A new double-stranded RNA mycovirus from Botrytis cinerea

A simple double-stranded RNA mycovirus was detected in a wild-type Botrytis cinerea 55k strain. The virus was located in the fungus cytoplasm as free particles of approximately 28 nm in diameter. The mycovirus possesses a single double-stranded genome segment of 1.8 kilobase pairs (kbp) encapsidated within an isometric protein coat whose main structural component is a polypeptide of 68 kDa. Cells infected with this virus showed an important degree of cellular degeneration.     

Exo-β-1,3-Glucanase from Yeast Inhibits Colletotrichum lupini and Botrytis cinerea Spore Germination

Yeast exo-β-1,3-glucanase (EXG1) was evaluated as an inhibitory agent of Colletotrichum lupini and Botrytis cinerea. Extracts obtained from yeast transformed with the exg1 gene, expressing high levels of EXG1 activity, or control untransformed yeast cultures that lacked EXG1 activity, were added to different starting concentrations of C. lupini fungal spore suspensions (2.5 × 10³ to 80 × 10³ spores per flask), and mycelial dry weight was measured after 5 days. Inhibition of C. lupini mycelial growth by EXG1 compared with control extracts ranged from 41 to 20% when added to starting fungal spore concentrations of 2.5 × 10³ to 80 × 10³, respectively. EXG1 activity in the extracts from the transformed yeast remained high over the 5-day incubation period. Addition of the EXG1 extract after C. lupini spore germination resulted in lower inhibition, indicating that the EXG1 targets the β-glucan in the cell walls of the fungal spores at an early stage of germination. Furthermore, the yeast EXG1 extracts were also shown to inhibit Botrytis cinerea spore germination and growth. Thus, the use of the yeast exg1 gene for protection of crops, such as lupin and pear in transgenic strategies against C. lupini and B. cinerea , respectively, could be considered.     

2-epi-botcinin A and 3-O-acetylbotcineric acid from Botrytis cinerea

Two metabolites, 2-epi-botcinin A and 3-O-acetylbotcineric acid, were isolated from Botrytis cinerea (AEM211). The former compound was new, and the latter was known but structurally revised by us. In a test for antifungal activity against Magnaporthe grisea, a pathogen of rice blast disease, 2-epi-botcinin A was 8 times less active than botcinin A (MIC 100 microM), and the MIC value for 3-O-acetylbotcineric acid being 100 microM.     

2-epi-Botcinin A and 3-O-Acetylbotcineric Acid from Botrytis cinerea

Two metabolites, 2-epi-botcinin A and 3-O-acetylbotcineric acid, were isolated from Botrytis cinerea (AEM211). The former compound was new, and the latter was known but structurally revised by us. In a test for antifungal activity against Magnaporthe grisea, a pathogen of rice blast disease, 2-epi-botcinin A was 8 times less active than botcinin A (MIC 100 μM), and the MIC value for 3-O-acetylbotcineric acid being 100 μM.     

Polyderm, a Barrier to Infection of Red Raspberry Buds by Didymella applanata and Botrytis cinerea

A histological study was made of the axillary region of raspberrycanes infected naturally by Didymella applanata (Niessl) Sacc.and Botrytis cinerea Pers.: Fr. The outer suberized phellemlayer of the polyderm and a primary protective layer of suberizedand lignified cells across the adaxial cortex of the petioleprecluded infection of the axillary buds by hyphae growing froma saprophytic base in the leaf. No protective layer formed throughthe abaxial cortex at the petiole base; consequently both fungicolonized the epidermis, primary cortex and outermost non-suberizedphelloid cells of the polyderm beneath the node. Red raspberry, Rubus idaeus, Didymella applanta, Botrytis cinerea, polyderm, periderm, suberin, lignin     

Host-Pathogen Interactions: VI. A Single Plant Protein Efficiently Inhibits Endopolygalacturonases Secreted by Colletotrichum Lindemuthianum and Aspergillus Niger

Endopolygalacturonases have been purified from the extracellular enzymes of Colletotrichum lindemuthianum and Aspergillus niger. A protein, purified from Red Kidney (Phaseolus vulgaris) beans for its ability to inhibit the endopolygalacturonase secreted by C. lindemuthianum, inhibits the A. niger endopolygalacturonase almost as efficiently as it inhibits the C. lindemuthianum enzyme.     

