2.2.40
CHARACTERIZATION OF PORTUGUESE PATHOTYPES OF ASCOCHYTA RABIEI BY RAPD

MT CARVALHO1, MJ GOMES2, PT AMARO2, A CLEMENTE3 J MATOS3

1Est. Nac. Melh. Plant., Elvas, Portugal; 2DGPC, Tapada da Ajuda, Edif. I 1300, Lisboa, Portugal; 3INETI/DB, 1699 Lisboa Codex, Portugal

Background and objectives
Chickpea is an economically valuable culture that is well adjusted to the Mediterranean climate. Traditionally, in Portugal, the plant is sown during springtime, which avoids Ascochyta blight infections but drastically decreases production rates. The autumn sowing is highly preferable, but these cultures tend to be much more vulnerable to Ascochyta rabiei infection which destroys a vast percentage of the crops.

The virulence of this pathogen has been studied and identified as the major restriction for the winter growth of chickpea [1]. The selection of A. ;rabiei-resistant chickpea genotypes is being performed at our Centre. However, there is no data available on the genetic variability of the pathogen, which is a prerequisite for a breeding programme focusing on resistance selection [2]. This work shows the existence of genetic variability of A. ;rabiei which was determined by RAPD analysis of different isolates of the fungus.

Materials and methods
Ten different isolates of A. ;rabiei were selected from five different regions from the south of Portugal. DNA was extracted from freeze-dried mycelia using standard procedures and PCR reactions were performed in an Hybaid Omn-E thermocycler. A set of 24 random 10-bp primers was tested from Operon and Advanced Biotechnology. Several different polymerases were tested for reproducibility analysis and electrophoresis agarose gels were analysed with BioCapture software (Vilber-Lourmat).

Results and conclusions
The 10 different A. ;rabiei isolates analysed by RAPD were found to present significant polymorphism that allows the identification of different classes within this pool of samples. No individual typing was made possible with a single random primer, but several classes have been identified with three different markers. Four primers (OPJ1, P3, P5, P6) have been tested which have already been used with this species by other authors. The results obtained with the Portuguese isolates have shown amplification patterns that greatly differ from the ones found by those authors. This seems to indicate that our isolates present a great degree of polymorphism when compared with isolates from other countries. The data shows the importance of pursuing the typing of these isolates and the advantages of such work in the selection of a broad-range resistant chickpea genotype.

The reproducibility of the technique has also been tested in the present work. It was found that under the same PCR conditions, results can differ to a great extent when using different polymerases.

References
1. Carvalho MT, 1988. Melhoramento 30, 151-164.
2. Porta-Puglia A, Tivoli B, 1997. Grain Legumes 16, 16-17.