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.     

Effects of indole-3-acetic acid on Botrytis cinerea isolates obtained from potted plants

We study the growth of different isolates of Botrytis cinerea collected from potted plants which were affected by Botrytis blight in southern Spain during recent years. These isolates, which show widely phenotypic differences when grown in vitro, are differentially affected by growth temperature, gibberellic acid applications and paclobutrazol, an efficient plant growth retardant and fungicide at the same time. In this work, we have evaluated the effect of the auxin indole-3-acetic acid (IAA) dose (0, 1, 10, and 100 mg/plate) on the growth of the collection of B. cinerea isolates obtained from the following potted plants: Cyclamen persicum, Hydrangea macrophylla, Lantona camara, and Lonicera japonica. B. cinerea produces indolacetic acid, but so far the precise biosynthetic pathway and some effects on this fungal species are still unclear, although recent studies have revealed an antifungal activity of IAA on several fungi, including B. cinerea isolated from harvested fruits. Mycelial growth curves and growth rates assessed from difference in colony areas during the both linear and deceleration phase, conidiation (measured as time of appearance), conidia length (microm), and sclerotia production (number/plate) were evaluated in the isolates, which were grown at 26 degrees C on Petri dishes containing potato dextrose agar for up to 35 days. Mycelial growth curves fitted a typical kinetic equation of fungi grown on solid media. B. cinerea isolates showed a high degree of variability in their growth kinetics, depending on the isolate and auxin dose. This plant growth substance delayed mycelial growth during the linear phase in an isolate-dependent manner, thus isolates from C. persicum, H. macrophylla and L. camara were more affected by IAA than L. japonica. On the other hand, 100 mg of IAA was the critical dose to significantly reduce the growth rate in all isolates and to promote brown-striped hyphae development, especially in isolate from C. persicum. 10 and 100 mg IAA delayed conidiation in isolates from H. macrophylla but scarcely effects were found in the conidia length. The sclerotia production process was blocked at IAA doses of 100 mg in isolates from L. camara and L. japonica, and was reduced in isolate from H. macrophylla. However, dose of 100 mg IAA had no effect on sclerotia production in isolate from C. persicum. It was concluded that the effect of IAA on B. cinerea growth depends on the isolate, thus isolates from H. macrophylla and L. camara were the most affected by IAA. B. cinerea reduced its development under IAA applications, depending on the isolate and dose. These results confirm those recently published on the inhibitory effect of IAA on Botrytris species growth.     

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.     

The D-galacturonic acid catabolic pathway in Botrytis cinerea

D-galacturonic acid is the most abundant component of pectin, one of the major polysaccharide constituents of plant cell walls. Galacturonic acid potentially is an important carbon source for microorganisms living on (decaying) plant material. A catabolic pathway was proposed in filamentous fungi, comprising three enzymatic steps, involving D-galacturonate reductase, L-galactonate dehydratase, and 2-keto-3-deoxy-L-galactonate aldolase. We describe the functional, biochemical and genetic characterization of the entire D-galacturonate-specific catabolic pathway in the plant pathogenic fungus Botrytis cinerea. The B. cinerea genome contains two non-homologous galacturonate reductase genes (Bcgar1 and Bcgar2), a galactonate dehydratase gene (Bclgd1), and a 2-keto-3-deoxy-L-galactonate aldolase gene (Bclga1). Their expression levels were highly induced in cultures containing GalA, pectate, or pectin as the sole carbon source. The four proteins were expressed in Escherichia coli and their enzymatic activity was characterized. Targeted gene replacement of all four genes in B. cinerea, either separately or in combinations, yielded mutants that were affected in growth on D-galacturonic acid, pectate, or pectin as the sole carbon source. In Aspergillus nidulans and A. niger, the first catabolic conversion only involves the Bcgar2 ortholog, while in Hypocrea jecorina, it only involves the Bcgar1 ortholog. In B. cinerea, however, BcGAR1 and BcGAR2 jointly contribute to the first step of the catabolic pathway, albeit to different extent. The virulence of all B. cinerea mutants in the D-galacturonic acid catabolic pathway on tomato leaves, apple fruit and bell peppers was unaltered.     