Botcinolide: A Biologically Active Natural Product from Botrytis cinerea

A novel biologically active natural product was isolated from a strain of Botrytis cinerea found on cultivated raspberry fruit (Rubus ideaus) upon fermentation in a liquid medium. Following a bioassay-directed purification process, the final product was an amorphous solid with the molecular formula C₂₀H₃₄O₈, and is trivially named botcinolide. It significantly inhibited etiolated wheat coleoptile growth at 10^?3 and 10^?4 M by 100 and 82% respectively, relative to the controls. Greenhouse-grown bean, corn, and tobacco plants were affected by treating with botcinolide, and exhibited chlorosis and severe necrosis at 10^?2 and 10^?3 M. The structure is a new hydroxylated nonalactone that is esterified with 4-hydroxy-2-octenoic acid.     

A double-stranded RNA mycovirus in Botrytis cinerea

In wild-type Botrytis cinerea CVg25 strain we have detected the presence of extrachromosomal genetic elements corresponding to double-stranded RNA molecules. These genetic elements have been designated L, M₁ and M₂ with molecular sizes of 8.3, 2.0 and 1.4 kb, respectively. The visualization by electron microscopy of mycelium ultrathin sections from B. cinerea CVg25 showed the presence of isometric virus-like particles of about 40 nm in diameter. Linear sucrose gradient centrifugation of mycelium-free extracts was done to determine if the double-stranded RNAs were associated with virus-like particles. The gradient profile obtained at 260 and 280 nm revealed a major peak that was analyzed by both agarose-gel electrophoresis and electron microscopy. It was observed that only the L-double-stranded RNA molecule copurified with isometric virus-like particles. These virus-like particles had a similar morphology and size as those detected by electron microscopy in the mycelium sections. These results suggest that only the L-double-stranded RNA would be encapsidated.     

灰葡萄孢BC7-3菌株除草活性组分的纯化与结构鉴定

[目的]植物病原真菌毒素是一类重要的微生物源除草剂,本研究旨在找到一个新的具有除草活性的化合物结构.[方法]在前期薄层层析法、柱层析法和高效液相色谱法分析的基础上对灰葡萄孢诱变菌株BC7-3的代谢产物中具有除草活性的5个不同组分分别进行了液相色谱制备.[结果]本研究得到了一个纯度达99.38%对单子叶杂草马唐具有较强杀除活性的纯组分,通过对纯组分的物理性状测定并结合紫外-可见吸收光谱、红外光谱以及核磁共振波谱等分析方法鉴定化学结构为10-顺-二氢化灰霉二醛.[结论]研究的结果为微生物除草剂的创新和开发奠定了理论基础.     

Mycoviruses of Botrytis cinerea isolates from different hosts

Botrytis cinerea (teleomorph Botryotinia fuckeliana) is a necrotrophic plant pathogenic fungus that causes grey mould and enormous economic losses worldwide in different crops. Control of B. cinerea is difficult due to the appearance of fungicide‐resistant isolates, and the diversity in virulence due to genetic variability and, perhaps, the infection with mycoviruses or fungal viruses. The discovery of mycoviruses and their possible application as biocontrol agents, as well as their use as tools to study the plant–pathogen interaction, has encouraged their study in B. cinerea. Herein, we have analysed the occurrence of mycoviruses in Spanish B. cinerea isolates to approach a better understanding of the interactions among viruses, fungi and plants in this pathosystem. Fifty‐five percent of the B. cinerea isolates analysed contained double‐stranded RNA (dsRNA) elements, and the number of dsRNA elements, their relative concentration and size were variable among isolates. Some of these dsRNAs were related to the presence of virus like rod or isometric particles, and to cellular degeneration and malformed mitochondria. We have also demonstrated that a 3 kb dsRNA present in 55% of the isolates having dsRNA elements was a mycovirus genome. Partial sequence of that mycovirus presented high identity in nucleotide and amino acid sequence with Botrytis cinerea mitovirus 1 (BcMV1). Analysis of the genetic distance within Spanish BcMV1 sequences showed the existence of different isolates of this mitovirus inside the Spanish B. cinerea population analysed. This is the first report of the variability of dsRNA elements and the partial genome sequence of a mitovirus associated with Spanish B. cinerea isolates and the genetic diversity within Spanish isolates of BcMV1.     

