Mycoflora and polyphenolics of variously coloured seeds of Bambara groundnut (Vigna subterranea L. Verdc.)

Abstract

Bambara groundnut (Vigna subterranea (L.) Verdc.) is a leguminous crop indigenous to Africa, which is grown by small-scale farmers. It is a protein rich legume crop, which is mostly grown to supply rural and urban poor communities with a nutritious source of food. In this study six cultivars of bambara groundnut seeds differing in seed coat colour were used to determine the seed-borne mycoflora present on the seed and whether this was related to surface topography and/or, the amount and localisation of polyphenolic compounds in the seed. Bambara groundnut seeds were surface sterilised with 1.5% sodium hypochlorite for 5min, plated on PDA petri dishes and incubated at ±25°C. After 7d of incubation, percentage of fungal infestation was determined. It was found that dark coloured seeds (Sb4- 4A and Sb 8-1) of bambara groundinut were less infected, with the exception of one dark cultivar (Swazi Vs A), than the light coloured seeds (As7 and Sbas 1-8). Aspergillus, Fusarium and Chaetomium spp. were found to be the dominant fungal species. Scanning electron microscopy was used to determine if seed infection of the various cultivars could be linked to seed surface topography. Seeds were cut in half, placed directly on stubs and coated with gold. Examination of surface topography indicated that there were no distinct morphological differences among the different cultivars. The seeds had smooth surfaces and although some showed a little shrinkage, none had structures like pores or pits. Hilum structures differed among cultivars and were either tightly or loosely packed. As seed fungal infection could not be linked directly to surface topography, the amount and localization of polyphenolic compounds was then studied to determine whether these factors may influence fungal infection. In the histochemistry study, 10μm seed coat sections were mounted in glycerine or vanillin-HCI. Other sections were stained with Neu reagent. Illumination was recorded at 420nm and 360nm for blue and UV light respectively. Thin layer chromatography (TLC) was performed to separate phenolic compounds. It was found that dark coloured seeds were stained dark red showing the presence of polyphenolic compounds in the epidermis and parenchymatous layer. Dark coloured seeds emitted a strong blue fluorescence under UV illumination indicating the presence of large quantities of polyphenolic compounds in the epidermis. This was not apparent in the light coloured seeds. Under blue illumination, the green colouration of the epidermis indicated the presence of flavonoids, which also appeared to be more apparent in darker seeds than in light coloured seeds. This difference is brought about by the different amounts of total phenolic compounds in the seeds. The amount of phenolic compounds was higher in the seed coats than in the embryo and cotyledons. A TLC study showed more compounds in the dark coloured seeds than in light coloured seeds. The results of this study indicated that the pdlyphenolic compounds present in the seed coats of bambara groundnut may indeed play a role in the resistance or susceptibility of different coloured seeds to fungal infection.