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PubMed1995

Adhesion of germlings of Botrytis cinerea.

Adhesion of conidia and germlings of the facultative plant parasite Botrytis cinerea occurs in two distinct stages. The first stage, which occurs immediately upon hydration of conidia and is characterized by relatively weak adhesive forces, appears to involve hydrophobic interactions (R. P. Doss, S. W. Potter, G. A. Chastagner, and J. K. Christian, Appl. Environ. Microbiol. 59:1786-1791, 1993). The second stage of adhesion, delayed adhesion, occurs after viable conidia have been incubated for several hours under conditions that promote germination. At this time, the germlings attach strongly to either hydrophobic or hydrophilic substrata. Delayed adhesion involves secretion of an ensheating film that remains attached to the substratum upon physical removal of the germlings. This fungal sheath, which can be visualized by using interference-contrast light microscopy, scanning electron microscopy, or atomic force microscopy, is 25 to 60 nm thick in the region immediately adjacent to the germ tubes. Germlings are resistant to removal by boiling or by treatment with a number of hydrolytic enzymes, 2.0 M periodic acid, or 1.0 M sulfuric acid. They are readily removed by brief exposure to 1.25 N NaOH. A base-soluble material that adheres to culture flask walls in short-term liquid cultures of B. cinerea is composed of glucose (about 30%), galactosamine (about 3%), and protein (30 to 44%).

Cell Adhesion↗
PubMed1995

Production and Characterization of Laccase from Botrytis cinerea 61-34.

An isolate of Botrytis cinerea (strain 61-34) constitutively expresses substantial amounts of extracellular laccase on a defined growth medium. The enzyme has been purified to homogeneity by a facile operational sequence, the last stage of which involves hydrophobic interaction chromatography. By these means, over 80 mg of laccase liter(sup-1) can be obtained from aerated fermentor reaction broths. The enzyme, with an estimated M(infr) of 74,000 and pI of 4.0, is a monomeric glycoprotein containing 49% carbohydrate predominantly as hexose. With 2,6-dimethoxyphenol, it exhibits a pH optimum of 3.5 and a temperature optimum of 60(deg)C, and its K(infm) is 100 (mu)M. The purified enzyme with this substrate has a specific activity of 9.1 mkat mg of protein(sup-1). Taken together with a broad substrate range and its stability in 4% sodium dodecyl sulfate or 2 M urea solutions, several biotechnology transfers are suggested.

Journal Article↗
PubMed1999

Composition and enzymatic activity of the extracellular matrix secreted by germlings of botrytis cinerea.

Germlings of Botrytis cinerea, an important fungal pathogen of plants, produce an extracellular matrix (ECM), or ensheathing film, that serves, in part, in their attachment (R. P. Doss, et al., Appl. Environ. Microbiol. 61:260-265, 1995). The composition of this film has been ascertained by using samples obtained by growing germlings on a glass surface, removing the fungal mycelium by vigorous washing, and collecting the tightly attached film by scraping the substratum with a razor blade. Slightly over half of the dry weight of the ECM was found to be carbohydrates (about 20%), proteins (about 28%), and lipids (about 6%). Hydrolysis of the carbohydrate portion of the ECM revealed that glucose was the most prominent monosaccharide present, comprising about 60% of the total monosaccharides. Also present were mannose (about 35%) and myo-inositol (about 5%). The proteinaceous fraction of the ECM was made up of a number of polypeptides separable by polyacrylamide gel electrophoresis. The lipid fraction of the ECM, analyzed by thin-layer chromatography, was made up of several simple lipid components, including free fatty acid, mono- and triacylglycerol, wax ester, fatty alcohol, and several unidentified components. No complex lipids were detected. Isolated ECM exhibited polygalacturonase and laccase activity and was able to catalyze the hydrolysis of p-nitrophenyl butyrate, a model substrate for assessing cutinase activity. Cellulase, pectin lyase, and pectin methyl esterase activities were noted with both heated and unheated ECM preparations. Proteinase activity was not detected.

Journal Article↗
PubMed1971

Oxidation of alcohols by Botrytis cinerea.

Crude cell-free preparations of Botrytis cinerea were found to oxidize straight-chain primary alcohols (except methanol), aromatic primary alcohols, and unsaturated primary alcohols. The resulting products were the corresponding aldehydes and an equal molar quantity of hydrogen peroxide.

Alcohol Oxidoreductases↗
PubMed2002

Highly informative nature of inter simple sequence repeat (ISSR) sequences amplified using tri- and tetra-nucleotide primers from DNA of cauliflower (Brassica oleracea var. botrytis L.).

