Secondary Metabolites of the Grapevine Pathogen <Emphasis Type="Italic">Eutypa lata</Emphasis> Inhibit Mitochondrial Respiration,Based on a Model Bioassay Using the Yeast <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis>

Acetylenic phenols and a chromene isolated from the grapevine fungal pathogen Eutypa lata were examined for mode of toxicity. The compounds included eutypine (4-hydroxy-3-[3-methyl-3-butene-1-ynyl] benzyl aldehyde), eutypinol (4-hydroxy-3-[3-methyl-3-butene-1-ynyl] benzyl alcohol), eulatachromene, 2-isoprenyl-5-formyl-benzofuran, siccayne, and eulatinol. A bioassay using the yeast Saccharomyces cerevisiae showed that all compounds were either lethal or inhibited growth. A respiratory assay using 2,3,5-triphenyltetrazolium (TTC) indicated that eutypinol and eulatachromene inhibited mitochondrial respiration in wild-type yeast. Bioassays also showed that 2-isoprenyl-5-formyl-benzofuran and siccayne inhibited mitochondrial respiration in the S. cerevisiae deletion mutant vph2, lacking a vacuolar type H (+) ATPase (V-ATPase) assembly protein. Cell growth of tsa1, a deletion mutant of S. cerevisiae lacking a thioredoxin peroxidase (cTPx I), was greatly reduced when grown on media containing eutypinol or eulatachromene and exposed to hydrogen peroxide (H₂O₂) as an oxidative stress. This reduction in growth establishes the toxic mode of action of these compounds through inhibition of mitochondrial respiration.     

A novel NADPH-dependent aldehyde reductase gene from Vigna radiata confers resistance to the grapevine fungal toxin eutypine

Eutypine, 4-hydroxy-3-(3-methyl-3-butene-1-ynyl) benzyl aldehyde, is a toxin produced by Eutypa lata, the causal agent of eutypa dieback of grapevines. It has previously been demonstrated that tolerance of some cultivars to this disease was correlated with their capacity to convert eutypine to the corresponding alcohol, eutypinol, which lacks phytotoxicity. We have thus purified to homogeneity a protein from Vigna radiata that exhibited eutypine-reducing activity and have isolated the corresponding cDNA. This encodes an NADPH-dependent reductase of 36 kDa that we have named Vigna radiata eutypine-reducing enzyme (VR-ERE), based on the capacity of a recombinant form of the protein to reduce eutypine into eutypinol. The strongest homologies (86.8%) of VR-ERE at the amino acid level were found with CPRD14, a drought-inducible gene of unknown function, isolated from Vigna unguiculata and with an aromatic alcohol dehydrogenase (71.7%) from Eucalyptus gunnii . Biochemical characterization of VR-ERE revealed that a variety of compounds containing an aldehyde group can act as substrates. However, the highest affinity was observed with 3-substituted benzaldehydes. Expression of a VR-ERE transgene in Vitis vinifera cells cultured in vitro conferred resistance to the toxin. This discovery opens up new biotechnological approaches for the generation of grapevines resistant to eutypa dieback.     

Control of anthocyanin biosynthesis pathway gene expression by eutypine,a toxin from Eutypa lata,in grape cell tissue cultures

Eutypine, 4-hydroxy-3-(3-methyl-3-butene-1-ynyl) benzaldehyde, is a toxin produced by Eutypa lata, the causal agent of Eutypa dieback in grapevine. The effect of the toxin on anthocyanin synthesis has been investigated in Vitis vinifera cv. Gamay cell cultures. At concentrations higher than 200 micromol/L, eutypine reduced anthocyanin accumulation in cells. The reduction in anthocyanin accumulation was proportional to the eutypine concentrations and HPLC analysis showed that eutypine affected the levels of all anthocyanins. The effect of eutypine application on the expression of five genes of the anthocyanin biosynthesis pathway, including chalcone synthase (CHS), flavonone-3-hydroxylase (F3H), dihydroflavonol 4-reductase (DFR), leucoanthocyanidin dioxygenase (LDOX), and UDP glucose-flavonoid 3-O-glucosyl transferase (UFGT) was determined. Expression of CHS, F3H, DFR and LDOXwas not affected by the addition of eutypine to grapevine cell cultures. In contrast, expression of the UFGT gene was dramatically inhibited by the toxin. These results suggest that in grapevine cell cultures, eutypine strongly affects anthocyanin accumulation by inhibiting UFGT gene expression. The mechanism of action of eutypine is discussed.     

