ofBotrytis Cinerea Research Articles

Effects of Two Salts Compounds on Mycelial Growth, Sporulation, and Spore Germination of Six Isolates of Botrytis cinerea in the Western North of Algeria.

Six isolates of Botrytis cinerea were isolated from leaves and stems of different tomato varieties taken from four areas in the northwest of Algeria where tomato is mostly grown in greenhouses and high tunnels. The purpose of this research was to determine the effect of two salts, NaCl and CaCl2, on three stages of Botrytis cinerea's life cycle. All isolates tested were stimulated in 50 to 150 ppm; NaCl was the most effective treatment to increase mycelial growth at two tested concentrations. However, at 300 ppm concentration, CaCl2 completely inhibited the growth of mycelium; they reach 34.78% for the isolate TR46 and 26.72% for isolate F27. The sodium and calcium salts stimulated conidia production in liquid culture. We noticed that the effect of calcium chloride on sporulation was average while sodium chloride. In the medium containing 50 ppm, calcium chloride and sodium chloride increased the germination capacity of most isolates compared with the control. Other calcium salts, at 100 or 300 ppm, decreased the germination percentage of the conidia. With the exception of sodium salts, the inhibitions of germination reduce at 150 or 300 compared with the control. Conidial germination was slightly inhibited by sodium chloride only when the concentration was over 300 ppm.

Effect of Chlorine Dioxide Gas Treatment on Surface Sterilization of Grape

Experiments were conducted to investigate the effect of concentration and duration of chlorine dioxide gas treatment on Surface sterilization of grape at 25°C. The results showed that the values of inactivate bacterial log reduction ofBotrytis cinerea,Penicilliumandalternariaincreased with the increasing of ClO2gas concentrations and treatment time. When the concentrations and treatment time was about 10 mg/m3and 30 minutes, respectively, more than 4 log reduction was obtained for the three spoilage bacteria on grape surface. Furthermore, the effect of chlorine dioxide gas treatment on quality of grape was investigated. It was found that the contents of vitamin C (Vc) and reducing sugar (RS) in grape also increased compared with grape without ClO2gas treatment.

Exploring Mechanisms of Resistance to Respiratory Inhibitors in Field Strains of Botrytis cinerea , the Causal Agent of Gray Mold

Respiratory inhibitors are among the fungicides most widely used for disease control on crops. Most are strobilurins and carboxamides, inhibiting the cytochrome b of mitochondrial complex III and the succinate dehydrogenase of mitochondrial complex II, respectively. A few years after the approval of these inhibitors for use on grapevines, field isolates of Botrytis cinerea, the causal agent of gray mold, resistant to one or both of these classes of fungicide were recovered in France and Germany. However, little was known about the mechanisms underlying this resistance in field populations of this fungus. Such knowledge could facilitate resistance risk assessment. The aim of this study was to investigate the mechanisms of resistance occurring in B. cinerea populations. Highly specific resistance to strobilurins was correlated with a single mutation of the cytb target gene. Changes in its intronic structure may also have occurred due to an evolutionary process controlling selection for resistance. Specific resistance to carboxamides was identified for six phenotypes, with various patterns of resistance levels and cross-resistance. Several mutations specific to B. cinerea were identified within the sdhB and sdhD genes encoding the iron-sulfur protein and an anchor protein of the succinate dehydrogenase complex. Another as-yet-uncharacterized mechanism of resistance was also recorded. In addition to target site resistance mechanisms, multidrug resistance, linked to the overexpression of membrane transporters, was identified in strains with low to moderate resistance to several respiratory inhibitors. This diversity of resistance mechanisms makes resistance management difficult and must be taken into account when developing strategies for Botrytis control.