Inter simple sequence repeat (ISSR) sequences as molecular markers can lead to the detection of polymorphism and also be a new approach to the study of SSR distribution and frequency. In this study, ISSR amplification with nonanchored primer was performed in closely related cauliflower lines. Fourty-four different amplified fragments were sequenced. Sequences of PCR products are delimited by the expected motifs and number of repeats, which validates the ISSR nonanchored primer amplification technique. DNA and amino acids homology search between internal sequences and databases (i) show that the majority of the internal regions of ISSR had homologies with known sequences, mainly with genes coding for proteins implicated in DNA interaction or gene expression, which reflected the significance of amplified ISSR sequences and (ii) display long and numerous homologies with the Arabidopsis thaliana genome. ISSR amplifications revealed a high conservation of these sequences between Arabidopsis thaliana and Brassica oleracea var. botrytis. Thirty-four of the 44 ISSRs had one or several perfect or imperfect internal microsatellites. Such distribution indicates the presence in genomes of highly concentrated regions of SSR, or "SSR hot spots." Among the four nonanchored primers used in this study, trinucleotide repeats, and especially (CAA)5, were the most powerful primers for ISSR amplifications regarding the number of amplified bands, level of polymorphism, and their nature.

Base Sequence↗
PubMed2003

Molecular mapping of Or, a gene inducing beta-carotene accumulation in cauliflower (Brassica oleracea L. var. botrytis).

The cauliflower (Brassica oleracea L. var. botrytis) Or gene is a semi-dominant, single-locus mutation that induces the accumulation of high levels of beta-carotene in various tissues of the plant, turning them orange. As part of a map-based cloning strategy, molecular mapping of the Or gene in the cauliflower genome was undertaken in a mapping population consisting of 195 F2 individuals. By using amplified fragment length polymorphism (AFLP) in conjunction with bulked segregant analysis, we identified 10 AFLP markers closely linked to the Or gene. Four of the most closely linked flanking markers were converted into restriction fragment length polymorphism (RFLP) markers. Mapping of these markers in the mapping population placed two of them at 0.5 cM from the Or locus on one side, while another marker flanked the Or gene at 1.6 cM on the other side. Three of these markers were also successfully converted into sequence-characterized amplified region (SCAR) markers. These PCR-based markers will be useful for a large-scale application in facilitating the positional cloning of the Or gene.

Brassica↗
PubMed1996

[Effect of monensin on the growth and secretion of exopolysaccharides in Botrytis cinerea Pers. and Sclerotium rolfsii Sacc].

The addition of various concentrations of monensin (1,5, and 10 micrograms/mL) to the culture medium inhibits the fungal growth and perturbs exopolysaccharides secretion, provoking a decrease of production in Botrytis cinerea and an increase in Sclerotium rolfsii. The ionophore induces also modifications in both polymer composition and structure. New monomers were observed in the two species and a decreased branching rate for Sclerotium rolfsii. These modifications show that monensin affects the enzymes responsible for normal wall synthesis and therefore vesicular traffic.

Antifungal Agents↗
PubMed1987

Botrytis cinerea: a study of the immunological properties during growth. Incidence of antibodies against B. cinerea in a group of patients with aspergillosis.

When growing Botrytis cinerea in Sabouraud medium, different phases of growth could be recognized using pH, mycelium yield, and culture filtrate antigens as parameters of growth. Immunological characteristics of the different phases (early, intermediate, and late) of growth were measured using IgE and IgG binding by enzyme allergosorbent test and enzyme-linked immunosorbent assay, respectively, and quantities of precipitating components (double immunodiffusion and rocket immunoelectrophoresis). The number of precipitating components was maximal in culture filtrate extracts during late phase (III) of growth. In contrast, both IgE and IgG binding were already maximal in the early phase of growth (9 days of cultivation). In a group of patients with elevated IgE antibodies against Aspergillus fumigatus, about half of them also showed elevated IgE antibodies against B. cinerea. This indicates that multiple exposition is a cause of multiple sensitization; however, cross-reactivity in some of these sera cannot be excluded.

Antibodies, Fungal↗
PubMed1993

S-methyl methane thiosulfonate, a new antimutagenic compound isolated from Brassica oleracea L. var. botrytis.

Though various antimutagens with desmutagenic activities have been found in our daily foods of plant origin, the numbers of antimutagens with bio-antimutagenic activities found so far are limited. In the present study, a compound with potential bio-antimutagenic activity to Escherichia coli B/r WP2 was newly isolated from cauliflower, Brassica oleracea L. var. botrytis, and its chemical structure was identified to be S-methyl methane thiosulfonate by NMR and MS analysis.