Purification and Characterization of a NADPH-Dependent Aldehyde Reductase from Mung Bean That Detoxifies Eutypine, a Toxin from Eutypa lata

Eutypine (4-hydroxy-3-[3-methyl-3-butene-1-ynyl] benzaldehyde) is a toxin produced by Eutypa lata, the causal agent of eutypa dieback in the grapevine (Vitis vinifera). Eutypine is enzymatically converted by numerous plant tissues into eutypinol (4-hydroxy-3-[3-methyl-3-butene-1-ynyl] benzyl alcohol), a metabolite that is nontoxic to grapevine. We report a four-step procedure for the purification to apparent electrophoretic homogeneity of a eutypine-reducing enzyme (ERE) from etiolated mung bean (Vigna radiata) hypocotyls. The purified protein is a monomer of 36 kD, uses NADPH as a cofactor, and exhibits a K_m value of 6.3 μm for eutypine and a high affinity for 3- and 4-nitro-benzaldehyde. The enzyme failed to catalyze the reverse reaction using eutypinol as a substrate. ERE detoxifies eutypine efficiently over a pH range from 6.2 to 7.5. These data strongly suggest that ERE is an aldehyde reductase that could probably be classified into the aldo-keto reductase superfamily. We discuss the possible role of this enzyme in eutypine detoxification.Many pathogenic bacteria and fungi produce toxins that interfere with various functions of plant cells and may affect plant defense mechanisms (Durbin, 1981). Toxin production is commonly associated with disease severity and can be involved in colonization or systemic invasion by the pathogen (Schäfer, 1994). Toxin resistance has been shown in most cases to be based on the ability of the plant to metabolically detoxify pathogen toxins (Meeley and Walton, 1991; Zhang and Birch, 1997; Zweimuller et al., 1997). Few cloned toxin-resistance genes that encode proteins involved in detoxification mechanisms have been described (Utsumi et al., 1988; Johal and Briggs, 1992; Zhang and Birch, 1997). In many cases a relationship exists between toxin tolerance and resistance to the disease (Anzai et al., 1989; Meeley et al., 1992). The availability of toxin-resistance genes will permit a greater understanding of the mechanisms causing plant disease and will also set the stage for engineering resistance to plant disease (Keen, 1993).Eutypine (4-hydroxy-3-[3-methyl-3-butene-1-ynyl] benzaldehyde) is a toxin produced by the ascomycete fungus Eutypa lata (Pers.: Fr.) Tul., the causal agent of eutypa dieback (Tey-Rulh et al., 1991). This disease is responsible for considerable loss in yield and is the most devastating disease of grapevine (Vitis vinifera) in many countries (Moller and Kasamitis, 1981; Munkvold et al., 1994). The fungus infects the stock through pruning wounds and is present in the xylem and phloem of the vine trunk and branches (Moller and Kasamitis, 1978; Duthie et al., 1991). After a long incubation period, a canker forms around the infected wound. The toxin synthesized by the fungus in the trunk is believed to be transported by the sap to the herbaceous parts of the vine (Fallot et al., 1997). Eutypine penetrates grapevine cells through passive diffusion and its accumulation in the cytoplasm has been explained by an ion-trapping mechanism related to the ionization state of the molecule (Deswarte et al., 1996b). In the cell the effects of eutypine include reduction of adenylated nucleotide content, inhibition of succinate dehydrogenase, uncoupling of oxidative phosphorylation, and mitochondrial swelling (Deswarte et al., 1996a).Symptoms of eutypa dieback in the herbaceous part of the plant lead to dwarfed and withered new growth of branches, marginal necrosis of the leaves, dryness of the inflorescence, and, finally, death of one or more branches (Moller and Kasamitis, 1981). The toxin appears to be an important virulence factor involved in symptom development of the disease (Deswarte et al., 1996a). However, the absence of toxin-deficient mutants of the fungus and its long incubation period in the trunk before symptom development have prevented a critical study of the toxin in vine plants. Determining the gene responsible for eutypine resistance would therefore be an important critical tool in determining the role of eutypine toxin in symptom development in the disease; and it has the potential to confer resistance to transgenic grapevines.Recently, Colrat et al. (1998) found detoxification to occur in grapevine cells through the enzymatic reduction of eutypine into its corresponding alcohol, eutypinol (4-hydroxy-3-[3-methyl-3-butene-1-ynyl] benzyl alcohol). We have determined that this derivative of the toxin is nontoxic for grapevine tissues. Furthermore, we have established a relationship between the susceptibility of grapevine to eutypa dieback and the ability of tissues to inactivate eutypine, suggesting that the detoxification mechanism plays an important role in defense reactions. Eutypine is enzymatically detoxified in numerous plant species and, among them, we found that the tissues of mung bean (Vigna radiata), a nonhost plant for the pathogen, exhibit an efficient detoxification activity. As a prerequisite for demonstrating the involvement of eutypine toxin in eutypa dieback, we report here the purification to homogeneity and the characterization of an ERE from etiolated mung bean hypocotyls.     