Sensitivity ofBotrytis cinerea from greenhouse vegetables to DMIs and fenhexamid

Two hundred isolates ofBotrytis cinerea were collected from greenhouse vegetables between 2003 and 2006 to determine their baseline sensitivity to triadimefone, penconazole, tebuconazole and fenhexamid. Mean values of 50% effective concentrations (EC50) of inhibiting growth were 4.853±5.102, 0.41±0.215, 0.19±0.099 and 0.36±0.891 mgl −1, respectively (mean±SD). Individuals ofB. cinerea in the population differed by a factor (EC50 of the least sensitive isolate/EC50 of the most sensitive isolate) of 6625, 20, 603 and 1800, respectively. Naturally fenhexamid-resistant isolates were detected with an unexpected high frequency of 10% although the pathogen population had never been exposed to this fungicide. The resistance level (mean EC50 of resistant isolates / mean EC50 of sensitive isolates) was 19.5. These naturally resistant isolates also were resistantin vivo, and there was no significant difference in growth rate, conidial production or pathogenicity ability between naturally resistant and wild sensitive isolates. These results indicated that there was a potential risk of practical resistance if fenhexamid was applied alone. Negative cross-resistance was observed between fenhexamid and tebuconazole in 90% of the naturally resistant isolates. Moreover, an obvious synergism of the antifungal activity of fenhexamid by tebuconazole was demonstrated in some of the naturally fenhexamid-resistant isolates.

BcSAK1, a Stress-Activated Mitogen-Activated Protein Kinase, Is Involved in Vegetative Differentiation and Pathogenicity in Botrytis cinerea

The gene bcsak1, encoding a mitogen-activated protein kinase (MAPK) of Botrytis cinerea, was cloned and characterized. The protein has high homology to the yeast Hog1 and to corresponding MAPKs from filamentous fungi, but it shows unique functional features. The protein is phosphorylated under osmotic stress, specific fungicides, and oxidative stress mediated by H(2)O(2) and menadione. Northern blot analyses indicate that only a subset of typical oxidative stress response genes is regulated by BcSAK1. In contrast to most other fungal systems, Deltabcsak1 mutants are significantly impaired in vegetative and pathogenic development: they are blocked in conidia formation, show increased sclerotial development, and are unable to penetrate unwounded plant tissue. These data indicate that in B. cinerea the stress-activated MAPK cascade is involved in essential differentiation programs.

Improvement of the Fungal Biocontrol Agent Trichoderma atroviride To Enhance both Antagonism and Induction of Plant Systemic Disease Resistance

Biocontrol agents generally do not perform well enough under field conditions to compete with chemical fungicides. We determined whether transgenic strain SJ3-4 of Trichoderma atroviride, which expresses the Aspergillus niger glucose oxidase-encoding gene, goxA, under a homologous chitinase (nag1) promoter had increased capabilities as a fungal biocontrol agent. The transgenic strain differed only slightly from the wild-type in sporulation or the growth rate. goxA expression occurred immediately after contact with the plant pathogen, and the glucose oxidase formed was secreted. SJ3-4 had significantly less N-acetylglucosaminidase and endochitinase activities than its nontransformed parent. Glucose oxidase-containing culture filtrates exhibited threefold-greater inhibition of germination of spores of Botrytis cinerea. The transgenic strain also more quickly overgrew and lysed the plant pathogens Rhizoctonia solani and Pythium ultimum. In planta, SJ3-4 had no detectable improved effect against low inoculum levels of these pathogens. Beans planted in heavily infested soil and treated with conidia of the transgenic Trichoderma strain germinated, but beans treated with wild-type spores did not germinate. SJ3-4 also was more effective in inducing systemic resistance in plants. Beans with SJ3-4 root protection were highly resistant to leaf lesions caused by the foliar pathogen B. cinerea. This work demonstrates that heterologous genes driven by pathogen-inducible promoters can increase the biocontrol and systemic resistance-inducing properties of fungal biocontrol agents, such as Trichoderma spp., and that these microbes can be used as vectors to provide plants with useful molecules (e.g., glucose oxidase) that can increase their resistance to pathogens.

