Molecular identification of ectomycorrhizal fungi associated with Dipterocarpaceae

Abstract

Typically, the species that are abundant below-ground will seldom produce fruiting bodies, conversely the species that commonly form fruiting bodies may not be well represented below-ground (Dahlberg, 2001). Therefore, fruiting body observation alone is insufficient for assessing the EM fungal communities. Molecular identification has been recently applied to characterize EM community structure (Buscot et al., 2000; Horton & Bruns, 2001). In this study, we collected and identified the dipterocarp EM roots for both dipterocarps and EM fungi using phylogenetic analysis. Our EM fungi identification was based on the ML region of the mitochondrial genome and the internal transcribed spacer (ITS) region of nuclear genome while Dipterocarpaceae identification was based on the matK and the trnL-trnF spacer region of the chloroplast genome. The objective of this study was to assess the presence and abundance of EM fungi associated with Dipterocarpaceae species in Malaysia. The roots from different species of Dipterocarpaceae were randomly sampled to investigate the diversity of associated EM fungi. EM roots were taken from soil not more than 60 cm away from the trees. We collected one or several root sample(s) from different places around each tree. The species of the trees growing close to the root sampling site were also identified and recorded. We collected 116 root samples in total. The roots were cleaned and examined under a dissecting microscope to confirm the presence of typical structures of the EM roots. The EM roots were kept in 100% ethanol at 4 until further analysis. In addition, we collected fruiting bodies of the following five fungi: Scleroderma sinnamariense, Scleroderma columnare, Amanita fritillaria, Russula alboareolata and Russula sect. Ilicinae. DNA extraction DNA was extracted from the root samples and from fruiting bodies with the CTAB method (Doyle & Doyle, 1987) with minor modifications. Samples were ground to a powder in liquid nitrogen and incubated in 2xCTAB buffer for 2 hours. After chloroform-isoamyl alcohol extraction and RNAse digestion, DNA was precipitated overnight with isopropanol.