Mycol. Res. 104 (8): 927–936 (August 2000). Printed in the United Kingdom. 927 Direct analysis of wood-inhabiting fungi using denaturing gradient gel electrophoresis of amplified ribosomal DNA Eeva J. VAINIO* and Jarkko HANTULA Finnish Forest Research Institute, P.O. Box 18, FIN-01301, Vantaa, Finland. E-mail: eeva.vainio!metla.fi. Received 13 August 1999; accepted 20 November 1999. Four different primer pairs were designed for the preferential PCR amplification of fungal SSU rDNA directly from environmental samples. Most of the amplification products obtained from a reference collection of 46 wood-decomposing fungi could be separated by denaturing gradient gel electrophoresis (DGGE) using 1650 bp rDNA fragments produced by primer pair FR1jNS1. A relatively high level of resolution was also achieved using 390 bp products amplified with primer pair FR1jFF390. In contrast, the separation of amplification products obtained by the remaining two primer pairs (FR1jFF700 and FR1jFF1100; 700 bp and 1100 bp, respectively) was inadequate when applied to our fungal collection. Differentiation between all the species tested was achieved by combined analysis of the rDNA fragments produced by primer pairs FR1jNS1 and FR1jFF390. The DGGE analysis of environmental samples collected from Norway spruce stumps showed that the analysis of fungal DNA extracted directly from wood was usually in accordance with the investigation of mycelial cultures isolated from the same decay column. In some cases, however, disagreement was observed, which suggests that these two fundamentally different techniques present different views about fungal diversity. The investigation of fungal species profiles directly from environmental samples using DGGE analysis of PCR-amplified SSU rDNA molecules can be used to improve the detection of fungal groups that are difficult to cultivate. INTRODUCTION et al. 1997, Ka/ re! n et al. 1997, Johanneson & Stenlid 1999, Jonsson et al. 1999a, Jonsson et al. 1999b). Many fungi, Ecological inventories of wood-inhabiting fungi have tra- however, contain within-species variation in their ITS ditionally been based on inspecting the occurrence of sequences (Anderson & Stasovski 1992, O’Donnel 1992, fruit bodies (Hintikka 1993, Renvall et al. 1995). Since, however, Kasuga et al. 1993, Feibelman, Bayman & Cibula 1994, fruit body development is affected by environmental factors Neuve! glise et al. 1994, Hallenberg, Larsson & Mahlapuu 1996, and many microfungi do not produce visible sporocarps, Norman & Egger 1996, Ka/ re! n et al. 1997, Fatehi & Bridge fruit body distribution may present a limited view of the 1998), which may cause overestimation of species diversity fungal diversity existing as vegetative mycelia (Gardes & when analysis is based on different ITS-types. A more Bruns 1996, Dahlberg, Jonsson & Nylund 1997, Johannesson conserved region of the ribosomal gene cluster might, & Stenlid 1999, Jonsson et al. 1999a, Jonsson et al. 1999b). therefore, be more suitable for the analysis of fungal The use of artificial cultivation media enables the isolation communities containing many unknown species. A promising of vegetative forms of fungi independently of sporocarp alternative would be the SSU (small subunit or 18S) rDNA formation (Rayner & Boddy 1988). The adequacy of culture- gene, which shows very limited within-species variation, but based methods is, however, limited by the selectivity of the different species can usually be separated from each other by isolation procedure used, mainly due to the lack of suitable their sequence (Berbee & Taylor 1993, Olsen & Woese 1993, culturing media for some species. This can be overcome by the Mitchell, Roberts & Moss 1995). analysis of total DNA extracted directly from an environ- RFLP analysis has been the most common method used to mental sample. In theory, the species composition of a mixed investigate sequence variation within the rRNA gene cluster DNA sample can be revealed by PCR-analysis, provided that from uncultured fungal communities (Gardes & Bruns 1996, the amplification efficiencies from different templates are Dahlberg et al. 1997, Ka/ re! n et al. 1997, Johannesson et al. similar. 