Further suppression ofBotrytis cinerea disease in cucumber seedlings by chitosan-copper complex as compared with chitosan alone

Chitosan-copper complex compared with chitosan alone enhanced suppression ofBotrytis cinerea rot development on four-true-leaf cucumber seedlings in controlled growth chambers. This paper constitutes the first report of such enhancement. The optimal concentrations for the most effective suppression ofBotrytis development were 0.2 gl −1 chitosan and 1.6 mmole copper. After 12 days’ incubation, marked and significantly better disease suppression was obtained with chitosan-copper complex (75% suppression) than with chitosan alone. The chitosan-copper complex could be a very promising decay control agent for use in both conventional and organic agriculture.

Development ofClonostachys rosea and interactions withBotrytis cinerea in rose leaves and residues

The effect of microclimate variables on development ofClonostachys rosea and biocontrol ofBotrytis cinerea was investigated on rose leaves and crop residues. C.rosea established and sporulated abundantly on inoculated leaflets incubated for 7–35 days at 10°, 20° and 30°C and then placed on paraquat—chloramphenical agar (PCA) for 15 days at 20°C. On leaflets kept at 10°C, the sporulation after incubation on PCA increased from 60% to 93% on samples taken 7 to 21 days after inoculation, but decreased to 45% on material sampled after 35 days. A similar pattern was observed on leaves incubated at either 20° or 30°C. The sporulation ofC. rosea on leaf disks on PCA was not affected when the onset of high humidity occurred 0, 4, 8, 12 or 16 h after inoculation. However, sporulation was reduced to 54–58% on leaflets kept for 20–24 h under dry conditions after inoculation and before being placed on PCA. The fungus sporulated on 68–74% of the surface of leaf disks kept for up to 24 h at high humidity after inoculation, but decreased to 40–51% if the high humidity period before transferral to PCA was prolonged to 36–48 h. The growth ofC. rosea on leaflets was reduced at low inoculum concentrations (103 and 104 conidia/ml) because of competition with indigenous microorganisms, but at higher concentrations (105 and 106 conidia/ml) the indigenous fungi were inhibited. Regardless of the time of application ofC. rosea in relation toB. cinerea, the pathogen’s sporulation was reduced by more than 99%. The antagonist was able to parasitize hyphae and conidiophores ofB. cinerea in the leaf residues. AsC. rosea exhibited flexibility in association with rose leaves under a wide range of microclimatic conditions, and in reducingB. cinerea sporulation on rose leaves and residues, it can be expected to suppress the pathogen effectively in rose production systems.

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.

Evaluation of pyrimethanil (Scala) for the control of Botrytis cinerea on harvested Geraldton waxflower

Pyrimethanil (Scala) is a fungicide that might be used for control of Botrytis cinerea infecting Geraldton waxflower. New treatments are sought to reduce the risk of developing strains of B. cinerea that are resistant to fungicides commonly used against it, such as benomyl (Benlate) or iprodione (Rovral). Scala was applied to waxflower as a postharvest dip at 1.5 or 2 mL product/L. Disease severity on and flower and leaf drop from waxflower bunches pre-inoculated with B. cinerea were significantly (P = 0.05) reduced by treatment with Scala. Further, flower and foliage vase lives were significantly (P = 0.05) longer as a result of postharvest treatment with Scala as compared with water-treated (control) stems. Similar degrees of disease control where achieved with either Scala or Rovral. The results demonstrate that Scala has real potential as an alternative fungicide for control of B. cinerea on waxflower.

Purification and characterization of glucose oxidase ofBotrytis cinerea

The H2O2-producing enzyme glucose oxidase of the phytopathogenic fungusBotrytis cinereawas purified to homogeneity by anion-exchange chromatography and chromatofocusing. The enzyme has its pH optimum at 7.5 and an isoelectric point of 4.2. The enzyme appears to be a tetrameric protein with a native molecular weight of about 160 kDa. Like the glucose oxidase ofPhanerochaete chrysosporium, the glucose oxidase ofB. cinereais not glycosylated. Analysis of substrate specificity confirmed that β-D-glucose is the specific substrate of the enzyme. The expression of the glucose oxidase ofB. cinereais induced by low glucose concentration in the culture medium. In this respect, the glucose oxidase ofB. cinereais also similar to the glucose oxidase ofP. chrysosporiumand differs from the glucose oxidases ofAspergillusandPenicillium,which require high glucose concentrations for expression.

