Rapid detection and estimation of low numbers of spoilage fungi

Two methods were established for the detection and estimation of low levels of some of the spoilage fungi which are responsible for severe crop losses during storage under tropical conditions. The fungi examined produce the enzyme pectinesterase (EC.3.1.1.11) and both methods described, made use of this fact. A modification of the Most-Probable-Number technique (used for estimating Coliform bacteria in liquid samples) was utilized and this enabled low numbers of Aspergillus flavus to be detected and estimated within 18 hours. This method was based on the use of a medium supplemented with pectin in which the pectinesterase produced by the fungus, hydrolyses the ester linkages of the pectin to produce methanol and polygalacturonic acid. This results in pH depression of the medium causing an indicator-dye to change colour. Counts by this method were generally higher than traditional Surface and Pour Plate methods but the method has the advantage of being capable of accommodating large sample volumes. In the course of the MPN work it was found that the amount of methanol liberated varied with the size of the spore inoculum. A dialysis-cell procedure was developed for the removal of the methanol from the medium followed by its estimation by Gas Liquid Chromatography and this formed the basis of the second method. A relationship Y = ce -ß/x was established; where y = log 10 of initial spore number/ml OR dry weight of spore material (mg/ml), x =amount of methanol (~g/ml) released after a fixed incubation time (15 hours) and c, e and e are constants. Linearization of this function resulted in a straight line relationship between spore numbers/ml of inoculum and methanol production (R = -0.963). When dry weight of spores was plotted against methanol production a better correlation (R = -0.988) was obtained. Either of these plots when used as a calibration graph enabled the detection and estimation of spores of some spoilage fungi belonging to the Aspergillus and Penicillium groups in the range 20 - 106 spores/ml within 20 hours. The accuracy obtained was comparable to traditional standard plating methods. The spoilage Yeast strains examined did not obey the above relationship, neither could a generalized equation be produced solely for the yeasts.