PCR assays that identify the grapevine dieback fungus Eutypa lata

Eutypa lata is the causal fungal agent of Eutypa dieback, a serious grapevine necrotic disease. The erratic and delayed (1 to 2 months) appearance of characteristic conidia on culture media and the presence of numerous microorganisms in decaying wood make it difficult either to identify or to detect E. lata in grapevine wood samples. We designed six pairs of PCR primers for diagnosis of E. lata. Three primer pairs were derived from ribosomal DNA internal transcribed spacer sequences, and three pairs were derived from randomly amplified polymorphic DNA fragments. The six primer pairs could be used to amplify DNAs extracted from all of the E. lata isolates tested. They did not amplify DNAs from fungi and bacteria representing more than 50 different species of microorganisms associated with grapevine. We developed a simple protocol, leading to a rapid release of DNA, that enabled us to identify E. lata from pure or mixed cultures as well as from grapevine wood samples. Identification of E. lata in wood was achieved within a few hours, instead of the several weeks required for classical cultures on agar medium. We believe that the procedure described here can be adapted to detect other microorganisms involved in woody plant diseases.     

Extracellular compounds produced by fungi associated with Botryosphaeria dieback induce differential defence gene expression patterns and necrosis in Vitis vinifera cv. Chardonnay cells

Three major grapevine trunk diseases, esca, botryosphaeria dieback and eutypa dieback, pose important economic problems for vineyards worldwide, and currently, no efficient treatment is available to control these diseases. The different fungi associated with grapevine trunk diseases can be isolated in the necrotic wood, but not in the symptomatic leaves. Other factors seem to be responsible for the foliar symptoms and may represent the link between wood and foliar symptoms. One hypothesis is that the extracellular compounds produced by the fungi associated with grapevine trunk diseases are responsible for pathogenicity. In the present work, we used Vitis vinifera cv. Chardonnay cells to test the aggressiveness of total extracellular compounds produced by Diplodia seriata and Neofusicoccum parvum, two causal agents associated with botryosphaeria dieback. Additionally, the toxicity of purified mellein, a characteristic toxin present in the extracellular compounds of Botryosphaeriaceae, was assessed. Our results show that the total extracellular compounds produced by N. parvum induce more necrosis on Chardonnay calli and induce a different defence gene expression pattern than those of D. seriata. Mellein was produced by both fungi in amounts proportional to its aggressiveness. However, when purified mellein was added to the culture medium of calli, only a delayed necrosis and a lower-level expression of defence genes were observed. Extracellular compounds seem to be involved in the pathogenicity of the fungi associated with botryosphaeria dieback. However, the doses of mellein used in this study are 100 times higher than those found in the liquid fungal cultures: therefore, the possible function of this toxin is discussed.     

