QUANTITATIVE DETERMINATION OF SELECTED ORGANIC ACIDS PRODUCED BY SCLEROTINIA SCLEROTIORUM FROM INFECTED SOILS AND SOYBEANS

Abstract

As the world’s population continues to increase, food supplies must grow to meet the nutritional requirements. One means of establishing the stability and adequate supply of food is to mitigate crop losses caused by pathogens. Soybean is the world’s most important legume in terms of production and due to its high content of protein (30-40%) w/w and oil (15-22%) w/w. The crop yield is however affected by Sclerotinia sclerotiorum, a ubiquitous phytopathogenic fungus which attacks a wide range of plants. Though the diseases reported in western, Nyanza, Eastern and Rift valley provinces, most data from developing countries such as Kenya is unavailable and this is one of the reasons for carrying out this study. In Kenya Sclerotinia white mould has been. How oxalate metabolism is regulated in plants is currently not well understood. Effective pathogenesis by the fungus requires the secretion of oxalic acid hence understanding the metabolism of oxalic acid is of great importance in the control of this fungus. The aim of this research was to qualitatively and quantitatively determine the selected organic acids suspected to be associated with oxalate metabolism by S. sclerotiorum isolates from infected soybean and soil. Infected soybean varieties (Nyala, Duicker, EAI 3600, and 931) and soil samples were collected from Kisii, Kakamega, Nakuru, and Machakos, and cultured in potato dextrose agar (PDA) on a petridish. The isolates were then sub-cultured in 250 ml flasks for 7days after which culture filtrates were obtained by vacuum filtration through a Buchner funnel containing Whatman no. 1 filter paper. Mycelia were freeze dried and weighed to obtain the dried fungal biomass. The pH of the culture filtrate was determined with a calibrated ion-selective pH meter and concentration of the oxalate, succinate, oxaloacetate, malate and acetate were determined using a high performance liquid chromatography (HPLC) coupled with UV-detector. The levels of organic acids were calculated from the equation of the standard calibration graph. From the fungal biomass weight it was clear that the isolates obtained were of different strains of S. sclerotiorum. The pH of the medium varied from pH 2.1 to pH 4.6 which is as a result of presence of organic acids. All the organic acids were found to be present in culture filtrates with their concentration ranging from 0.002±0.000 to 1.44±0.460 mM. The concentrations of organic acid were found to vary from one region to another and from one soybean variety to another. Oxalate level ranged between 0.008±0.001 and 0.436±0.133 mM. This could suggest presence of different strains of S. sclerotiorum in the regions where the samples were collected. There was a relationship between the amounts of oxalate produced with the pH of the culture filtrate. The results in this study show that all soybean varieties were found to be susceptible to S. sclerotiorum.