Fungal Pathogen Botrytis Cinerea Research Articles

Interplay of the Arabidopsis nonhost resistance gene NHO1 with bacterial virulence.

It is poorly understood why a particular plant species is resistant to the vast majority of potential pathogens that infect other plant species, a phenomenon referred to as "nonhost" resistance. Here, we show that Arabidopsis NHO1, encoding a glycerol kinase, is required for resistance to and induced by Pseudomonas syringae isolates from bean and tobacco. NHO1 is also required for resistance to the fungal pathogen Botrytis cinerea, indicating that NHO1 is not limited to bacterial resistance. Strikingly, P. s. pv. tomato DC3000, an isolate fully virulent on Arabidopsis, actively suppressed the NHO1 expression. This suppression is abolished in coi1 plants, indicating that DC3000 required an intact jasmonic acid signaling pathway in the plant to suppress NHO1 expression. Constitutive overexpression of NHO1 led to enhanced resistance to this otherwise virulent bacterium. The presence of avrB in DC3000, which activates a cultivar-specific "gene-for-gene" resistance in Arabidopsis, restored the induction of NHO1 expression. Thus, NHO1 is deployed for both general and specific resistance in Arabidopsis and targeted by the bacterium for parasitism.

Five components of the ethylene-response pathway identified in a screen for weak ethylene-insensitive mutants in Arabidopsis.

Five ethylene-insensitive loci (wei1-wei5) were identified by using a low-dose screen for "weak" ethylene-insensitive mutants. wei1, wei2, and wei3 seedlings showed hormone insensitivity only in roots, whereas wei4 and wei5 displayed insensitivity in both roots and hypocotyls. The genes corresponding to wei1, wei4, and wei5 were isolated using a positional cloning approach. The wei1 mutant harbored a recessive mutation in TIR1, which encodes a component of the SCF protein ubiquitin ligase involved in the auxin response. wei4, a dominant mutant, resulted from a mutation in the ethylene receptor ERS, whereas wei5, a semidominant mutant, was caused by a mutation in the EIN3-related transcription factor gene EIL1. The simultaneous loss of functional WEI5EIL1 and EIN3 nearly completely abolished the ethylene response in etiolated seedlings, and adult plants were highly susceptible to infection by the necrotrophic fungal pathogen Botrytis cinerea. Moreover, wei5eil1 ein3 double mutants were able to fully suppress constitutive signaling caused by ctr1, suggesting a synergistic interaction among these gene products. Unlike previously known root ethylene-insensitive mutants, wei2 and wei3 were not affected in their response to auxin and showed a normal response to gravity. Genetic mapping studies indicate that wei2 and wei3 correspond to previously unidentified ethylene pathway genes that may control cell-elongation processes functioning at the intersection of the ethylene and auxin response pathways.

Differential regulation by ambient pH of putative virulence factor secretion by the phytopathogenic fungus Botrytis cinerea

The fungal pathogen Botrytis cinerea is capable of developing on a wide variety of host plants that differ greatly in their pH values and biochemical defences. To evaluate whether the pH of the host tissue can regulate the production of pathogenicity factors by this fungus, we examined the ability of two isolates of B. cinerea that originated from different plant species to secrete putative virulence elements on synthetic media buffered at pH 2.0 to pH 7.0. Even though differing in the intensity of their responses, both isolates reacted similarly to their ambient pH. The production of extracellular polysaccharides and oxalic acid was detectable above pH 4.0 and pH 5.0 respectively. Conversely, the production of aspartic acid proteases could only be seen between pH 3.0 and 4.0. Finally, the secretion of polygalacturonase and laccase activity was found to exhibit two maxima, one around pH 3.1 and one around pH 6.0. Thus, pathogenicity factor production was found to be minimal between pH 4.5 and 5.5 and a different set of factors was produced at pH 3.1 and 6.0, two values that were found to correspond respectively to the average host fruit and leaf pH. These results demonstrate that ambient pH differentially regulates the synthesis of pathogenicity factors by Botrytis and may act as a novel regulatory element to assist this fungus in tuning its virulence machinery to the composition of its host tissue.

Detoxification of terpinolene by plant pathogenic fungus Botrytis cinerea.

Detoxification of an antifungal monoterpene terpinolene (1) by the plant pathogenic fungus Botrytis cinerea afforded hydroxlyated metabolites 2,3-dihydro-3beta,6beta-dihydroxy-terpinolene (2) (39%) and 2,3-dihydro-1alpha,3alpha-dihydroxy-terpinolene (3) (20%), respectively. Terpinolene showed good levels of antifungal activity while both the metabolites were inactive against another plant pathogenic fungus Cladosporium herbarun.