Cloning and partial characterization of endopolygalacturonase genes from Botrytis cinerea

Botrytis cinerea is a plant-pathogenic fungus infecting over 200 different plant species. We use a molecular genetic approach to study the process of pectin degradation by the fungus. Recently, we described the cloning and characterization of an endopolygalacturonase (endoPG) gene from B. cinerea (Bcpg1) which is required for full virulence. Here we describe the cloning and characterization of five additional endoPG-encoding genes from B. cinerea SAS56. The identity at the amino acid level between the six endoPGs of B. cinerea varied from 34 to 73%. Phylogenetic analysis, by using a group of 35 related fungal endoPGs and as an outgroup one plant PG, resulted in the identification of five monophyletic groups of closely related proteins. The endoPG proteins from B. cinerea SAS56 could be assigned to three different monophyletic groups. DNA blot analysis revealed the presence of the complete endoPG gene family in other strains of B. cinerea, as well as in other Botrytis species. Differential gene expression of the gene family members was found in mycelium grown in liquid culture with either glucose or polygalacturonic acid as the carbon source.     

Cloning and Partial Characterization of Endopolygalacturonase Genes from Botrytis cinerea

Botrytis cinerea is a plant-pathogenic fungus infecting over 200 different plant species. We use a molecular genetic approach to study the process of pectin degradation by the fungus. Recently, we described the cloning and characterization of an endopolygalacturonase (endoPG) gene from B. cinerea (Bcpg1) which is required for full virulence. Here we describe the cloning and characterization of five additional endoPG-encoding genes from B. cinerea SAS56. The identity at the amino acid level between the six endoPGs of B. cinerea varied from 34 to 73%. Phylogenetic analysis, by using a group of 35 related fungal endoPGs and as an outgroup one plant PG, resulted in the identification of five monophyletic groups of closely related proteins. The endoPG proteins from B. cinerea SAS56 could be assigned to three different monophyletic groups. DNA blot analysis revealed the presence of the complete endoPG gene family in other strains of B. cinerea, as well as in other Botrytis species. Differential gene expression of the gene family members was found in mycelium grown in liquid culture with either glucose or polygalacturonic acid as the carbon source.     

Generation and analysis of expressed sequence tags from Botrytis cinerea

Botrytis cinerea is a filamentous plant pathogen of a wide range of plant species, and its infection may cause enormous damage both during plant growth and in the post-harvest phase. We have constructed a cDNA library from an isolate of B. cinerea and have sequenced 11,482 expressed sequence tags that were assembled into 1,003 contigs sequences and 3,032 singletons. Approximately 81% of the unigenes showed significant similarity to genes coding for proteins with known functions: more than 50% of the sequences code for genes involved in cellular metabolism, 12% for transport of metabolites, and approximately 10% for cellular organization. Other functional categories include responses to biotic and abiotic stimuli, cell communication, cell homeostasis, and cell development. We carried out pair-wise comparisons with fungal databases to determine the B. cinerea unisequence set with relevant similarity to genes in other fungal pathogenic counterparts. Among the 4,035 non-redundant B. cinerea unigenes, 1,338 (23%) have significant homology with Fusarium verticillioides unigenes. Similar values were obtained for Saccharomyces cerevisiae and Aspergillus nidulans (22% and 24%, respectively). The lower percentages of homology were with Magnaporthe grisae and Neurospora crassa (13% and 19%, respectively). Several genes involved in putative and known fungal virulence and general pathogenicity were identified. The results provide important information for future research on this fungal pathogen.     

