Arora, Mary Kathleen Mera Kumari; (1996) Phytoalexin and aflatoxin accumulation in groundnuts differing in susceptibility to Aspergillus flavus. Doctoral thesis (Ph.D.), University College London (United Kingdom).

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Abstract

Imbibed cotyledons of groundnut accumulated phytoalexins in response to wounding by slicing. Those found in high concentrations were isolated by semi-preparative HPLC and identified by their UV and mass spectra as 3, 5, 4'-trihydroxystilbene (resveratrol), 4-(3- methyl-but-l-enyl)-3, 5, 4'-trihydroxystilbene (arachidin III) and 3- isopentadienyl-4, 3', 5'-trihydroxystilbene (arachidin IV). Techniques were compared for the extraction of phytoalexins from cotyledons. Facilitated diffusion in acetonitrile extracted arachidins III and IV but not resveratrol; in contrast, homogenisation in 30[percent] acetone and 95[percent] ethanol extracted resveratrol as well as arachidins III and IV. Phytoalexin accumulation varied according to sample maturity, cv. (cultivar), seed viability and method of elicitation. The amounts accumulated 48 h after slicing, in umoles/g. f. wt., varied from 0. 0 - 0.59 resveratrol, 0.02 - 0.98 arachidin III and 1.11 - 4. 39 arachidin IV. Inclusion of cycloheximide at 50 [mu]g/ml or 100) [mu]g/ml in the water of imbibition reduced phytoalexin accumulation by 69[percent] and 96[percent], respectively. Groundnut development and phytoalexin production were investigated. Immature pods and pegs of cv. TMV2, wounded by slicing, accumulated 0.0 - 0.14 [mu]moles/g.f. wt. arachidin III and 0.17 - 0. 90 [mu]moles/g.f. wt arachidin IV, the amounts varying according to the stage of maturity. Cotyledons of resistant cvs. J11, UF71513 and Ah7223 inoculated with Aspergillus parasiticus, accumulated higher concentrations of phytoalexins than the susceptible cv. TMV2. Phytoalexins in all cvs. reached maximum concentrations 5-8 days after inoculation. Ten days after inoculation, cv. J11 contained 168 ppb aflatoxin whereas cv. TMV2 contained 5,803 ppb. In order to separate phytoalexins from each other and other components in extracts of infected cotyledons a solvent optimisation software package was used. Separation was carried out on a column of ODS silica with ten solvent systems on the same isoeluotropic plane; eluant was monitored by diode array detection. The optimum solvent composition was 9.1[percent] methanol, 1.5[percent] acetonitrile, 11.8[percent] tetrahydrofuran, 77.6[percent] water. Spore topography and the infection process were studied by scanning electron microscopy. Air drying, critical point drying and fixation with osmium tetroxide were compared. Fixation with osmium tetroxide enabled the verrucose nature of conidia of A. flavus to be differentiated from the echinulate nature of those of A. parasiticus. Extracts of groundnut pod and testa were antifungal. The main component of the extract was isolated by flash chromatography, semi-preparative TLC and HPLC. It had a UV spectrum ([lambda] max of 275 and 311 nm) that was similar to 7, 2'-dihydroxy-4'-methoxy isoflavanone.

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