Inmaculada Vallejo

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.

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.