P PATTANAMAHAKUL and R STRANGE

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

Background and objectives
Dark leaf spot is a serious disease of the brassica species cabbage (Brassica oleracae var. capitata), cauliflower (B. oleracae var. botrytis), Chinese kale (B. alboglabra), and Choi-Sum (B. chinensis var. parachinensis), all of which are widely grown in Thailand. The disease is thought to be responsible for considerable losses and is favoured by the high temperatures and humidity that prevail there. A morphological examination of the spores suggested that the causal organism(s) was a species of Alternaria, a genus which is notorious for the production of numerous secondary metabolites that are toxic to plants and animals. The objectives of the research were therefore to identify the pathogen to the species level and to determine the structures of any phytotoxic compounds that it produced.

Materials and methods
Spores from lesions on the four plant species were isolated under the microscope, transferred to acidic potato dextrose agar and incubated. The resulting colonies were single-spored and the spores stored in 10% glycerol in liquid nitrogen. Koch's postulates were proved on the plants from which the isolates were obtained.

The isolates were identified by their spore morphology and by sequencing the 5.8S gene and ITS regions of ribosomal DNA. For the latter, the fungi were grown on Czapek-Dox-V8 broth for 48 h and the DNA extracted by a commercial kit (Nucleon Phytopure, Scotlab, UK). The ribosomal DNA was polymerized using the universal fungal primers ITS1 and ITS4 and sequenced using the same primers and fluorescently labelled dideoxy nucleotides in an ABI Prism 310 Genetic Analyzer.

To test for the ability of the isolates to produce phytotoxic compounds, they were grown on Czapek-Dox liquid medium supplemented with eight cations. Culture filtrates were freeze-dried and the products dissolved in buffer. The solutions were tested for toxicity by a cell assay on cells isolated from the four plant species, using fluorescein diacetate as a vital dye.

Toxins were isolated from culture filtrates of a cabbage strain of the pathogen. The filtrates were dialysed against distilled water and the diffusible fraction partitioned against ethyl acetate. Further fractionation was achieved by solid phase extraction using C18 cartridges and by thin layer chromatography.

Results and conclusions
Spores of all isolates appeared identical and were produced in chains (6-12 spores per chain) which were seldom branched. They were nearly cylindrical (15-45 µm long, 10-16 µm wide), had no beak and were olivaceous brown with 1-8 transverse and a few longitudinal septa. These characteristics are typical of Alternaria species. The ITS2 sequences of all isolates showed complete identity and they were also identical to that reported for an authentic culture of A. brassicicola.

Filtrates from cultures grown on Czapek-Dox nutrients, supplemented with cations, were toxic to cells isolated from all four host plants. An isolate from cauliflower was the least toxic and one from cabbage the most. About half the activity of filtrates from the cabbage isolate was retained by a dialysis membrane and the remainder was diffusible and partitioned into ethyl acetate. Purification of the ethyl acetate fraction by solid-phase extraction on C18 cartridges and thin-layer chromatography on silica gel led to the isolation of two toxic compounds with characteristic UV spectra.