Biological control ofBotrytis cinerea in residues and flowers of Rose (Rosa hybrida)

Microbial isolates from living petals, petal residues and leaf residues of rose, and from laboratory collections, were evaluated for control ofBotrytis cinerea in rose. In leaf residues artificially infested withB. cinerea, isolates of the filamentous fungiGliocladium roseum, FR136 (unidentified) andTrichoderma inhamatum reduced sporulation of the pathogen by >90%, other filamentous fungi were 25–90% effective, and those of yeasts and bacteria were 75%, but isolates ofCladosporium oxysporum and four yeasts were 51–75% effective, and three filamentous fungi, eight yeasts andBacillus subtilis isolates were 26–50% effective. Isolates ofT. inhamatum, C. oxysporum andG. roseum performed best againstB. cinerea among isolates evaluated in leaf residues naturally infested with the pathogen and indigenous microorganisms. Totals of ten isolates of filamentous fungi (includingC. oxysporum andC. cladosporioides), two of yeasts and five ofBacillus subtilis completely prevented lesion production byB. cinerea in detached petals, and a further six isolates of filamentous fungi (includingG. roseum) and six yeasts were 90–99% effective. Isolates ofC. oxysporum, C. cladosporioides andB. subtilis, the most effective microorganisms againstB. cinerea in flower buds, reduced number of lesions in the range of 42–65% compared with 59–89% for a standard fungicide (vinclozolin). It is suggested that application of leading antagonists Jo living rose leaves and flowers should optimize control of inoculum production byB. cinerea when the tissues die. Optimal biocontrol of lesion production in flower buds requires a better understanding of the microenvironment of petals.

Catalase A fromBotrytis cinereais not expressed during infection on tomato leaves

Catalase mediates the enzymatic breakdown of hydrogen peroxide to water and molecular oxygen. During the infection of broad bean (Vicia faba) byBotrytis cinereathe release of hydrogen peroxide by fungal oxidase activity was proposed to facilitate penetration by the pathogen. Catalase activity might play a role in protecting the fungus against the damaging effects of hydrogen peroxide.A cDNA clone encoding catalase was isolated from a library ofBotrytis cinerea. Southern blot analysis of genomic DNA indicated the presence of a single copy gene, denoted ascatA. The cDNA clone encoded a protein, CAT-A, of 480 amino acids showing 56–65% similarity to fungal catalases. Detailed analysis of sequence homologies between other fungal catalases enabled grouping of catalases according to their cellular location. CAT-A ofB. cinerearesembled most closely the peroxisomal catalases. Northern blot analysis showed induction of the gene by H2O2in vitro. In planta, nocatAexpression could be detected by northern blot analysis, whereas a constitutively expressed β-tubulin mRNA ofB. cinereawas detectable and symptom development on the inoculated leaves was very clear. However, a catalase genecat1of tomato appeared to be induced from the time of fungal penetration onwards, suggesting that H2O2is produced during the interaction.

Mechanisms involved in the biological control ofBotrytis cinerea incited diseases

Mechanisms involved in the biological suppression of infection and inoculum potential ofBotrytis cinerea are numerous and variable and the involvement of two or more mechanisms has been demonstrated in several systems. Reported combinations include antibiosis with enzyme degradation ofB. cinerea cell walls; competition for nutrients followed by interference with pathogenicity enzymes of the pathogen or with induced resistance; and alteration of plant surface wettability combined with antibiosis. Since germinatingB. cinerea conidia are dependent on the presence of nutrients, competition for nutrients is regarded as important in systems where biocontrol is involved. Conidial viability and germination capacity are also potentially affected by the presence of antibiotics produced by biocontrol agents and present in the phyllosphere. Slower in action are mechanisms involving induced resistance in the host plant and production of hydrolytic enzymes that degradeB. cinerea cell walls. The latter has been demonstrated much more convincinglyin vitro than in the phyllosphere. Biocontrol in established lesions and reduction of sporulation on necrotic plant tissues is a means to minimize the pathogen inoculum.

