plates only at concentrations much higher than their EC(50)s for inhibition of mycelial growth. Similarly, in P. recondita the ergosterol biosynthesis inhibitors myclobutanil and fenbuconazole acted after germination and did not inhibit spore adhesion. The assays provide a rapid, high-throughput alternative to traditional spore germination assays and may be applicable to other fungi.

Basidiomycota↗

PubMed2004

The P450 monooxygenase BcABA1 is essential for abscisic acid biosynthesis in Botrytis cinerea.

The phytopathogenic ascomycete Botrytis cinerea is known to produce abscisic acid (ABA), which is thought to be involved in host-pathogen interaction. Biochemical analyses had previously shown that, in contrast to higher plants, the fungal ABA biosynthesis probably does not proceed via carotenoids but involves direct cyclization of farnesyl diphosphate and subsequent oxidation steps. We present here evidence that this "direct" pathway is indeed the only one used by an ABA-overproducing strain of B. cinerea. Targeted inactivation of the gene bccpr1 encoding a cytochrome P450 oxidoreductase reduced the ABA production significantly, proving the involvement of P450 monooxygenases in the pathway. Expression analysis of 28 different putative P450 monooxygenase genes revealed two that were induced under ABA biosynthesis conditions. Targeted inactivation showed that one of these, bcaba1, is essential for ABA biosynthesis: DeltaBcaba1 mutants contained no residual ABA. Thus, bcaba1 represents the first identified fungal ABA biosynthetic gene.

Abscisic Acid↗