Antimutagenic Agents↗
PubMed1996

Homobotcinolide: a biologically active natural homolog of botcinolide from Botrytis cinerea.

A novel natural product exhibiting biological activity was isolated from a strain of Botrytis cinerea that had infected raspberry fruit (Rubus ideaus). Liquid fermentation and bioassay-directed fractionation of the organism yielded a compound with molecular formula C22H38O8 that is trivially named homobotcinolide. It significantly inhibited etiolated wheat coleoptile growth. Greenhouse-grown bean, corn, and tobacco plants were also affected by exogenous application of homobotcinolide, severe chlorosis and necrosis being exhibited in corn. The compound is a polyhydroxylated nonalactone esterified with 4-hydroxy-2-decenoic acid.

Biological Assay↗
PubMed2001

Osmotic dehydration of apple slices using a sucrose/CaCl2 combination to control spoilage caused by Botrytis cinerea, Colletotrichum acutatum, and Penicillium expansum.

The efficacy of sucrose combined with CaCl2 during osmotic dehydration (OD) was tested for the control of Botrytis cinerea, Colletotrichum acutatum, and Penicillium expansum growth on lightly processed apple slices. The objective of this work was to determine whether the addition of CaCl2 in the osmotic solutions would limit the proliferation of fungal decay organisms. Slices were submitted to OD for 1 h at 25 degrees C in solutions containing 5 to 65% sucrose. Calcium chloride was added to a similar set of sucrose solutions at 0 to 8%. Control slices were made of untreated slices, and slices were processed in water. The mass ratio of the slices did not vary when fruit pieces were processed in solutions containing 5 to 65% sucrose. These slices showed a high susceptibility to spoilage compared to the control slices not submitted to OD: a significant twofold and 60% increase in decay area caused by B. cinerea and P. expansum, respectively, was observed when slices were processed in 50% sucrose/0% CaCl2; C. acutatum showed a significant 50% increase in decay area when slices were processed in 20% sucrose/0% CaCl2. Calcium uptake was significantly increased when slices were processed in CaCl2 solutions, and the highest Ca content was observed when processed in 8% CaCl2, reaching 40 times that of the control slices processed in water. Calcium-treated slices were less susceptible to spoilage by all three pathogens, and the most effective combination in reducing apple slice spoilage was 20 to 30% sucrose combined with 2% CaCl2.

Calcium Chloride↗
PubMed2002

Osmotic dehydration of apple slices with CaCl2 and sucrose limits decay caused by Penicillium expansum, Colletotrichum acutatum, and Botrytis cinerea and does not promote Listeria monocytogenes or total aerobic population growth.

The interaction of Penicillium expansum Link, Colletotrichum acutatum, and Botrytis cinerea Pers.:Fr. with Listeria monocytogenes on osmotically dehydrated apple slices was evaluated. In mineral analyses of the slices, the calcium content of the peel and flesh tissues increased by 4- and 11-fold, respectively, when processed in 2% CaCl2. These slices also exhibited less decay by P. expansum, C. acutatum, and B. cinerea. Inoculation of slices with P. expansum resulted in a decrease in the pH of the flesh tissue at the infection site, while the pHs of slices infected with C. acutatum and B. cinerea increased and remained stable, respectively. Total mold population increased in wounds inoculated with P. expansum or C. acutatum. The presence of L. monocytogenes in the wounds did not significantly affect mold growth. The association of P. expansum and L. monocytogenes on apple slices resulted in a decrease in the bacterial population, whereas L. monocytogenes survived when slices were inoculated with C. acutatum. When associated with B. cinerea, there was a fourfold decrease in the L. monocytogenes population when slices were treated with 2% CaCl2. The total aerobic population was not significantly affected by the type of microorganism added to the wounds or by the osmotic treatment. These data show that osmotic dehydration with 2% CaCl2 combined with 20% sucrose limits decay of apple slices and does not promote bacterial or total aerobic population growth.

Calcium Chloride↗
PubMed2000

[Genetic analysis and relationship to pathogenicity in Botrytis cinerea].

Botrytis cinerea is a plant-pathogenic fungus that produces the disease known as grey mould in a wide variety of agriculturally important hosts in many countries. Ten strains from different locations collected on different years have been isolated and characterized by several methods (morphological, biochemical, genetical and molecular). Results showed that clear morphological differences exist between strains, and showing a relationship between the presence of sclerotia and pathogenicity. The conidial size and the nuclear number were highly variable between different strains. Pulsed-field gel electrophoresis showed a unique karyotype for each strain, highly polymorphic between strains and with a number of bands ranging from 4 to 8. An efficient transformation system has been achieved through the plasmid pAMPF21, containing the region AMA1 of Aspergillus nidulans. Lastly, from a genomic library the gdhA gene has been cloned. This gene produces an RNAm of 1.7 Kb and complements the deficiency on glutamate dehydrogenase activity of A. nidulans.