Investigations on the destruxin production of the entomopathogenic fungus Metarhizium anisopliae

The dynamics of cyclic peptide destruxins (dtxs) produced by Metarhizium anisopliae strains V245 and V275 were monitored both on solid and in liquid media. The results showed that both strains did not produce dtxs in large-scale fermenter cultures or solid Czapek Dox (CD) agar. Production of the major dtxs A and B could be determined in both strains when grown on rice for up to 10-30 days. The main dtxs A, B, E, and E diol were detected in CD liquid culture filtrate from both strains after three days post-inoculation on. Parallel decrease of dtx E and increase of E diol in the culture medium were found, indicating that the latter is the hydrolytic product from the former. Production of dtxs A and B was significantly positively correlated. A negative correlation was observed between the production of the metabolites and pH value of the medium. The influence of different nutrient sources on dtx production was evaluated by using media with different carbon and nitrogen ratios as well as with different insect homogenates. The findings showed that the amount of dtxs A, B, and E increased with the increasing content of peptone in the medium. When insect homogenate was used as single nutrient source or added to CD medium, no toxins were detected in the culture filtrate. The potential risk posed by the toxic metabolites during mass production is discussed.     

Enzymatic detoxification of eutypine, a toxin from Eutypa lata, by Vitis vinifera cells: partial purification of an NADPH-dependent aldehyde reductase

Eutypine, 4-hydroxy-3-(3-methyl-3-butene-1-ynyl) benzaldehyde, is a toxin produced by Eutypa lata (Pers.: Fr.) Tul., the causal agent of dying arm disease of Vitis vinifera L. (grapevine). Previously, we have shown that eutypine is involved in the development of disease symptoms. In the present study, the effects of V. vinifera cell-suspension cultures on the biological activity of the toxin were investigated. Eutypine was converted by grapevine tissues into a single compound, identified by mass spectrometry and nuclear magnetic resonance as 4-hydroxy-3-(3-methyl-3-butene-1-ynyl) benzyl alcohol, designated eutypinol. This compound was found to be non-toxic for grapevine tissues. Unlike eutypine, eutypinol failed to affect the oxidation rate or membrane potential of isolated mitochondria. In grapevine cells, reduction of eutypine into the corresponding alcohol is an NADPH-dependent enzymatic reaction. An enzyme which reduced eutypine was partially purified, over 1000-fold, using a five-step purification procedure. By gel filtration and sodium dodecyl sulfate-polyacrylamide gel electrophoresis, the protein was found to have a molecular mass of 54–56 kDa. The enzyme exhibited an apparent K _m for eutypine of 44 μM, and was active between pH 6.8 and 7.5 with a maximum at pH 7.0. The eutypine reductase activity was improved by Mn²⁺ and Mg²⁺ and inhibited by disulfiram and p-hydroxymercuribenzoate. The possible role of the eutypine-detoxification mechanism in the defense reactions of V. vinifera cells is discussed. Received: 20 April 1998 / Accepted: 22 September 1998     

L'eutypiose de la vigne : isolement d'une molécule synthétisée par Eutypa lata et toxique pour la vigne

Eutypa dieback, caused by the ascomycet fungus Eutypa lata, is currently the most serious disease of the grapevine. This disease now affects a great number of vineyards throughout the world, its economic impact is very important, and there is no remedy available for the destruction of the parasitic fungus. In this article, we describe the dieback symptoms, the management practices, the economic impact and the present knowledge on the interactions between E. lata and grapevine. We also present findings concerning the role of a toxic compound, hydroxy-4(methyl-3 butene-3 ynyl-1)-3 benzaldehyde, name eutypine, synthesized by the parasitic fungus and which was shown to be involved in the expression of the disease symptoms. The recent progress made in understanding eutypine's mechanism of action has opened new prospects regarding development of efficient tools for stopping this disease.     

