Department of Biology, Darwin Building, University College London, Gower Street, London WC1E 6BT, UK

Background and objectives
Blight, caused by Ascochyta rabiei, is one of the most serious diseases of chickpea, causing losses that may be total under cool and wet conditions. The fungus attacks all aerial parts of the plant causing epinasty of petioles and young branches, followed by water-soaking and necrosis. Such symptoms are consistent with the production of toxins by the pathogen. Three toxins were isolated from cultures of the fungus grown on Czapek-Dox medium supplemented with chickpea extract or a mixture of cations [1]. The objectives of the present investigation were to devise a facile method for the production and separation of the solanapyrone toxins, to determine the sensitivity of a range of chickpea genotypes to the compounds, and to investigate the means by which the plant may metabolize them.

Materials and methods
The fungus was grown on Czapek-Dox nutrients supplemented with calcium, cobalt, copper, manganese and zinc cations for 12 days at 20C without shaking. Mycelium and spores were removed by filtration and centrifugation and the filtrate partitioned into ethyl acetate. The ethyl acetate fraction was evaporated to dryness, dissolved in a small volume of dichloromethane and separated by flash chromatography on a silica column (Biotage UK Ltd). The column was first washed with cyclohexane and then eluted with dichloromethane, cyclohexane, ethyl acetate (3:3:1), followed by the same solvents in the proportion (1:1:1) and finally ethyl acetate.

Chickpea cultivars were grown in John Innes compost no. 2 in the greenhouse. Leaflets were cut from 2-3-week-old plants bisected transversely and infiltrated under vacuum with an enzyme solution consisting of Macerozyme (15 mg/ml) and Pectolyase (0.05 mg/ml) dissolved in citrate buffer with glucose as osmoticum. Individual cells were separated from debris by filtration through muslin and washed free of enzymes in buffered osmoticum. They were added to serial dilutions of the toxins in microtest plates and their viability scored after incubation for 3 h using fluorescein diacetate as a vital dye. Results were converted to units of activity where a unit was defined as the concentration of toxin required to kill 50% of the cells. In comparing the sensitivity of cultivars, results were corrected for day-to-day variation by inclusion of cultivar ILC 3279 as a standard in all assays.

Degradation of the solanapyrone toxins was monitored by reversed-phase HPLC using a C18 column and water 56.3%, methanol 23.1% and tetrahydrofuran 20.6% as mobile phase.

Results and conclusions
Solanapyrone toxins A, B and C were produced on the defined medium and were separated from each other and other constituents of the medium by solvent partitioning and flash chromatography on silica gel. When shoots of the plant were placed in solutions of solanapyrone A their stems became shrivelled, whereas when they were placed in solutions of solanapyrone B the stems remained turgid but the leaflets became desiccated and chlorotic - symptoms typical of Ascochyta blight.

The range of sensitivity of cells isolated from leaflets of 12 cultivars of chickpea to solanapyrone A was five-fold with an LD50 value of 3.4 g/ml for the most sensitive. Solanapyrone A was 2-12 times more toxic than solanapyrone B, depending on cultivar. The most sensitive cultivars have also been reported as developing the most severe disease symptoms (Alam, personal communication). Both solanapyrone A and solanapyrone B were metabolized by shoot cuttings, cells isolated from leaflets and by a protein preparation from shoots of the plant.

1. Chen Y-M, Strange RN, 1991. Plant Pathology 40, 401-407.