María Carbú

2-DE proteomic approach to the Botrytis cinerea secretome induced with different carbon sources and plant-based elicitors

Botrytis cinerea is a phytopathogenic fungus infecting a number of crops (tomatoes, grapes and strawberries), which has been adopted as a model system in molecular phytopathology. B. cinerea uses a wide variety of infection strategies, which are mediated by a set of genes/proteins called pathogenicity/virulence factors. Many of these factors have been described as secreted proteins, and thus the study of this sub‐proteome, the secretome, under changing circumstances can help us to understand the roles of these factors, possibly revealing new loci for the fight against the pathogen. A 2‐DE, MALDI TOF/TOF‐based approach has been developed to establish the proteins secreted to culture media supplemented with different carbon sources and plant‐based elicitors (in this study: glucose, cellulose, starch, pectin and tomato cell walls). Secreted proteins were obtained from the culture media by deoxycholate‐trichloroacetic acid/phenol extraction, and 76 spots were identified, yielding 95 positive hits that correspond to 56 unique proteins, including several known virulence factors (i.e. pectin methyl esterases, xylanases and proteases). The observed increases in secretion of proteins with established virulence‐related functions indicate that this in vitro‐induction/proteome‐mining approach is a promising strategy for discovering new pathogenicity factors and dissecting infection mechanisms in a discrete fashion.

Proteomic analysis of phytopathogenic fungus Botrytis cinerea as a potential tool for identifying pathogenicity factors, therapeutic targets and for basic research

Botrytis cinerea is a phytopathogenic fungus causing disease in a substantial number of economically important crops. In an attempt to identify putative fungal virulence factors, the two-dimensional gel electrophoresis (2-DE) protein profile from two B. cinerea strains differing in virulence and toxin production were compared. Protein extracts from fungal mycelium obtained by tissue homogenization were analyzed. The mycelial 2-DE protein profile revealed the existence of qualitative and quantitative differences between the analyzed strains. The lack of genomic data from B. cinerea required the use of peptide fragmentation data from MALDI-TOF/TOF and ESI ion trap for protein identification, resulting in the identification of 27 protein spots. A significant number of spots were identified as malate dehydrogenase (MDH) and glyceraldehyde-3-phosphate dehydrogenase (GAPDH). The different expression patterns revealed by some of the identified proteins could be ascribed to differences in virulence between strains. Our results indicate that proteomic analysis are becoming an important tool to be used as a starting point for identifying new pathogenicity factors, therapeutic targets and for basic research on this plant pathogen in the postgenomic era.

Development of Proteomics-Based Fungicides: New Strategies for Environmentally Friendly Control of Fungal Plant Diseases

Proteomics has become one of the most relevant high-throughput technologies. Several approaches have been used for studying, for example, tumor development, biomarker discovery, or microbiology. In this “post-genomic” era, the relevance of these studies has been highlighted as the phenotypes determined by the proteins and not by the genotypes encoding them that is responsible for the final phenotypes. One of the most interesting outcomes of these technologies is the design of new drugs, due to the discovery of new disease factors that may be candidates for new therapeutic targets. To our knowledge, no commercial fungicides have been developed from targeted molecular research, this review will shed some light on future prospects. We will summarize previous research efforts and discuss future innovations, focused on the fight against one of the main agents causing a devastating crops disease, fungal phytopathogens.

Isolation and pathogenicity of Colletotrichum spp. causing anthracnose of strawberry in south west Spain

Anthracnose, caused by Colletotrichum spp., is a major disease of cultivated strawberry, Fragaria × ananassa. This study identifies the Colletotrichum spp. which causes strawberry anthracnose in the southwest of Spain. A survey of the region was carried out, and the strains isolated were identified as C. acutatum by using the polymerase chain reaction (PCR) with genus and species-specific primers, demonstrating that this species is currently the causal agent of strawberry anthracnose in the studied region. The pathogenicity of C. acutatum and C. gloeosporioides strains was evaluated on two principal strawberry cultivars (cvs Camarosa and Ventana) under field conditions, the latter being more pathogenic than the former.

