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PubMed2001

The potential biocontrol agent Pseudomonas antimicrobica inhibits germination of conidia and outgrowth of Botrytis cinerea.

AIMS: Antifungal metabolites of Pseudomonas antimicrobica have previously been shown to inhibit conidial germination of the grey mould pathogen Botrytis cinerea. In this study, metabolites of the bacterium have been tested at different stages of Botrytis germination to determine their effects on germ tube production and extension. METHODS AND RESULTS: Metabolites were added to conidia that had been pre-incubated for either 120 or 255 min. Pseudomonas antimicrobica inhibited B. cinerea conidial germination and caused a significant reduction in germ tube extension, irrespective of the stage of germination. Abnormal germination and a reduction in the frequency of lateral branching of the germ tubes in the presence of the metabolites were also reported, suggesting interference with normal hyphal development. CONCLUSION: The bacterium can inhibit germination of conidia and extension of germ tubes at different stages of Botrytis development. SIGNIFICANCE AND IMPACT OF THE STUDY: The antagonistic activity of the bacterium has promising implications for its use as a biocontrol agent.

Antifungal Agents↗

PubMed2002

Characterization of molecular markers for specific and sensitive detection of Botrytis cinerea Pers.: Fr. in strawberry (Fragariaxananassa Duch.) using PCR.

Random amplified polymorphic DNA (RAPD) assays were applied on 34 fungal strains isolated from strawberry and other host plants, in order to detect polymorphism to consequently identify and isolate molecular markers specific to Botrytis cinerea. Among the 26 10-mer primers tested, one primer mainly amplified a 750-bp product present in all the B. cinerea strains and absent in the other species and genera examined. This product was cloned and sequenced in order to design a specific 20-mer primer pair, which was tested on the 34 fungal isolates by polymerase chain reaction (PCR). A single 0.7-kb band was amplified in all the 13 strains of B. cinerea isolated from different host plants. Moreover, a 0.6-kb band was amplified in both Botrytis fabae strains. No band was observed for the nine other Botrytis species and 12 fungal genera isolated from strawberry plants. A comparison between the 0.7-kb B. cinerea sequence and the 0.6-kb B. fabae sequence revealed 98% homology and one 122-bp deletion for B. fabae, including an EcoRI site. Hybridization of Southern blots with RAPD and EcoRI-digested DNA confirmed the specificity of the marker. The limit of detection of B. cinerea genomic DNA was approximately 0.2 pg. The PCR procedure was able to amplify the 0.7-kb B. cinerea fragment form mixed samples of DNA as low as 2 pg B. cinerea genomic DNA and 1 microg plant DNA. Thus this PCR-based detection procedure is a powerful tool for diagnosis of B. cinerea in symptomless strawberry plants, and should allow infection and latency sites to be localized in order to improve knowledge of the epidemiology of the pathogen under field conditions.

Base Sequence↗

PubMed2006

Necrotrophic mycoparasitism of Botrytis cinerea by cellulolytic and ligninocellulolytic Basidiomycetes.

Twenty-six isolates representing 17 species of aphyllophoraceous, wood-decaying Basidiomycetes and five species of agaricoid, turf-borne, thatch-decaying Basidiomycetes were screened for their abilities to degrade cellulose, lignin, and melanin by using colorimetric degradation assays on agar media. Selected ligninocellulolytic Basidiomycetes capable of degrading melanin were screened for antagonism of Botrytis cinerea Per.:Fr. The greatest inhibition of Botrytis colony and hyphal growth in vitro was observed in confrontations with Irpex lacteus (Fr.) Fr., Trametes versicolor (L.:Fr.) Pilat, and Chondrostereum purpureum (Pers.:Fr.) Pouzar. Hyphal interference and necrotrophic mycoparasitism by these ligninocellulolytic Basidiomycetes were recognized microscopically as coagulation and degeneration of Botrytis cytoplasm and as coiling and invasion of hyphae, conidiophores, and conidia, respectively. Sclerotia of B. cinerea were killed and parasitized in agar media, straw mulch, or moist sand infested separately with these three mycoparasites.

Basidiomycota↗

PubMed2005

Induction of resistance to the pathogenic agent Botrytis cinerea in the cultivation of the tomato by means of the application of the protein "Harpin"(Messenger).