English Abstract↗
PubMed2000

[Pathogenicity factors in Botrytis cinerea].

Botrytis cinereais an important plant pathogenic fungi with a wide host range, which can make use of different infection mechanisms. Although genetic variation for resistance to B. cinereahas been observed within some species, no gene-for-gene relationship has been found. The development of resistant genotypes is, therefore, complicated. Any attempt to develop control strategies makes it necessary a detailed knowledge of both the fungal infection mechanisms and the plant defence mechanisms. The application of different experimental approaches allows the analysis of the infection process in different hosts, the description of the elements that participate in each stage of the process and the identification of those pathogenicity factors which are essential for the establishment of the interaction. The characterisation of the latter will provide information about key elements of the infection process as the basis for the development of effective, long term and environmentally friendly control strategis.

English Abstract↗
PubMed2005

[Identification of PCR markers associated with cytoplasmic male sterility in Brassica oleracea var Botrytis].

The homology-based candidate gene method was used to identified the specific PCR markers linked to cytoplasmic male sterility (CMS) in cauliflower( Brassica oleracea var botrytis.). Searching the DNA and protein data-base of NCBI , correlative genes or open reading frames were identified . Analysis of biosoft, based on the conservative regions ,five primers were designed . Among them, only primer P9/P10 produced a 313- bp specific fragment. Identified by individual plant testing , analysis of RT-PCR and dot blot ,this fragment was only existed in CMS cauliflower knxd612. Analysis of the sequence indicated it was high homologous(98%) with orf138 of Ogura CMS radish. Primary result suggested that the cytoplasmic type of CMS cauliflower knxd612 may belong to Ogura type. This research offered a good foundation to further investigate the CMS mechanism of cauliflower in molecular level.

Amino Acid Sequence↗
PubMed1981

Hormone effects on Cosmarium botrytis cell division.

The synchronous generation time for Cosmarium botrytis Menegh. was 48 h at pH 8.0, and 72 h at pH 5.3. Cells divided 2 to 3 prior to the end of the dark phase in a 15 h light/9 h dark cycle at 21 degrees C. Kinetin at concentrations of 0.01 to 1.0 mg/ml, added to cells at the beginning of the light cycle, eliminated the initial lag phase at both pH 5.3 and pH 8.0, and all cells divided until 24 h. Indole-acetic acid in concentrations of 0.03 to 0.06 mg/ml decreased the lag phase to less than 48 h at pH 5.3, and eliminated the lag phase at pH 8.0. Normal generation time was re-established in 6 to 12 days. Daily addition of identical amounts of both IAA and kinetin maintained the 24 h division cycle up to 14 days. Kinetin was lethal at 3 mg/ml and IAA at 0.05 mg/ml. IAA and kinetin in combinations of previously effective and non-effective concentrations did not have synergistic or additive effects. Cell numbers did not correlate with optical density at 678 nm. Medium pH dropped in steps coinciding with cell division.

Cell Division↗
PubMedDate not supplied

Antifungal compounds induced in the dual culture with Phytolacca americana callus and Botrytis fabae.

In order to investigate new metabolites which are only induced in a plant callus infected by a pathogenic fungus, dual cultures with combinations of 10 species of fungi and 6 plant cell lines from different species were established. Among the combinations tested, the methanolic extract of a dual culture consisting of a plant cell line, Phytolacca americana and a fungus, Botrytis fabae showed a marked antifungal activity to Cladosporium herbarum. The main active constituent of this extract was identified to be phytolaccoside B (Pls B) by the spectroscopic analyses.

Antifungal Agents↗
PubMed1995

Polygalacturonase produced in apple tissue decayed by Botrytis cinerea.

An exo-polygalacturonase with an isoelectric point of 4.6 and an apparent molecular weight of 45 kDa was isolated from apple tissue decayed by Botrytis cinerea. This isozyme had a similar isoelectric point, optimum pH, and mode of action as an isozyme produced in liquid culture by B. cinerea. The enzyme produced in the decayed tissue was less sensitive to lower pH and less inhibited by CaCl2, MgCl2, or NaCl than the enzyme produced in culture. Such changes in the properties of the enzyme produced in infected tissue could have been essential for the pathogen's successful colonization of the host tissue. Among the cations studied, calcium was the best inhibitor of PG activity.

Chlorides↗