Inheritance of chromosome-length polymorphisms in the phytopathogenic ascomycete Botryotinia fuckeliana (anam. Botrytis cinerea )

The electrophoretic karyotype (EK) of 24 strains of Botryotinia fuckeliana was resolved to determine the extent of chromosomal polymorphism among them. Based on EK variation, nine different profiles with a number of bands ranging from 5 to 8 were found. Those profiles were not related to the origins of the strains, hosts, or year of isolation. Inheritance of chromosomal bands was studied by analyzing the EK of the progeny from crosses between sexually compatible B. fuckeliana strains that had different karyotype. The EKs of 44 monoascospore strains showed 12 new EK profiles, suggesting that chromosomal rearrangements generated after meiotic recombination provides EK variability in this fungus. Also, we report here that the degree of EK variability indicates that sexual reproduction under natural conditions might be quite important in the life cycle of B. fuckeliana. Hybridization analysis showed that, in most strains, the rDNA is located in both a-chromosomal band of high molecular weight and different size depending on the strain and a band of low molecular weight, revealing evidence for chromosome rearrangements in this plant pathogen.

Study on fungicide resistance of botrytis cinerea isolates from diseased strawberry plants

Southwestern Spain is one of the main areas for strawberry culture in the world. Large losses are due to grey mould caused by Botrytis cinerea, a plant pathogen that affects a wide variety of crops. Resistance to synthetic fungicides is one of the more important problems that hamper control of grey mould on strawberry. We have characterized the relative response to benzimidazole (benomyl and carbendazaim) and dicarboximide (vinclozolin) among 36 isolates of B. cinerea obtained from different plots in six counties of the province of Huelva during a 2-year period. Three phenotypes with resistance to benzimidazole were detected: (i) BenR1 having phenotype resistant (R) to benomyl and high-resistant (HR) to carbendazim; (ii) BenR2 with phenotype HR to both benomyl and carbendazim; and (iii) BenR3 with phenotype HR to benomyl and R to carbendazim. Strains were either resistant or sensitive to the dicarboximide vinclozolin (DicR or DicS, respectively), but high-resistance to this fungicide (DicHR strains) was not found. The low fitness of the DicHR mutants and the possibility of heterokaryosis indicated by the number of nuclei within fungal conidia indicates that DicHR mutants might not become established in field populations in southwestern Spain.

Sulfur free red wines through the use of grapevine shoots: Impact on the wine quality

Following a preliminary study to determine the possibility of using a grapevine shoot extract (VIN) as a sustainable alternative to sulfur dioxide (SO2), in this study, the chromatic features, phenolic composition, and sensory analysis of wines treated with VIN at two concentrations were studied during storage in bottle for the first time. The highest differences were found in phenolic compounds after 12months of storage in bottle. The VIN wines had a low content of free anthocyanins and were high in vinyl-pyranoanthocyanins, and B-type vitisins. Consequently, they showed better chromatic characteristics. Moreover VIN, especially at high dose, preserved non-anthocyanin phenolic compounds better than SO2. However, at this high dose some organoleptic properties were affected. VIN, when used at a low dose, is able to preserve wine composition without loss of quality.

Molecular Microbiology Applied to the Study of Phytopathogenic Fungi

Fungi is an extensive group of eukaryotic microorganisms, generally they are microscopic and usually filamentous. It is estimated that there are between 70,000 and 1.5 millions species of fungi, most of them are being discovering and describing (Agrios, 2005). Most of the known hundred thousand fungal species are strictly saprophytic, living on decomposing dead organic matter. About fifty species cause disease in human, and more than ten thousand species can cause disease in one (obligate parasite) or many kinds of plants (nonobligate parasites) (Fernandez-Acero et al., 2007a).