Harpin(Ea), an active component in Messenger, was originally isolated from the bacterial plant pathogen Erwinia amylovora based on its ability to elicit a hypersensitive response (HR). This bacterial pathogen causes "fire blight", a disease in apples and other members of the Rosaceae. Harpin(Ea) also induces resistance in a variety of plants against a wide array of pathogens. The objective of this investigation was to determine indications of resistance induction to Botrytis cinerea in the plants treated with the formulated Harpin(Ea) protein. This pathogenic agent is responsible for important economic losses in the cultivation of both greenhouse and field grown tomatoes. Plants with and without Messenger applications were inoculated with Botrytis cinerea in the same way. In addition, some plants with and without Messenger applications were not inoculated. Inoculated plants were symptomatologically checked for local and systemic symptoms. Evaluations of the number of tomatoes produced and their growth were also carried out. The results obtained demonstrate that there were active defence systems in the plant, producing a reduction in the damage caused by the pathogenic agent Botrytis cinerea. A greater growth also took place in the fruit obtained. These results show that the formulated Harpin(Ea) protein could reduce the use of chemical agents and be a new tool to serve as the basis for an Integrated Pest Management system.

Bacterial Outer Membrane Proteins↗

PubMed2005

Botrytis infection warnings in strawberry: reduced enhanced chemical control.

The fungal pathogen Botrytis cinerea is the causal agent of grey mould, the most important fungal fruit rot disease in strawberry in Europe. Currently disease control for grey mould is based on preventive spraying every five to seven days during flowering and harvest. Replacing preventive spraying with applications based on infection warnings can optimize performance and reduce the amount of sprays needed. Success of this approach will depend on the accuracy of the model used to predict disease outbreak. For this reason three infection models (BOTEM, BoWaS, DSS-Italy) were evaluated during the growth seasons of 2003 and 2004. The experiments included June bearing, retarded June bearing and ever bearing strawberries. In all experiments the use of infection models leaded to a reduced number of fungicide applications. However the efficacy of the different models towards the control of B. cinerea also decreased compared to the efficacy obtained with a standard 7 day schedule. Best results were obtained with BOTEM, developed by HRI (Horticultural Research International, East-Malling, UK): 17-60% reduction in fungicide use and an efficacy between 66-93 depending on the growth season, culture practice and the fungicides used. Compared with routine preventive spraying, the Botrytis fruit rot percentage is slightly higher. A higher efficacy with Botrytis infection warnings can only be obtained if infection warnings change from curative to preventive. A retroactive evaluation of a preventive warning system was included. Making use of the 48h weather forecasts supplied by the Royal Meteorological Institute of Belgium (KMI) based on ALADIN for the region of Haspengouw, it was possible to replace 30 up to 100% of the curative application by preventive spraying depending on the experiment and the threshold set for the preventive model.

Botrytis↗

PubMed2005

Biological control of Botrytis gray mould and Sclerotinia drop in lettuce.

Research was carried out to evaluate the effectiveness of the biological control of two most important fungal diseases of lettuce (Lactuca sativa L.): 1) Botrytis Gray Mould caused by Botrytis cinerea Pers. ex Fr.; 2) Sclerotinia Drop caused by two pathogenic fungi, Sclerotinia sclerotiorum (Lib.) De Bary and/or Sclerotinia minorJagger. Biological control in lettuce was carried out: 1) using Coniothyrium minitans Campbell, an antagonist fungus that attacks and destroys sclerotia within the soil; 2) identifying lettuce genotypes showing less susceptibility or tolerance. The object of this research was to find control strategies to reduce chemical treatments. The use of resistant varieties is one of the most economical ways to control vegeTable diseases. The lettuce genotypes showing in preliminary trials the best behaviour to the sclerotial diseases were compared in a randomized complete block experiment design and replicated four times. Observations were carried out from February up to April registering the number of diseased plants and yield. The pathogens were isolated on PDA medium and identified. The isolates grown onto PDA plates, after incubation for 6 weeks, allowed obtaining sclerotia that were the target of C. minitans in biological control trials. In laboratory, in controlled conditions, 27 small plots (30 cm in diameter each) with disinfected soil were performed. In 18 plots 9 sclerotia were inoculated (per plot, three of each fungus) and in 9 plots of them a suspension of the antagonist fungus was added. Subsequently, three lettuce varieties were transplanted. For each variety were compared: 1) untreated plots; 2) treated plots with sclerotia only; 3) treated plots with sclerotia and C. minitans suspension. The number of diseased plants was recorded. According to symptom evaluation scale, ranged from 0 (no disease) up to 10 (100% necrotic leaves or dead plants) the plants were grouped into infection classes, calculating the McKinney index. In greenhouse trials, "LM 1307" genotype showed less significant susceptibility to Botrytis Gray Mould (0-2% of affected plants), while "Ninja" and "Charmy" showed 4-11% and 16-26% of diseased plants, respectively. The yields were 69.7, 62.7, 55.3 t/Ha, respectively. In laboratory tests, the McKinney index gave the following results: no disease in all untreated plants; 38.3, 54.2 and 89.2% in "LM 1307", "Ninja" and "Charmy" treated with sclerotia only, respectively; 2.5, 7.5 and 20.8% in "LM 1307", "Ninja" and "Charmy" treated with sclerotia and C. minitans, respectively. In conclusion, the less susceptibility of the genotypes to sclerotial diseases and the use of hyperparasites of sclerotia of phytopathogenic fungi exhibited best results.