L-amino acid oxidase-induced apoptosis in filamentous Botrytis cinerea

As opposed to single-cell yeast and mammalian cell lines, apoptosis has not been greatly investigated in filamentous fungi because antibodies to the relevant fungal apoptosis-related proteins are not available commercially and because multicellular organisms cannot be studied using flow cytometry. Here we demonstrate how antibodies from a nonfungal source could be used to investigate this pathway. We show that apoptosis in the filamentous fungus Botrytis cinerea is triggered by the mitochondria-mediated caspase pathway, with release of the apoptotic factors cytochrome c, caspase 3, and caspase 9, on treatment with Trichoderma harzianum-derived L-amino acid oxidase.     

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.     

抗灰葡萄孢霉菌乳酸菌的筛选

从80株乳酸菌中筛选出45株具有抗灰葡萄孢霉菌活性的乳酸菌菌株,其中10株具有较强抗灰葡萄孢霉菌活性。对这10株乳酸菌菌株的抗植物致病真菌谱进行了研究,这10株乳酸菌对番茄早疫病菌,甜瓜疫霉菌,甜瓜枯萎病菌,苹果炭疽病菌的生长均有较强的抑制作用。其中1株具有广谱抗植物致病真菌活性的乳酸菌菌株BX6-4为植物乳杆菌。经番茄离体叶片接种试验发现,植物乳杆菌BX6-4的发酵液能够在体外强烈地抑制灰葡萄孢霉菌的生长。     

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.     

A copper-transporting ATPase BcCCC2 is necessary for pathogenicity of Botrytis cinerea

Copper is an essential trace element that serves as a cofactor for numerous enzymes. In eukaryotes, copper-transporting ATPases deliver copper to various copper-containing proteins in the trans-golgi network. This study identified a copper-transporting ATPase gene BcCcc2 in a fungus pathogenic to plants, Botrytis cinerea. We investigated the biological roles of BcCCC2 by generating null mutants for BcCcc2. Melanization, conidiation and the formation of sclerotia were severely affected in ∆BcCcc2 mutants. Moreover, a pathogenicity assay using tomato leaves and carnation petals revealed the mutants to be nonpathogenic. Further analysis indicated that they formed fewer appressoria and infection cushions than the wild-type. These structures were aberrant in morphology and in many cases had a significantly reduced ability to penetrate the plant epidermis. An assay also indicated that ∆BcCcc2 mutants were defective in infection through wounds. BcCCC2 is necessary not only for penetrating a host but also for fungal growth within plant tissues. Our results also imply that B. cinerea requires copper-containing proteins for infection that are inactive in the absence of the copper-transporting ATPase BcCCC2.     

The Botrytis cinerea early secretome

The extracellular proteome, or secretome, of phytopathogenic fungi is presumed to be a key element of their infection strategy. Especially interesting constituents of this set are those proteins secreted at the beginning of the infection, during the germination of conidia on the plant surfaces or wounds, since they may play essential roles in the establishment of a successful infection. We have germinated Botrytis cinerea conidia in conditions that resemble the plant environment, a synthetic medium enriched with low molecular weight plant compounds, and we have collected the proteins secreted during the first 16 h by a double precipitation protocol. 2‐D electrophoresis of the precipitated secretome showed a spot pattern similar for all conditions evaluated and for the control medium without plant extract. The proteins in 16 of these spots were identified by PMF and corresponded to 11 different polypeptides. Alternative determination of secretome composition by LC‐MS/MS of tryptic fragments rendered a much larger number, 105 proteins, which included all previously identified by PMF. All proteins were functionally classified according to their putative function in the infection process. Key features of the early secretome include a large number of proteases, the abundance of proteins involved in the degradation of plant defensive barriers, and plenty of proteins with unknown function.     

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.