Bioactivity of intra and extracellular substances from cyanobacteria and lactic acid bacteria on “wood blue stain” fungi

Cyanobacteria (photoautotrophic prokariota) have potential for the control of pathogenic bacteria and fungi. The effect of intra and extracellular products from cyanobacterial strains on the growth of fungi isolated from “wood blue stain,” was tested. Extracellular products were obtained by concentration and sterilization of the culture medium where cyanobacteria were grown. Cyanobacterial substances promoted or inhibited fungal growth according to the fungal and cyanobacterial strains tested. Extracellular products from Nostoc muscorum 79a and the methanolic extract from Microchaete tenera 84b biomass inhibited growth of Sphaeropsis sapinea 2157 (64.7 and 775.6%, respectively). Extracellular products of Nostoc piscinale 59 and biomass methanolic extract from N. muscorum 79a produced the highest growth promotion of Trichoderma boningii 452 (105.0%) and T. viride 993 (136.7%). Extracellular products of the heterotrophic lactic acid bacterium Streptococcus termophilus were also tested and strongly inhibited (64–92%) all the fungal strains. The tested fungi have different sensitivity to the bioactive substances present in the biomass and/or the culture medium of the studied cyanobacteria and lactic acid bacterium. N. muscorum 79a, M. tenera 84b, and S. termophilus have potential to control the wood blue stain fungi by a friendly environmental alternative.     

Fuscoside E: a strong anti-inflammatory diterpene from Caribbean octocoral Eunicea fusca

The screen of 10 soft coral extracts collected from the Colombian Caribbean Sea in the TPA-induced ear edema model allowed us to identify Eunicea fusca extract among others as an interesting source of active compounds. The new diterpene, fuscoside E (1), along with the known fuscoside B (2), fuscol (3), (+)-germacrene D (4) and a mixture of six sterols (5-10), were isolated from this soft coral. Their structures were elucidated by 1D and 2D NMR spectroscopy techniques. Fuscoside E (1) absolute stereochemistry was determined by chiroptical methods. Fuscoside E (1) and B (2) showed strong anti-inflammatory in the above mentioned bioassay. Additionally, fuscoside E (1) and the sterol mixture (5-10) presented antifouling activity against bacterial strains involved in surface colonization.     

Three isocoumarins and a benzofuran from the cultured lichen mycobionts of Pyrenula sp

The spore-derived mycobionts of the lichen Pyrenula sp. were cultivated on a malt-yeast extract medium supplemented with 10% sucrose. The investigation of their metabolites resulted in isolation of four compounds, three isocoumarins and a biogenetically related benzofuran; their structures were determined by spectroscopic methods.     

Antifungal activity of <Emphasis Type="Italic">Bacillus subtilis</Emphasis> 355 against wood-surface contaminant fungi

A strain of Bacillus subtilis was examined for antifungal activity against phytopathogenic and wood-surface contaminant fungi. The bacterium was grown in five culture media with different incubation times in order to study cell development, sporulation, and the production of metabolites with antifungal activity. The anti-sapstain and anti-mould activity of the bacterium grown in yeast extract glucose broth (YGB) medium in wood was also evaluated. In YGB, the bacterium inhibited the growth of several fungi and displayed a broader spectrum of activity than in the other media tested. A relationship between bacterial spore production and the formation of metabolites with antifungal activity was detected. YGB medium displayed effective control in wood block tests. YGB medium was extracted with solvents of increasing polarity and the dry residues were applied to silicagel plates, resolved with the appropriate solvent and sprayed with different solutions, detecting the presence, of amines, and higher alcohols. The bioautographic method revealed the presence of at least two active compounds against the blue-stain fungus Cladosporium cucumerinum.     