Antibiosis↗

PubMed1976

Antibiotic-induced changes of mycelial growth of Botrytis cinerea.

The effect of antibiotics and metabolic inhibitors on mycelial growth of Botrytis cinera was followed. Inhibitors of protein synthesis, chloramphenicol, erythromycin and tetracycline inhibit growth or sporulation of Botrytis cinera. Ethidium bromide, 5-fluorouracil, phenylethylalcohol and K 20 cause granulation, vacuolization and undulation of hyphase. 2,4-Dinitrophenol, boromycin, macrotetrolides, monensin, scopathricin and TX2 at subfungistatic concentrations induce intensive branching of hyphal tips i.e. at the site of synthesis of the cell wall. In older hyphase grown in the absence of the antibiotics the branching begins after their addition, particularly in the septum region. When comparing the results referred to here with those obtained previously and on the basis of literature data it may be assumed that the changes in polarity of growth of Botrytis cinerea might be caused primarily or secondarily by impairing membrane functions and formation of cell walls.

Anti-Bacterial Agents↗

PubMed1998

Bacillus isolates from the spermosphere of peas and dwarf French beans with antifungal activity against Botrytis cinerea and Pythium species.

A range of isolation procedures including washing, sonication and incubation in nutrient broth were used separately and in combination to obtain potential bacterial antagonists to Botrytis cinerea and Pythium mamillatum from the testae and cotyledons of peas and dwarf French beans. Heat treatment was also used to bias this selection towards spore-forming bacteria. Ninety-two bacterial isolates were obtained, 72 of which were provisionally characterized as species of Bacillus. Four of these Bacillus isolates (B3, C1, D4 and J7) displayed distinct antagonism in vitro against Botrytis cinerea and P. mamillatum when screened using dual culture analysis. Further characterization of these antagonists using API 50CHB biochemical profiling identified isolate D4 as Bacillus polymyxa and isolates B3, C1 and J7 as strains of B. subtilis. In vitro screening techniques, using cell-free and heat-killed extracts of liquid cultures against Botrytis cinerea, demonstrated the production of antifungal compounds by these four Bacillus antagonists. With each isolate the antifungal activity was found not to be either exclusively spore-bound nor released entirely into the medium but present in both fractions. The antifungal compounds produced by these isolates were shown to be heat-stable. Their identification, production and release require further study for exploitation as biocontrol systems.

Antibiosis↗

PubMed2002

The symbiosis of Bacillus subtilis L-forms with Chinese cabbage seedlings inhibits conidial germination of Botrytis cinerea.

AIMS: To establish whether germination of Botrytis cinerea was affected by the symbiosis of Bacillus subtilis L-form bacteria with Chinese cabbage. METHODS AND RESULTS: Germinating seeds of Chinese cabbage were co-cultivated with either L-forms of Bacillus subtilis or 5% (w/v) mannitol by soaking for 3 h. Seeds were then washed in sterile water, sown on a minimal medium and incubated in controlled conditions. L-form symbiosis was detected over a time course by ELISA. Conidial germination of Botrytis cinerea was significantly reduced on cotyledonous leaves of L-form-treated plants compared with controls. CONCLUSIONS: Symbiosis of B. subtilis L-form bacteria during seed germination of Chinese cabbage inhibits conidial germination in plants on subsequent exposure to Botrytis cinerea. SIGNIFICANCE AND IMPACT OF THE STUDY: This is the first account of plant symbiosis with L-form bacteria showing antagonism to a fungal plant pathogen. This has promising implications for the use of this L-form as a biocontrol agent.

Bacillus subtilis↗

PubMed1980

[Occurrence of an extracellular beta-(1 to 3)-glucanase from Botrytis cinerea].

