Pattanamahakul, Preprame; (1999) Identification and toxigenicity of Alternaria brassicicola, the casual agent of dark leaf spot of Brassica species grown in Thailand. Doctoral thesis (Ph.D), UCL (University College London).

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Abstract

Fungi, with spores characteristic of the genus Alternaria, were isolated from necrotic lesions on leaves of cabbage, cauliflower, Chinese kale and choi-sum, growing in Thailand, and were proved by Koch's postulates to be the causal agents of a disease known as dark leaf spot. All isolates corresponded in morphology to descriptions of Alternaria brassicicola and the identification was confirmed by analysis of the internal transcribed spacer (ITS)1, 5.8S gene and ITS2 regions of rDNA, the nucleotide sequences of all isolates tested (two isolates from each of the host plants) being identical to each other and to the published sequence of a known isolate of A. brassicicola. Comparisons of the ITS1 and ITS2 sequences of A. brassicicola with published sequences of these regions from other species of Alternaria showed that the fungus was closely related to two other pathogens of brassicas, A. brassicae and A. raphani. Culture filtrates of isolates of the fungus from each host, grown on a defined medium consisting of Czapek-Dox nutrients supplemented with cations, were toxic to cells isolated from the four host plants. Filtrates from the cabbage isolate tested on cabbage cells gave the highest toxin titres and those from the cauliflower isolate the least on all cell types. Among the other combinations filtrates of the choi-sum isolate were most toxic to choi-sum cells while those of the Chinese kale and cauliflower isolates were most toxic to cells of choi-sum and cabbage, respectively. Since the highest titres were obtained for the combination of filtrates from cabbage isolates and cabbage cells, this experimental system was used in studies of the toxic factors. Up to half the activity of filtrates 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 a C-18 cartridge, flash chromatography on a pre-packed C-18 cartridge, high performance liquid chromatography on an ODS column and thin layer chromatography on silica gel led to the isolation of four toxic compounds with characteristic UV and mass spectra.

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