1Institut National Agronomique de Tunisie, Laboratoire d'Entomologie, 1082 Tunis Mahrajene, Tunisia; 2Department of Biology, Darwin Building, University College London, Gower Street, London WC1E 6BT, UK

Background and objectives
Ascochyta rabiei causes serious losses of chickpea (Cicer arietinum) wherever the crop is grown under cool and moist conditions. The fungus attacks the aerial parts of the plant causing chlorosis, water-soaking and necrosis. Stems may break when girdled by lesions and finally the whole plant may blacken and die. Alam et al. [1] were the first to describe two toxins, solanapyrone A and solanapyrone C, which were produced when the fungus was grown on a medium consisting of Czapek Dox nutrients and chickpea extract. Subsequently, other isolates were found to produce these compounds as well as solanapyrone B. Sensitivity of chickpea genotypes to the most toxic of the solanapyrones, solanapyrone A, was related to susceptibility to Ascochyta blight (see poster by Hamid and Strange).

The objective of the work described here was to determine the relationship between virulence of Tunisian and Algerian isolates of A. rabiei and their production of the solanapyrone toxins.

Materials and methods
Nine isolates of A. rabiei from Tunisia and five from Algeria were grown on chickpea agar medium supplemented with glucose. When the medium was covered with pycnidia, they were scraped from the plate and suspended in sterile distilled water. The resulting suspension was diluted to 100,000/ml and used to spray to run-off 12-day-old plants of eight chickpea cultivars: Amdoun 1, ILC 482, ILC 72, ILC 3279, ILC 195, ILC 182, FLIP 8346C and FLIP 8479C. Plants were incubated at high humidity for 2 days and for a further 16 days at 20C in a growth chamber. Infection was scored on a 1-9 scale and by the linear infection index [2].

The same isolates of A. rabiei were grown on modifed Czapek Dox medium supplemented with chickpea extract for 12 days at 20-25C. After filtering to remove fungal mycelium and spores, the solanapyrones were partially purified by partitioning into ethyl acetate, the ethyl acetate fraction dried over anhydrous sodium sulphate, and the ethyl acetate evaporated. Samples were dissolved in acetonitrile and injected into an HPLC. The mobile phase consisted of methanol/acetonitrile/tetrahydrofuran/water 21.9/2.0/ 19.8/ 56.3 (v/v/v/v) and the stationary phase was a Spherisorb ODS column 250x4.6 mm. The solanapyrones were identified by their characteristic UV spectra recorded by the diode array detector and quantified by reference to external samples of the authentic compounds.

Results and discussion
The correlation coefficient for production of soalanpyrone A and the mean virulence for the eight genotypes when measured on the 1-9 scale was 0.725 and, when measured by the more objective linear infection index method, 0.716. The six most virulent isolates as measured on the 1-9 scale gave values of 7.8-8.1 and produced 6.08-19.44 micromoles of solanapyrone A per ml culture filtrate, whereas the three least virulent did not produce any solanapyrone. However, these three isolates did produce lesions on all eight cultivars with average scores on the 1-9 scale of 5.1, 4.5 and 3.0, suggesting that either the conditions for production of the solanapyrones in culture were inappropriate, or that other pathogenicity or virulence factors are involved.

1. Alam SS, Bilton J, Slawin AMZ et al., 1989. Phytochemistry 28, 2627-2630.
2. Riahi H, Harrabi MM, Halila MH, Strange RN, 1990. Canadian Journal of Botany 68, 2736-2738.