COLLETOTRICHUM ACUTATUM DETECTION BY DIRECT PCR IN STRAWBERRY PLANTS
I GRONDONA1, MB SUAREZ1, P MARTINEZ-CULEBRAS2,
A QUEROL3, MD GARCIA2 and E MONTE1
1Department of Microbiology and Genetics, University of Salamanca, Spain; 2Colección Española de Cultivos Tipo, University of Valencia, 46100 Burjassot, Valencia, Spain; 3Department of Biotechnology, IATA-CSIC,
46100 Burjassot, Valencia, Spain
Background and objectives
Colletotrichum species cause major economic damage to strawberry crops, inducing fruit, stem- and crown-rots, and also as post-harvest pathogens. In Europe, most outbreaks are attributed to C. acutatum, considered to be a wide-spectrum
pathogen. In North America many of the problems are caused by C. fragariae, considered to be a separate, equally strongly pathogenic species, although C. acutatum is also widespread. C. fragariae is not distinguishable in morphological
terms from the C. gloeosporioides (the asexual state of Glomerella cingulata) aggregate, and has been considered as a strongly pathogenic 'race'. The identification and circumscription of species of Colletotrichum pathogenic to strawberry were confused, and Buddie et al.  have recently proposed a molecular approach in order to obtain a more consistent classification of these species. With the aim of developing safer, more rapid and reliable diagnostic tests for C. acutatum, a quarantine pathogen in the EU, different PCR primers were designed based on Buddie's molecular classification.
Materials and methods
Strawberry plants were artificially inoculated with different isolates of Colletotrichum. Genomic DNA was extracted from 72-h-infected plants, and purified for PCR amplification with C. acutatum-specific primers. Primers were designed on the region ITS1-ITS4 including the gene 5.8S rDNA for every molecular group within C. acutatum. PCR amplification was performed with two different primer pairs in the same reaction. PCR products were separated by agarose gel electrophoresis.
Results and conclusions
C. acutatum latent infections in strawberry plants were detected, by PCR with selected specific primers, on DNA extracted from infected plant material. Depending on the bands obtained, four subspecific molecular groups  were detected in C.
acutatum pathogenic to strawberry. This method constitutes a diagnostic tool for a rapid and reliable detection of this quarantine pathogen in strawberry plants without the need for pathogen isolation in pure culture.
This research has been funded by the EU (project AIR3-CT94-1322).
1. Buddie AG, Martinez-Culebras P, Bridge PD et al., 1998. Mycological Research (in press).