The role of ethylene and wound signaling in resistance of tomato to Botrytis cinerea.

Ethylene, jasmonate, and salicylate play important roles in plant defense responses to pathogens. To investigate the contributions of these compounds in resistance of tomato (Lycopersicon esculentum) to the fungal pathogen Botrytis cinerea, three types of experiments were conducted: (a) quantitative disease assays with plants pretreated with ethylene, inhibitors of ethylene perception, or salicylate; (b) quantitative disease assays with mutants or transgenes affected in the production of or the response to either ethylene or jasmonate; and (c) expression analysis of defense-related genes before and after inoculation of plants with B. cinerea. Plants pretreated with ethylene showed a decreased susceptibility toward B. cinerea, whereas pretreatment with 1-methylcyclopropene, an inhibitor of ethylene perception, resulted in increased susceptibility. Ethylene pretreatment induced expression of several pathogenesis-related protein genes before B. cinerea infection. Proteinase inhibitor I expression was repressed by ethylene and induced by 1-methylcyclopropene. Ethylene also induced resistance in the mutant Never ripe. RNA analysis showed that Never ripe retained some ethylene sensitivity. The mutant Epinastic, constitutively activated in a subset of ethylene responses, and a transgenic line producing negligible ethylene were also tested. The results confirmed that ethylene responses are important for resistance of tomato to B. cinerea. The mutant Defenseless, impaired in jasmonate biosynthesis, showed increased susceptibility to B. cinerea. A transgenic line with reduced prosystemin expression showed similar susceptibility as Defenseless, whereas a prosystemin-overexpressing transgene was highly resistant. Ethylene and wound signaling acted independently on resistance. Salicylate and ethylene acted synergistically on defense gene expression, but antagonistically on resistance.

Functional analysis of an extracellular catalase of Botrytis cinerea

Summary There is evidence that the necrotrophic fungal pathogen Botrytis cinerea is exposed to oxidative processes within plant tissues. The pathogen itself also generates active oxygen species and H(2)O(2) as pathogenicity factors. Our aim was to study how the pathogen may defend itself against cellular damage caused by the accumulation of H(2)O(2) and the role of an extracellular catalase in its detoxification during the infection of tomato and bean plants by B. cinerea. Chloronaphthol staining followed by light microscopy showed that H(2)O(2) accumulates in the infection zone in tomato and bean leaves. An extracellular catalase gene (denominated Bccat2) was cloned from B. cinerea. Exposure of mycelium to H(2)O(2) in liquid culture resulted in increased Bccat2 mRNA levels in a concentration-dependent manner. Bccat2 mRNA was detected at early stages of tomato leaf infection, suggesting that B. cinerea experiences oxidative stress. Bccat2-deficient mutants were generated by transformation-mediated gene disruption. Mutants were more sensitive then the wild-type strain to H(2)O(2)in vitro, but they partly compensated for the absence of BcCAT2 by activating other protective mechanisms in the presence of H(2)O(2). Bccat2-deficient mutants did not display a consistent reduction of virulence on bean and tomato leaves. Cerium chloride staining of infected leaf tissue for ultrastructural studies showed that Bccat2-deficient mutants were exposed to H(2)O(2) comparably to the wild-type. The results suggest that B. cinerea is a robust pathogen adapted to growing in hostile oxidizing environments in host tissues.

Competitive substrate colonisation by Botrytis cinerea and Ulocladium atrum in relation to biological control of B. cinerea in cyclamen

The dynamics of competitive colonisation of necrotic cyclamen tissue by the plant pathogenic fungus Botrytis cinerea and the saprophytic fungal antagonist Ulocladium atrum were studied immuno-histologically, while sporulation was studied macroscopically. The effect of different time intervals between inoculation of both fungi on resource capture by each species was explored. Colonisation and sporulation were used as indicators for competitive resource capture and the effectiveness of biological control of B. cinerea, using U. atrum. Mycelial biomass and sporulation showed logistical time courses in both species, in monocultures as well as in mixed cultures. Final colonisation and sporulation levels were lowered by competition, indicating competitive resource capture. Analysis of the extent to which sporulation of either fungus could be reduced by co-inoculation with the other fungus at different times, showed that B. cinerea can be completely excluded by ‘early’ pre-inoculation with U. atrum, but not vice versa, indicating that U. atrum can exploit resources in the leaf that are not accessible to B. cinerea. A model of competitive substrate colonisation and resource capture was developed on the basis of the experimental results. Model results confirm that competition for resources provides a sufficient biological explanation for the dynamic interactions between the fungi. The model provides a tool to optimise dose and timing of U. atrum applications providing effective biological control of B. cinerea.