Chitinase Activity ofXenorhabdusandPhotorhabdusSpecies, Bacterial Associates of Entomopathogenic Nematodes

Xenorhabdus nematophilus(three strains),Xenorhabdus bovienii(one strain), andPhotorhabdus luminescens(one strain) showed both exo- and endochitinase activity usingp-nitrophenyl-N-acetyl-β-d-glucosaminide andp-nitrophenyl-β-d-N,N′,N″- triacetylchitotriose, respectively, as substrates. One to three bands were detected on PAGE gel with glycol chitin after electrophoresis. Variation in exo- and endochitinase activity among different species and strains was detected with the strongest activity inX. nematophilusand the weakest inP. luminescens.The primary form ofX. bovieniihad significantly greater chitinase activity than the secondary form, whereas their growth rate and total protein released into culture medium were similar. The partially purified chitinase ofX. bovieniishowed significant antimycotic activity against conidial germination and germ tube elongation ofBotrytis cinerea.

Production of laccase byBotrytis cinerea and fermentation studies with strain F226

After induction, seven strains ofBotrytis cinerea released into the culture broth considerable amounts of laccase in a brief production time. The set-up of a suitable production process was studied with a selected strain in a 10-L fermenter. The optimum fermentation conditions were a 3% inoculum with a high degree of sporulation, a simple medium containing 20 g L−1 of glucose and 2 g L−1 of yeast extract at pH 3.5, 2 g L−1 gallic acid as inducer, added after 2 days of growth, an agitation speed of 300 rpm, an aeration rate of 1.2 vvm and a temperature of 24°C. By optimizing the culture conditions, the enzyme activity reached 28 U ml−1 in 5 days with a specific activity of 560 U mg−1 protein. The best procedure to obtain a suitable crude enzyme preparation was concentration of the supernatant medium to 10% of the initial volume by ultrafiltration, followed by a fractional precipitation with ethanol. The optimum pH and temperature for laccase activity were 5.5 and 40°C, respectively, with syringaldazine as the substrate.

Lipid Peroxidation and Membrane Disruption by Vinclozolin in Dicarboximide-Susceptible and -Resistant Isolates ofBotrytis cinerea

A dicarboximide-susceptible (DS) isolate and a dicarboximide-resistant (DR) isolate ofBotrytis cinereawere compared with regard to spore germination, mycelial growth, cellular leakage, and lipid peroxidation upon treatment with the dicarboximide fungicide vinclozolin. The fungicide inhibited spore germination and mycelial growth of the DS isolate, but not those of the DR isolate. The inhibitory effect of the fungicide was greater on mycelial growth than on spore germination of the DS isolate. Significant cellular leakage from the fungicide-treated DS or DR isolate began to increase after 24 hr incubation, depending on the concentration of the fungicide and the duration of incubation time. However, the magnitude of cellular leakage was much greater from the DS isolate than from the DR isolate. Vinclozolin caused considerable lipid peroxidation in the DS isolate, whereas little or no lipid peroxidation occurred in the DR isolate treated with the fungicide. Lipid peroxidation preceded cellular leakage from the DS isolate following fungicide treatment. The effects of the fungicide on mycelial growth, cellular leakage, and lipid peroxidation of the DS isolate were all reversed by the addition of α-tocopherol to the incubation medium. These results demonstrate that vinclozolin causes significant lipid peroxidation and subsequent cellular leakage from a DS isolate, but not from a DR isolate. Furthermore, they suggest that the mechanism of action of dicarboximide fungicides is associated with membrane lipid peroxidation.

