Toxigenicity of Fusarium species causing wilt of chickpea

The toxigenicity of isolates of Fusarium for chickpea, Cicer arietinum, the third most important legume crop in the world was studied. Fungi were grown in liquid culture and the culture filtrates assayed on cells isolated from leaflets of the plant. One isolate, designated FOC 5, produced cultures that were predominately red (70-80% of the cultures). When the culture filtrates of all isolates over time were assayed, the red cultures of FOC 5 were much more toxic than those of the other isolates and were also about 10 times more toxic than the colourless cultures of FOC 5. Toxic titres of the red FOC 5 cultures peaked at 12 days when grown at 20 C. The toxin from these red cultures were purified by solvent partitioning, solid phase extraction (SPE), thin layer chromatography (TLC) and high performance liquid chromatography (HPLC) using the assay to monitor the stages in purification. Shaking of culture filtrates of FOC 5 with ethyl acetate resulted in about half the toxic activity (50-55%) partitioning into the organic phase and 25-30% remaining in the aqueous phase. The activity of the aqueous phase was lost on freeze-drying suggesting a volatile compound. When the ethyl acetate phase was dissolved in aqueous acetonitrile and applied to C18 SPE cartridges, about 9% was not adsorbed and 35% could be eluted with methanol. Greater affinity was shown for cyano SPE cartridges with 6% not adsorbed and 45 % recoverable by elution in acetonitrile. Attempts at purification of the toxin(s) of adsorbed and non-adsorbed fractions from these reversed phase cartridges by HPLC did not yield pure products. Recovery of activity of the ethyl acetate phase from flash chromatography on silica gel was 61-110%. However, HPLC demonstrated that several compounds were present in the active fractions Separation of components of the ethyl acetate phase or the fraction adsorbed by cyano cartridges of culture filtrates by TLC on silica gel rather than using SPE, flash or reversed phase HPLC was more successful. Red bands corresponding to the active compound were scraped from TLC plates and eluted in acetonitrile. HPLC of the eluents on a cyano column with 10% acetonitrile as the mobile phase demonstrated a single homogeneous peak with absorption maxima of 224 and 281 nm. The purified fraction is, at the time of writing, being studied by Professor Mike Beale at Rothamsted Research using nuclear magnetic resonance techniques in order to determine its structure. Four other isolates, identified by the International Crops Research Institute for the Semi-Arid Tropics as F. oxysporum f. sp. ciceri did not produce the red, toxic compound, throwing doubt on the correct identification of the isolates. When the sequences of ribosomal DNA of all five isolates were determined, the isolate that produced the red toxic compound most closely matched Fusarium acutatum (99%), in a BLAST search and this accorded with its morphology. A BLAST search showed that three of the other isolates matched the sequence of cotton pathogen, F. oxysporum f. sp. vasinfectum (100%, 100% and 97%) and one closely matched F. oxysporum f. sp. vanillae (99%) These results suggest that a re-evaluation of the taxonomy of Fusarium species causing wilt of chickpea is required.