Ethylene biosynthesis in Botrytis cinerea

Ethylene is often released during plant pathogenesis. Enhanced ethylene biosynthesis by the attacked plant, and formation of ethylene by the attacking pathogen may be involved. We defined the biosynthetic pathway of ethylene in the pathogenic fungus Botrytis cinerea, and characterized the conditions that affect ethylene production in vitro. During the first 48 h of culture the fungus uses methionine to produce alpha-keto gamma-methylthiobutyric acid (KMBA) and secretes it to the medium. In darkness, KMBA accumulates in the medium. In light KMBA is photo-oxidized and ethylene is released. The photo-oxidation reaction is spontaneous and does not involve any enzymatic activity. Low levels of ethylene are produced in darkness between 48 and 96 h of culture. Adding peroxidase to dark-grown cultures induced ethylene formation. The results suggest that formation and secretion of KMBA by B. cinerea may affect ethylene levels during plant infection.

Ethylene biosynthesis in Botrytis cinerea

Ethylene is often released during plant pathogenesis. Enhanced ethylene biosynthesis by the attacked plant, and formation of ethylene by the attacking pathogen may be involved. We defined the biosynthetic pathway of ethylene in the pathogenic fungus Botrytis cinerea, and characterized the conditions that affect ethylene production in vitro. During the first 48 h of culture the fungus uses methionine to produce α-keto γ-methylthiobutyric acid (KMBA) and secretes it to the medium. In darkness, KMBA accumulates in the medium. In light KMBA is photo-oxidized and ethylene is released. The photo-oxidation reaction is spontaneous and does not involve any enzymatic activity. Low levels of ethylene are produced in darkness between 48 and 96 h of culture. Adding peroxidase to dark-grown cultures induced ethylene formation. The results suggest that formation and secretion of KMBA by B. cinerea may affect ethylene levels during plant infection.

Effects of procymidone, fludioxonil and pyrimethanil on two non-target aquatic plants

Procymidone, fludioxonil, and pyrimethanil are widely used to control the pathogenic fungus Botrytis cinerea in Champagne's vineyards. These fungicides may end up in surface waters and present potential risks for aquatic vascular plants and algae. Therefore, their toxicity was evaluated on Lemna minor and Scenedesmus acutus in six-day or 48-h tests, respectively. Based on growth and chlorophyll (Chl) content of L. minor and S. acutus cultures, the results showed that the alga was the most sensitive to the fungicides. Among the fungicides, pyrimethanil was the most toxic for L. minor, its nominal IC 50 was 46.16 mg l −1 and that of the other two was >100 mg l −1. In contrast, pyrimethanil appeared the least toxic for S. acutus at low concentration, nominal IC 50 were 22.81, 4.85, and 4.55 mg l −1 for pyrimethanil, fludioxonil, and procymidone, respectively. Fate of the fungicides in the media was also investigated and acute toxicity of the agrochemicals is discussed in regard to concentration in the culture media. Poor solubility of procymidone and fludioxonil appeared to be partly responsible for the low toxicity of these fungicides. Based on these toxicity data and the concentrations found in ponds collecting vineyard runoff water, these pesticides should not impair the establishment of pioneer plants.

Markers for oxidative stress associated with soft rots in French beans (Phaseolus vulgaris) infected by Botrytis cinerea.

The role of active oxygen species has been studied in spreading soft-rot lesions caused by the necrotrophic fungal pathogen Botrytis cinerea Pers.:Fr. in leaves of four genotypes of French bean (Phaseolus vulgaris L.). Large increases were observed for the aldehydic end-products of oxidative damage, malondialdehyde and 4-hydroxy-2-nonenal, as a result of infection in each of the genotypes studied. Similar increases were found in a stable free radical and g=4.27 Fe(III) signals, but not Mn(II) signals, in electron paramagnetic resonance spectra. These changes were accompanied by large decreases in ascorbic acid levels, with changes in the antioxidant glutathione being genotype dependent.

Biotransformation of two cytotoxic terpenes, alpha-santonin and sclareol by Botrytis cinerea.