Relations between glasshouse climate and dry weight of petals, epicuticular wax, cuticle, pre-harvest flowering period and susceptibility toBotrytis cinerea of gerbera and rose flowers

Studies were conducted on the effects of seasonal levels of relative humidity, temperature, and total radiation, on dry weight of petals, on fresh weight of epicuticular wax and of cuticle of petals, on numbers ofBotrytis cinerea lesions in petals, and on preharvest flowering periods in gerbera and rose. No temporal relationships or significant correlations were found among dry weight of petals, weight of wax and cuticle of petals, and numbers of lesions on the petals. Temperature, relative humidity and total radiation did not correlate significantly with dry weight of petals, or with fresh weights of wax and cuticle of petals, except for a positive correlation between relative humidity and cuticle weight in the gerbera cultivar Delphi. No relation was found between weight of epicuticular wax and cuticle of petals and susceptibility of gerbera and rose petals toB. cinerea. The thickness of wax and cuticle on flowers did not seem to be an important factor influencing the susceptibility of flowers toB. cinerea. The seasonal pattern in number of lesions produced on the flowers byB. cinerea was related to the effects of relative humidity and radiation on infectivity of conidia of the pathogen on the flower surface but not to the effects on the susceptibility of flowers.

Sensitivity of populations ofBotrytis cinerea to triazoles, benomyl and vinclozolin

Sensitivity of field isolates (121) ofBotrytis cinerea from France (1992), Germany (1979–1992), Israel (1990) and the Netherlands (1970–1989) to the triazoles tebuconazole and triadimenol, the benzimidazole benomyl and the dicarboximide vinclozolin were tested in radial growth experiments. Resistance to benomyl (in 21 to 100% of isolates tested) and vinclozolin (in 25 to 71% of isolates tested) was common in most countries. EC50s (concentrations of fungicides inhibiting radial mycelial growth ofB. cinerea on B5-agar by 50%) for tebuconazole and triadimenol ranged between 0.01–1.64 and 0.4–32.6Μg ml−1, respectively, and were log-normally distributed. The variation factor (ratio between EC50s of the least and most sensitive isolate tested) amounts 164 and 82 for tebuconazole and triadimenol, respectively. These values are comparable to those for azole fungicides applied in control of other pathogens. Hence, variation in sensitivity to triazoles can probably not explain limited field performance of triazoles towardsB. cinerea. Isolates from south west Germany (1992) were significantly less sensitive to tebuconazole than isolates collected earlier in Germany, Israel and the Netherlands. Such less sensitive populations may contribute to the limited field performance of DMI fungicides towardsB. cinerea. The sensitivity of isolates from south west Germany to tebuconazole was similar to that of DMI-resistant mutants generated in the laboratory. These mutants displayed stable resistance with Q-values (ratio between EC50 of resistant mutant and wild type isolate) between 5 and 20. Sensitivity of field isolates and laboratory mutants to tebuconazole and triadimenol was correlated.

Interactions of air temperature, relative humidity and biological control agents on grey mold of bean

The interactions ofBotrytis cinerea and seven biological control agents (BCAs) were examined in controlled environments to determine the influence of selected relative humidities (RH) (90,95, and 100%) and air temperatures (20,24 and 28 ‡C) on grey mold of bean. All main effects and interactions were significant (P≤0.05) among the 72 treatments. In the control, lesions of grey mold developed under all environmental conditions but were largest at 24 ‡C×95 and 100% RH, and 28 ‡C×95% RH. Interactions of environment, BCAs and grey mold were complex.Alternaria alternata, Drechslera sp.,Myrothecium verrucaria, Trichoderma viride, Gliocladium roseum and an unidentified pink yeast were all highly dependent on environment for biological control efficacy, and changes of 4 ‡C or 5% RH were associated with variability in disease suppression that ranged from ≤ 15 to 100%. Efficacy ofEpicoccum purpurascens appeared independent of environment and this BCA suppressed disease by 100% in all of the environmental treatments. Suppression of grey mold by many of the BCAs was most effective under environmental conditions least conducive to disease. Therefore, evaluations of potential BCAs in environmental conditions that are marginal for disease can overestimate their efficacy in field environments. Assessments of biological control efficacy in various environments can be used to more accurately assess the potential of BCAs.