1999, Jonsson et al. 1999a, Jonsson et al. 1999b). This The ITS region of the rRNA gene cluster has been technique as well as sequence determination, requires a successfully used for studying fungal species profiles directly laborious cloning step in situations where each sample is from environmental samples (Gardes & Bruns 1996, Dahlberg inhabited by more than one species (Helgason et al. 1998). This can be circumvented by the use of denaturing gradient * Corresponding author. gel electrophoresis (DGGE), that separates DNA-fragments Direct analysis of fungal diversity 928 according to their sequences (in addition to length poly- plates (Difco, Detroit MI, USA) checking each sample daily in morphisms) and allows the simultaneous analysis of several order to detect all the emerging fungi. The resulting fungal different sequences PCR-amplified from a single environmental cultures were transferred to MOS (modified orange serum) sample (Muyzer, De Waal & Uitterlinden 1993). Among agar plates (Mu$ ller, Kantola & Kitunen 1994) supplemented several bacterial studies (Ferris, Muyzer & Ward 1996, Heuer with cellophane membranes to facilitate the removal of et al. 1997, Kowalchuk et al. 1997b, Vallaeys et al. 1997, Smalla mycelia for DNA extraction. et al. 1998), DGGE analysis has recently been applied also for eukaryotic communities represented by protozoan (Ciliophora) DNA extraction populations from activated sludge (Marsh et al. 1998), and fungal inhabitants of Marram grass roots (Kowalchuk, Gerards Extraction of DNA directly from wood samples was carried & Woldendorp 1997a) and the wheat rhizosphere (Smit et al. out using a multistep procedure beginning with the homo- 1999). genization of the wood chips using a glass rod and quartz The aim of this investigation was to compare fungal species sand (granulation size 0n1–0n5 mm; Riedel-deHae$ n, Seelze, profiles detected from Norway spruce stumps using two Germany) and disrupting the cells in extraction buffer fundamentally unrelated methods: (i) isolation of mycelial (50 m Tris\HCl, pH 7n2; 50 m EDTA; 3% SDS; 1% beta- pure cultures or (ii) analysis of fungal DNA extracted directly mercaptoethanol) at 65 mC for 1 h. The cell lysate was from wood. In order to test whether these techniques present extracted for five times with phenol\chloroform\isoamyl different views about fungal diversity fungus-specific primers alcohol (25:24:1, by vol.) and twice with chloroform\isoamyl were designed for the amplification of partial SSU rDNA alcohol (24:1, by vol). Further purification was carried out fragments and the DGGE technique was optimised for their using the High Pure PCR Template Preparation Kit (Boehringer analysis. In addition, a DNA purification procedure was Mannheim, GmbH, Indianapolis, USA) according to the developed to enable the PCR-amplification of fungal rDNA manufacturer’s instructions. Finally, the DNA was selectively directly from wood extracts. precipitated by adding 0n6 vol. of a solution containing 20% (w\v) polyethylene glycol (PEG 6000) and 2n5 NaCl and incubating on ice for 20 min. The samples were pelleted by MATERIALS AND METHODS centrifugation in a microcentrifuge (14000 rpm for 20 min), Reference organisms washed with 70% ethanol, dried under vacuum and resus- pended in TE-buffer (6 m Tris\HCl, pH 8n0; 1 m EDTA). The fungal isolates used for primer testing and DGGE The same extraction procedure was used for the fungal pure optimisation are listed in Table 1. This reference collection cultures, except omitting the use of the High Pure PCR includes common inhabitants of coniferous logging residues in Template Preparation Kit. boreal forests. Axenic tissues of Scots pine (Pinus sylvestris), birch (Betula sp.) and Norway spruce (Picea abies), and three different bacteria (Escherichia coli DH5α, Agrobacterium Primer design tumefaciens and Anabaena sp.) were used for the primer Potential primer target regions were located by comparing the specificity testing. complete SSU rDNA sequences of a phylogenetically diverse collection of organisms including 13 ascomycetes, six basidio- mycetes, nine animals and four plants. Sequences with the Field samples following GenBank accession numbers were used: D14165, Sample discs were collected from seven Norway spruce L37537, L37539, L37735, M83257, M83258, M83263, stumps two growth seasons after felling (autumn 1996) in a U00975, X58056, X69845, X69848, X69850, Z27393 single forest stand located in Nummi-Pusula (southern Finland). (ascomycetes); D13460, L22259, L36658, M94337, M94339, The outermost layer (ca 5 cm) of the stumps was discarded U00973 (basidiomycetes); D14365, D15067, L10826, L10827, and discs cut below this surface were visually examined to L49053, U29494, U36270, X53047, Z19562 (multicellular select distinct uniformly coloured decay columns of different animals); and D38245, U18632, X16077, X56105 (plants). The sizes to be used for cultivation of mycelia and direct extraction sequences were aligned using GCG (Genetics Computer of DNA. Group, University of Wisconsin, Madison, USA) software. Three adjacent samples were taken from each decay column The sequence alignment revealed a region near the 3h end as follows: the surface wood was removed aseptically and of the SSU rDNA, which was invariant in the fungi, but discarded, and the first cultivation sample (c1, wood fragment contained differences compared to all the other organisms