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Population diversity and pathogenicity mechanisms of Colletotrichum spp. causing olive anthracnose

Pedro Talhinhas1,2, S. Sreenivasaprasad2, João Neves-Martins1 and Helena Oliveira1

1 Instituto Superior de Agronomia, Tapada da Ajuda, 1349-017 Lisboa, Portugal

2 Warwick HRI, University of Warwick, Wellesbourne, CV35 9EF, Warwickshire, UK

Olive anthracnose, caused by Colletotrichum acutatum and C. gloeosporioides, is a disease currently becoming important in the Mediterranean region, frequently being responsible for major olive yield losses and poor oil quality. Very little information is available on the genetic diversity of the fungi causing olive anthracnose and the molecular mechanisms regulating pathogenic lifestyles in C. acutatum, although it is known that C. acutatum exhibits pathogenic and non-pathogenic lifestyles on target hosts, non-target crops and weeds. The objectives of this work were to analyse the genetic diversity found among a collection of Colletotrichum spp. isolates representing the causal agents of this disease in Portugal and to generate tools for studying the role of genes involved in the developmental and pathogenic cycles of C. acutatum. A total of 131 isolates were obtained representing 75 different locations throughout Portugal. This collection was analysed using a range of molecular markers namely, species-specific PCR, ISSR and RAPD markers and rDNA ITS and -tubulin 2 sequences. Morphology (shape and size of conidia), cultural characteristics (growth rate, colony characters and response to benomyl) and pathogenicity/virulence (in olives and other hosts) of isolates representing the diversity were also assessed. The species C. acutatum was more commonly isolated from olives, but C. gloeosporioides was also identified. The vast majority of isolates (87%) belong to a single sub-group within C. acutatum, but isolates belonging to four other sub-groups were also identified. We have detected the presence of C. acutatum in symptomless olive stems, leaves and fruits, which suggests the existence of either a latent or non-pathogenic stage of the fungus in olive. A number of approaches are being used to investigate the pathogenicity mechanisms. Degenerate PCR based on conserved sequences in other Colletotrichum spp. and various ascomycetes has been used to successfully amplify fragments of candidate pathogenicity genes in C. acutatum. PCR amplicons of cAMP-dependent protein kinase catalytic subunit, cutinase, endopolygalacturonase, G protein α sub-unit, GTPase CDC42 and MAP kinase genes, some of which are known for being essential for conidial germination, appressoria formation/host penetration or switch to necrotrophy, have been cloned and sequence confirmed. A genomic library has recently constructed to aid in the complete characterisation of pathogenicity genes. Agrobacterium tumefaciens – mediated transformation (ATMT) of C. acutatum has been developed and more than 600 putative T-DNA insertional mutants generated so far to test for their pathogenicity. ATMT was also successfully used for generating C. acutatum transformants expressing the Green Fluorescent Protein gene, which will be an important tool for investigating the pathogen epidemiology and host colonisation process.

Barley genomics and plant defence responses

David B. Collinge1, Sisse Gjetting1, Torben Gjetting2, Per L. Gregersen1,3, Peter Hagedorn2, Michael Krogh Jensen1, Michael Lyngkjær2 and Jesper H. Rung1

1Department of Plant Biology, Royal Veterinary and Agricultural University, Thorvaldsensvej 40, 1871 Frederiksberg C, Copenhagen, Denmark   dbc@kvl.dk

2Risoe National Laboratory, 4000 Roskilde, Denmark  

3Danish Institute of Agricultural Sciences, Flakkebjerg, Denmark.

The defence response of barley to the powdery mildew fungus Blumeria graminis f.sp. hordei was the first system involving an intact plant inoculated with a fungus to be studied at the molecular level by Ken Scott and colleagues 20 years ago – See (Collinge et al., In: R. R. Belanger and W. R. Bushnell (eds.), The Powdery Mildews: A Comprehensive Treatise. APS Press, St. Paul, Minnesota, USA., 2002) for review. They were the first to demonstrate, using 2D-gel electrophoretic analysis of in vitro translation products, that fungal infection caused the disappearance of translatable mRNA’s for certain proteins associated with photosynthesis and the appearance of novel products. This approach provided valuable information of the timing (within hours) and spatial distribution of the defence response. Thus there is a difference in tissue specificity; both the infected epidermis and mesophyll, which is not invaded by the pathogen, exhibit a rapid alteration in the population of translatable transcripts. This system was also the first where the identity of transcripts accumulating following pathogen attack was made. Although many represent pathogenesis-related proteins and other defence related gene products, many remain unidentified to this day. More recently, differential display of mRNA extracted from the epidermal layer has led to the isolation of 27 transcripts which accumulate in the epidermis. Interestingly, none of these represent transcripts identified by classical differential or subtractive hybridisation, through which over 30 up-regulated transcript families were identified. Furthermore, 21 cannot be identified in the sequence data bases

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