Two cytotoxic terpenes, alpha-santonin (1) and sclareol (3) were biotransformed by a plant pathogenic fungus Botrytis cinerea to produce oxidized metabolites in high yields. Alpha-Santonin (1) on fermentation with the fungus for ten days afforded a hydroxylated metabolite identified as 11beta-hydroxy-alpha-santonin (2) in a high yield (83%), while sclareol (3) was metabolized to epoxysclareol (4) (64%) and a new compound 8-deoxy-14,15-dihydro-15-chloro-14-hydroxy-8,9-dehydrosclareol (5) (7%), representing a rare example of microbial halogenation.

Oxidative metabolism of ambrox and sclareolide by Botrytis cinerea.

Ambrox (1), a perfumery diterpene, was oxidatively metabolised by a plant pathogenic fungus Botrytis cinerea in a xenobiotic fashion to afford a major product, i.e., 1beta-hydroxy-8-epiambrox (13) (60%) along with three minor metabolites 3beta-hydroxyambrox (2), sclareolide (5) and 3beta-hydroxysclareolide (7). Sclareolide (5), a cytotoxic diterpenoidal lactone was fermented with the same fungus to yield 3beta-hydroxysclareolide (7) (59%) as a major metabolite together with two minor metabolites characterised as 1-ketosclareolide (15), and 3beta,14-dihydroxysclareolide (16).

Structure and expression in planta of Botrytis cinerea ubiquitin genes

To identify genes of the necrotrophic pathogenic fungus Botrytis cinerea that are expressed during infection of tomato leaves, a differential screening of a genomic library with radioactively labelled cDNA was performed. This resulted in the identification of a B. cinerea gene, denominated Bcubi4, which encodes a precursor protein consisting of four identical head-to-tail repeats of a 76 aa ubiquitin unit. Subsequently a gene denominated Bcubi1CEP79, encoding a single ubiquitin unit joined to a Carboxyl Extension Protein of 79 amino acids, was isolated. The expression of the two ubiquitin genes was studied during pathogenesis of B. cinerea on tomato. Bcubi1CEP79, but not Bcubi4, mRNA was transiently induced at 16 h after inoculation. The increased expression of the Bcubi1CEP79 gene at this stage of pathogenesis might be required for enhanced ribosomal biogenesis.

Dimerization of resveratrol by the grapevine pathogen Botrytis cinerea.

Resveratrol (trans-3,4',5-trihydroxystilbene) is produced by grapes (Vitis spp.) in response to microbial attack by the fungal grapevine pathogen Botrytis cinerea. Several reports indicate that pathogenic B.cinerea strains are capable of biotransforming resveratrol into an assortment of unidentified oxidized metabolites as a means of reducing the antifungal effects of resveratrol and facilitating Botrytis invasion into host-plant tissues. Studies utilizing growing incubations of Botrytis cinerea ATCC 11542 with resveratrol resulted in the production of three new (restrytisols A-C) (1-3) and three known (resveratrol trans-dehydrodimer, leachinol F, and pallidol) oxidized resveratrol dimers. All of the metabolites were evaluated for their anti-HIV-1, cytotoxic, and cyclooxygenase (COX) I and COX II inhibitory activities.

Chloride and ethyl ester morpholine thiourea derivatives and their Ni(II) complexes. Crystal and molecular structures of the thiourea derivative L-leucine methyl ester and its complexes with Cu(II) and Pt(II). Growth of the pathogenic fungus Botrytis cinerea

We have synthesized a series of ligands ( 1, 3, 4, 6 and 7) and some of their complexes with Ni (II), Cu (II) and Pt (II) ( 2, 5, 8 and 9). These compounds were studied and characterized by elemental analysis, IR and UV–Vis spectra, conductivity measurements in solution, FAB +/MS, 1H and 13C NMR, ESR, etc. Compound 7 crystallized in the orthorhombic space group P2 12 12 1, with Z=4. Unit cell parameters were as follows: a=21.307(2) Å, α=90°, b=12.498(1) Å, β=90°, c=7.7232(4) Å, γ=90°. For seven of these compounds, the antifungal activity of a major pathogen responsible for important crop damage was studied. In general, inhibition by the ligands was higher than that of the complexes. When the thiourea was linked to some diethyl groups, the compounds showed higher antifungal activity than the morpholine groups. Compound 3 achieved total inhibition (100%).