Constituents of two Flourensia species

The MeOH extract of aerial parts of Flourensia riparia Grisebach (Asteraceae) afforded a sesquiterpene lactone, 4beta-hydroxy-4,10alpha-dimethyl-7alphaH,8alphaH-eudesman-11-ene-8,12-olide, together with septuplinolide, its isomer at positions C-5 and C-10. In addition, known flavonoids, p-hydroxyacetophenone derivatives, carabrone and isoalantolactone were identified. Three known flavonoids and a benzofuran were isolated from Flourensia campestris Wedd.     

An improved bioassay for the detection of Bacillus thuringiensis β-exotoxin

Some Bacillus thuringiensis strains secrete β-exotoxin, which is an insecticidal, thermostable adenine nucleotide analogue. Discrepancies between detection of β-exotoxin by high-performance liquid chromatography and insect bioassays have shown the importance of bioassays in the determination of β-exotoxin production. With the aim of improving the fly β-exotoxin bioassay, a range of fly diets were evaluated and the best performing diet was incorporated into a novel β-exotoxin bioassay. The improved bioassay is characterised by good control pupation percentages, low variability, easy setup and monitoring. The bioassay allowed unambiguous differentiation between β-exotoxin producing and non-producing strains, and is suitable for the routine screening of B. thuringiensis strains for β-exotoxins.     

Antioxidants and iron chelators inhibit oxygen radical generation in fungal cultures of plant pathogenic fungi

Eutypa dieback and Esca are serious fungal grapevine trunk diseases (GTDs). Eutypa dieback is caused by Eutypa lata (Elata), and is often associated Phaeoacremonium minimum (Pmin), and Phaeomoniella chlamydospora (Pch) which are also important contributors to Esca disease.Understanding the complex pathogenesis mechanisms used by these causative fungi may potentially lead targeted treatments for GTDs in the future. Elata has been reported as a wood decay “soft rot” fungus and understanding of Elata’s pathogenesis chemistries can aid in controlling GTDs. Recent work that suggests that Pmin and Pch may contribute to pathogenesis by stimulating hydroxyl radical generation via secretion of low molecular weight phenolic metabolites. Building on these findings, we tested a hypothesis that antioxidants and chelators, and biocontrol agents that have been reported to secrete antioxidants and low molecular weight chelators, may inhibit the growth and activity of these fungi. Butylated hydroxy anisole (BHA) and butylated hydroxytoluene (BHT) were tested as antioxidant/chelators. BHA was found to be a highly effective control measure for the three pathogenic fungi tested at concentrations >0.5 mM. The biocontrol species Bacillus subtilis and Hypocrea (Trichoderma) atroviride were also tested, with both H. atroviride and B. subtilis effectively inhibiting growth of the three GTD fungi.     

Differential Behaviour of Mycelial Growth of Several Botrytis cinerea Strains on either Patchoulol- or Globulol-amended Media

Botrydial and dihidrobotrydial are two characteristic metabolites of the phytopathogenic fungus Botrytis cinerea , which are involved in the development of necrotic lesions on grapevine and tobacco. Patchoulol and globulol, two natural products which are analogues to precursors of botrydial and dihidrobotrydial, were tested on 10 B. cinerea strains which were isolated from different hosts and varied in aggressiveness on grapevine leaves. Mycelial growth of all strains was prevented when they were grown on either patchoulol- or globulol-amended malt agar media (200  μ g/ μ l). Each strain displayed a specific response pattern to those products, according to the high variability previously described for this species. Furthermore, strains were different from one another with regard to their level of aggressiveness against leaves detached from sherry grapevine vineyards.