Purification and Properties of Benzyl Alcohol Oxidase from Botrytis cinerea

A benzyl alcohol oxidase (BAO) was purified to homogeneity from Botrytis cinerea. The enzyme was found to have a molecular mass of 214 kD with a trimeric structure, and optimal pH and temperature of 5.0 and 30°C, respectively. The enzyme activity was not sensitive to metal ions or to metal ion chelators, while thiol blocking reagents strongly inhibited BAO activity. Sulfur dioxide irreversibly inhibited the enzyme activity and the inhibitory effect of ethanol was weak and reversible. Benzyl alcohol was the most effective alcohol substrate for BAO. Para or meta monosubstituted benzyl alcohol with methyl or methoxy groups were good substrates. BAO also oxidized cinnamyl alcohol, furfuryl alcohol, and some terpenic alcohols· with an alkenyl group near the reactive carbinol. Secondary alcohol, methanol and phenol were not substrates. Product inhibition studies suggested that benzaldehyde and benzyl alcohol were bound at different places to the active site. O₂ was the only electron acceptor identified and Botrytis cinerea benzyl alcohol oxidase was classified .as EC 1.1.3.7 according to stoichiometrical studies. We discuss the metabolic role of BAO in the Botrytis cinerea-grape host-parasite relationship.     

Blossom–end Rot of Pears: Systemic Infection of Flowers and Immature Fruit by Botrytis cinerea1

A histological study was made of the systemic growth of Botrytis cinerea from styles, stamens and sepals to the flower receptacle and mesocarp of immature pear fruit. In most styles, hyphal growth ceased in the upper portion at the onset of stylar senescence, which occurred at about 1 wk after full bloom. Hyphae never passed through styles into the carpel. Unlike the styles, hyphae in filaments grew without restriction and progressed within 4 days, via vascular tissue, through sepals into tissues of the upper end of the flower receptacle, or of the mesocarp adjoining the sepals, without causing symptoms. Filaments remained green to partly green until harvest. B. cinerea entered filaments and spread into the receptacle or mesocarp at any time between blossoming and harvest and then became latent in these tissues. Filaments were, however, more susceptible at the flowering stage. After 2 months floral tubes were closed, and the stamens protected from infection. Careful inspection of ripe, cold–stored fruit showed that decay invariably spreads from mesocarp tissue adjoining the sepals, outward along the vascular bundles, but not from secondary inoculum in the floral tube. The behaviour of the pathogen suggests that control of blossom–end rot could be achieved if pears are sprayed with fungicide at 75—100% petal fall (when most stamens are exposed) and a month later (before floral tubes started to close).     

Production and Characterization of Laccase from Botrytis cinerea 61-34

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

Benomyl tolerance in isolates of Botrytis cinerea from tomato plants

Three hundred and forty-nine isolates of Botrytis cinerea were collected from tomato crops on forty-one nurseries and 173 (40/6 %) were found to be tolerant to benomyl. There was no obvious association between disease incidence and the occurrence of tolerance. In a fungicide comparison experiment on tomatoes in 1973, twenty of the sixty-four (31 %) isolates examined were benomyl tolerant, the majority of these were from benomyl sprayed plants. In 1974 in a similar experiment, 384 of the 394 (97-5 %) isolates examined were tolerant. Tolerance was monitored in two tomato experiments in relation to a spray programme in which benomyl and dichlofluanid were used in various combinations. There was no marked effect of the spray programmes on the incidence of tolerance on either site. In the experiments B. cinerea was controlled and significant increases in yield were obtained with benomyl in 1973 but not in 1974. This difference is attributed to the change in the pathogen population with a large increase in the incidence of tolerance on the experimental site in 1974.     

A New Isoflavone with Antifungal Activity from Immature Fruits of Lupinus luteus

A new isoflavone having antifungal activity was isolated from immature fruits of Lupinus luteus (Leguminosae), and named luteone. The structure was shown to be 5,7,2′,4′-tetrahy-droxy-6-(3,3-dimethylallyl)-isoflavone by degradative and spectroscopic studies.