USING TERMINAL RESTRICTION FRAGMENT LENGTH POLYMORPHISM TRFLP TO MONITOR CHANGES IN FUNGAL POPULATIONS ASSOCIATED WITH PLANTS
M. Yu, J. Hodgetts, S. Rossall, M. Dickinson
Terminal restriction fragment length polymorphism (T-RFLP) has been widely used as a method for analysing changes in microbial populations. Here, the technique has been further developed using Gaeumannomyces graminis var. tritici (Ggt) as a model system, to show that it can be used as a semi-quantitative measurement of fungal pathogens associated with the roots of plants, and to monitor the survival of pathogens in the soil. To provide an internal reference for measuring fluxes in Ggt populations, primers were designed to amplify products from the fungal ITS2 region and simultaneously from wheat root DNA. Peak height ratios for the fungus relative to the wheat control were then calculated and these ratios were then compared between samples to measure changes in populations. The results showed that the ratio of the Ggt TRF peak height to the wheat TRF peak height could be used as a measure of the relative amount of Ggt in a sample. These ratios correlated well with disease index scores and also with fungal biomass as assessed by determination of ergosterol content using HPLC. T-RFLP can therefore be used directly as a tool for comparison of the relative levels of Ggt and other fungi on different root samples, and by incorporating fixed amounts of wheat DNA as an internal standard into soil samples prior to DNA extraction, the same method could be used to measure fluxes in soil populations of fungi.