Like other fungi, Botrytis cinera produces an exocellular beta-(1 leads to 3)-glucanase. This constitutive enzyme is produced only when Botrytis is cultivated in a glucose depleted medium. The enzyme is precipitated by ammonium sulfate from 20 to 80% saturation. Maximum activity is at pH between 5 and 6 and temperature between 40 and 50 degrees C; stability is good up to 30 degrees C. This enzyme cannot be applied to the treatment of wines containing Botrytis glucan.

Glucan 1,3-beta-Glucosidase↗

PubMed2002

Secretion of beta-1,3-glucanases by the yeast Pichia membranifaciens and its possible role in the biocontrol of Botrytis cinerea causing grey mold disease of the grapevine.

Pichia membranifaciens strain FY-101, isolated from grape skin, was found to be antagonistic to Botrytis cinerea, the causal organism of the grey mold disease of the grapevine. When grown together on solid as well as in liquid media, the yeast brings about the inhibition of Botrytis cinerea, which in turn loses its ability to produce the grey mold symptoms on the grapevine plantlets. The secretion of beta-1,3-glucanases by P. membranifaciens is one of the possible mechanisms related to this antagonism. In vitro experiments confirm that this yeast can be used as a biological control organism against B. cinerea. An account of this antagonism and the production of beta-1,3-glucanases by P. membranifaciens is given here.

Botrytis↗

PubMed2004

Yeasts as biological agents to control Botrytis cinerea.

Yeasts, isolated from different sources, were identified and tested for inhibition using YMA-MB plates seeded with Botrytis cinerea strains. A total of 42 yeast strains of 20 different species were tested in vitro for antagonism against 18 pathogenic B. cinerea strains. Pichia membranifaciens, P. anomala and Debaryomyces hansenii displayed the most important inhibitory effect against Botrytis strains. In small-scale trials, post-harvest application of P. membranifaciens CYC 1106 to apple wounds inhibited B. cinerea CYC 20010. Purified killer toxin from P. membronifaciens CYC 1106 inhibited B. cinerea CYC 20010. Results indicated that certain yeasts, or their toxins such us P. membranifaciens CYC 1106 killer toxin, might have potential as novel agents to control B. cinerea.

Antibiosis↗

PubMed2003

Transformations of testosterone and related steroids by Botrytis cinerea.

Botrytis cinerea (strain AM235) was used to investigate the transformations of testosterone and related steroids. It was found that the position and stereochemistry of the introduced hydroxyl group, as well as the yield of products, depended on the structure of the substrate. Botrytis cinerea converts the examined substrates mainly to 7 alpha-hydroxy derivatives. 1-Dehydrotestosterone was also significantly hydroxylated at a 14 alpha-position.

Biotransformation↗

PubMed2003

Chemical composition and antifungal activity of essential oils of seven Moroccan Labiatae against Botrytis cinerea Pers: Fr.

Essential oils of seven Moroccan Labiatae were chemically analysed by GC-MS and evaluated for their in vitro antifungal activity against Botrytis cinerea. Among them, Origanum compactum and Thymus glandulosus greatly inhibited the growth of the mycelium. The inhibition of Botrytis cinerea was 100% for both oils at 100 ppm, while the IC(50)s were 35.1 and 79.2 ppm, respectively. Mentha pulegium exhibited moderate activity at 250 ppm since the inhibition of the mycelial growth was 58.5% and the IC(50) was 233.5 ppm. The main constituents of the studied oils were also examined. Thymol and carvacrol that are the two main constituents of Thymus glandulosus and Origanum compactum exhibited the strongest antifungal activity with 100% of inhibition at 100 ppm, respectively.

Antifungal Agents↗

PubMed2004

Enniatins of Fusarium sp. strain F31 and their inhibition of Botrytis cinerea spore germination.

A spectrum of enniatins was isolated from Fusarium sp. strain F31 by bioassay-guided isolation directed against Botrytis cinerea. Two new enniatins, J(2) (7) and J(3) (8), were co-isolated and both contained, in addition to three hydroxyisovaleric acid units, N-methylated l-alanine, l-valine, and l-isoleucine units, differing only in their primary sequence. Two other enniatins, named enniatin J(1) (1) and enniatin K(1) (6), each containing two N-Me-l-Val units and one N-Me-l-Ala or alpha-N-Me-l-butyric acid unit, respectively, were isolated for the first time without directed biosynthesis. The enniatin structures were elucidated by spectroscopic and chemical methods, and the absolute configuration of the amino acids (l) and hydroxyisovaleric acid (d) was consistent with all previously isolated enniatins. The known enniatins B (2), B(1) (4), B(2) (5), and B(4) (3) were also isolated. The minimum inhibitory concentration of pure enniatins against Botrytis cinerea was 75 microg/mL.