Nitrogen availability and susceptibility of tomato leaves to Botrytis cinerea

The aim of this study was to investigate the effect of nitrogen availability on susceptibility of tomato leaves to the fungal pathogen Botrytis cinerea. Plants with varying nitrogen availability were grown by adding N daily in exponentially increasing amounts to a nutrient solution at different rates. Leaves of plants grown at low nitrogen availability had a high leaf C/N ratio (21 g g-1) and were about 2.5 times more susceptible to primary lesion formation by B. cinerea compared to plant grown at high nitrogen availability, which had a low leaf C/N ratio (11 g g-1). Leaf C/N ratio accounted for 95% of variation in susceptibility. This relationship between C/N ratio and susceptibility persisted when plants were grown with exponential P addition and optimal N supply, and was thus independent of plant growth rate or related factors. We could not explain the effect of nitrogen availability by variation in the most obvious N-based resistance compound α-tomatine because more susceptible leaves with a high C/N ratio contained more α-tomatine. These leaves also contained more soluble carbohydrates. The level of soluble carbohydrates correlated positively with susceptibility, independent of the growth method. We therefore suggest that the effect of N availability on susceptibility must be explained by variation in levels of soluble carbohydrates and speculate about the role of these carbohydrates in the infection process.

Production et propriétés du complexe cellulasique du Botrytis cinerea

The production of cellulasic complex by the fungal pathogen Botrytis cinerea is induced by addition of cellulose in the culture medium. The amount of activity increases greatly when the culture medium is supplemented with pectins, whereas glucose and cellobiose strongly inhibit the production of the complex. The cellulasic complex is constituted by three enzymes, separated and identified as β-glucosidase, exo 1,4-β-D-glucanase and endo 1,4-β-D-glucanase. The catalytic activity of the whole cellulasic complex is low and the inhibitors glucose and cellobiose act differently on this activity. Keywords: fungal pathogen, Botrytis cinerea, cellulasic complex, β-glucosidase, 1,4-β-D-glucanases.

High field NMR microscopic imaging of cultivated strawberry fruit.

The experimental conditions required for discrimination of various types of tissue in fruits of cultivated strawberry (Fragaria x Ananassa) at high fields (ca. 7 T) have been investigated. In marked contrast to soft fruits of other species, from which informative images have been derived at high fields using a variety of pulse sequences and acquisition parameters, appreciable image intensities from parenchymal and vascular tissues in healthy strawberry fruits were obtained only with a spin-echo imaging sequence using large sweep widths (ca. 100,000 Hz), and consequently small values for TE ( < 5 ms), indicating predominantly short T2 values for these tissues. Damage caused by infection by the fungal pathogen Botrytis cinerea is readily seen as a result of a large increase in T2 in the infected tissue, whereas ripening processes appear to be characterized primarily by small variations in the T2-weighted contrast and in the relative magnitudes of T1 between vascular and parenchymal tissue. In addition, it was possible selectively to enhance the contributions to images from the achenes ("seeds") by using very short relaxation delays, thereby enhancing T1-dominated contrast mechanisms.

Suppression of botrytis blight in rose flowers with gibberellic acid. Possible formation of endogenous inhibitory compounds

Summary Elongation of conidial germ tubes as well as linear growth of colonies of the fungal pathogen Botrytis cinerea were inhibited when growth media in vitro were supplemented with leachates from GA 3 -treated petals of rose ( Rosa × hybrida cv. Mercedes) flowers. Content of phenolic substances extracted from petals 6 days after the GA 3 treatment was higher than that in the control petals whereas no differences in content of phenols were found between the GA 3 -treated and the control petals examined 8 days after the GA 3 treatment (9 days after harvest). Spore germination, germ tube elongation and mycelium growth were also inhibited when growth media in vitro were supplemented with phenolic extracts from petals treated with GA 3 6 days prior to extraction but not when they were extracted 8 days after the GA 3 treatment. Botrytis blight development in vivo was almost completely inhibited by phenolics extracted 6 days after GA 3 treatment and was also practically inhibited by extracts from older petals. The content of sugars and amino acids in leachates from GA 3 -treated petals was lower than that in leachates from control petals. The inhibition of germ tube elongation in leachates from GA 3 -treated petals was eliminated when the growth media were supplemented with excessive amounts of glucose. The inhibition of Botrytis blight development in GA 3 -treated flowers in vivo was also eliminated when conidial suspensions were supplemented with glucose. The possible GA 3 -imposed formation of inhibitory compounds with a phenolic moiety is discussed.