Amino Acid Sequence↗

PubMed2000

The BMP1 gene is essential for pathogenicity in the gray mold fungus Botrytis cinerea.

In Magnaporthe grisea, a well-conserved mitogen-activated protein (MAP) kinase gene, PMK1, is essential for fungal pathogenesis. In this study, we tested whether the same MAP kinase is essential for plant infection in the gray mold fungus Botrytis cinerea, a necrotrophic pathogen that employs infection mechanisms different from those of M. grisea. We used a polymerase chain reaction-based approach to isolate MAP kinase homologues from B. cinerea. The Botrytis MAP kinase required for pathogenesis (BMP) MAP kinase gene is highly homologous to the M. grisea PMK1. BMP1 is a single-copy gene. bmp1 gene replacement mutants produced normal conidia and mycelia but were reduced in growth rate on nutrient-rich medium. bmp1 mutants were nonpathogenic on carnation flowers and tomato leaves. Re-introduction of the wild-type BMP1 allele into the bmp1 mutant restored both normal growth rate and pathogenicity. Further studies indicated that conidia from bmp1 mutants germinated on plant surfaces but failed to penetrate and macerate plant tissues. bmp1 mutants also appeared to be defective in infecting through wounds. These results indicated that BMP1 is essential for plant infection in B. cinerea, and this MAP kinase pathway may be widely conserved in pathogenic fungi for regulating infection processes.

Amino Acid Sequence↗

PubMed2003

Disruption of Botrytis cinerea pectin methylesterase gene Bcpme1 reduces virulence on several host plants.

The pectinolytic enzyme pectin methylesterase (PME) hydrolyses pectin in methanol and polygalacturonic acid. In the expressed sequence tag library of Botrytis cinerea T4, we identified a 1,041 bp Bcpme1 cDNA potentially encoding a 346-amino acid protein of 37 kDa showing 46.8% identity with Aspergillus sp. PMEs. Bcpme1 is a single copy gene and is similarly expressed in glucose and pectin containing media. To evaluate the role of Bcpme1 in Botrytis cinerea virulence, a mutant in Bcpme1 was generated by gene disruption. The Bcpme1 mutant showed similar growth on rich medium but reduced growth on pectin medium. Two isozymes of pI 7.4 and 7.1 were detected in pectin liquid-culture supernatants of wild-type strain Bd90 analyzed by isoelectric focusing-polyacrylamide gel electrophoresis, while those of Bcpme1 mutant possessed only the pI 7.1 isozyme. BCPME1, the pI 7.4 isozyme, is the major PME activity, as PME activity is 75% reduced in Bcpme1 mutant. Moreover, the Bcpme1 mutant was less virulent on apple fruits, grapevine, and Arabidopsis thaliana leaves. Those phenotypes were complemented by reintroducing a Bcpme1 copy in the Bcpme1 mutant. These results showed that B. cinerea possessed more than one PME-encoding gene and that BCPME1 is an important determinant of B. cinerea virulence.

Amino Acid Sequence↗

PubMed2003

The endopolygalacturonase 1 from Botrytis cinerea activates grapevine defense reactions unrelated to its enzymatic activity.

A purified glycoprotein from Botrytis cinerea (strain T4), identified as endopolygalacturonase 1 (T4BcPG1) by mass spectrometry analysis, has been shown to activate defense reactions in grapevine (Vitis vinifera cv. Gamay). These reactions include calcium influx, production of active oxygen species, activation of two mitogen-activated protein kinases, defense gene transcript accumulation, and phytoalexin production. Most of these defense reactions were also activated in grapevine in response to purified oligogalacturonides (OGA) with a degree of polymerization of 9 to 20. In vivo, these active OGA might be a part of the released products resulting from endopolygalacturonase activity on plant cell walls. Nevertheless, the intensity and kinetics of events triggered by OGA were very different when compared with T4BcPG1 effects. Moreover, chemical treatments of T4BcPG1 and desensitization assays have allowed us to discriminate enzymatic and elicitor activities, indicating that elicitor activity was not due to released oligogalacturonides. Thus, BcPG1 should be considered as both an avirulence and a virulence factor. The role of the secreted BcPG1 in the pathogenicity of Botrytis cinerea is discussed.

Amino Acid Sequence↗