Identification and Differentiation of Mycorrhizal Isolates of Black Alder by Sequence Analysis of the ITS Region

Mycorrhiza (2000) 10:87–93 Q Springer-Verlag 2000 ORIGINAL PAPER Karin Pritsch 7 Jean-Charles Munch 7 François Buscot Identification and differentiation of mycorrhizal isolates of black alder by sequence analysis of the ITS region Accepted: 25 May 2000 Abstract Twenty isolates of black alder ectomycorrhi- (Agerer 1998). In the framework of the German pro- zas were characterized on the basis of internal tran- gram “Ecosystem research in the Bornhöved Lakes dis- scribed spacer (ITS) DNA sequences and colony mor- trict” (Northern Germany), 16 different EM morpho- phology in pure culture. The isolates were obtained types of black alder were characterized by morphologi- from individual, surface-sterilized mycorrhizas morpho- cal-anatomical studies and PCR/RFLP patterns of the logically identified as the mycorrhizal type “Alnirhiza rDNA internal transcribed spacer (ITS) region (Pritsch cystidiobrunnea”. Analysis of ITS sequences allowed et al. 1997a,b). During these studies, eight morphotypes differentiation into four groups; three were closely re- were identified by corresponding fruit body RFLP pat- lated, while one isolate (BEh-Uw1) was separated by terns, while eight isolates remained unidentified. Of high sequence dissimilarity within the ITS1 and ITS2 these eight unidentified morphotypes, “Alnirhiza cys- spacer regions. Culture morphology was not a satisfac- tidiobrunnea” (Pritsch et al. 1997b) was one of the EM tory differentiating feature for these four groups. An most regularly occurring at all investigated plots, with Ncbi GenBank DNA database search revealed that iso- an estimated proportion of 3–10% per year of all my- lates within the three closely related ITS groups dis- corrhizal types at the two main experimental locations played high homology to ITS sequences of Tomentella at Lake Belau (Pritsch 1996). This morphotype showed sublilacina and Thelephora terrestris, whereas BEh- some color variation with mostly light to dark brown Uw1 had the highest sequence similarity to an ITS but also with almost grayish and whitish forms. As a DNA sequence of a basidiomycete DNA isolated from precondition for physiological studies, isolation of the bamboo leaves. fungal partner into pure culture was successfully performed from mycorrhizal roots of “Alnirhiza cystidio- Key words ITS sequence 7 Ectomycorrhizal isolates 7 brunnea”. Colony morphology varied among different “Alnirhiza cystidiobrunnea” 7 Tomentella sublilacina 7 isolates, again indicating some genetic variability within Thelephora terrestris this morphotype. Because of the varying culture morphology and color of collected mycorrhizal tips, a detailed investigation was started of whether the isolates Introduction of “Alnirhiza cystidiobrunnea” represent a single species or a species group. Although identification of many field-sampled ectomy- The ITS region of the ribosomal DNA was used as a corrhizas (EM) has been achieved, a considerable num- target sequence because the variability of this region is ber of mycorrhizal types described on the basis of mor- suited for phylogenetic differentiation at the species phological and anatomical features remain unidentified and intraspecies level (Bruns et al. 1991). Although the number of published ITS sequences is still restricted, a K. Pritsch (Y) 7 J-C Munch range of sequence data is available from EM fungi. In GSF-National Research Center for Environment and Health, this paper, a comparison of sequences from mycorrhizal Institute of Soil Ecology, Ingolstaedter Landstrasse 1, isolates with the databases is presented. 85764 Neuherberg, Germany e-mail: pritsch6gsf.de Fax: c49-89-31873376 Materials and methods F. Buscot University of Jena, Department of Environmental Sciences, Sampling sites Dornburger Strasse 159, Samples of mycorrhizal roots were taken from an alder forest at 07743 Jena, Germany Lake Belau (30 km south of Kiel, Schleswig-Holstein, Germany) 88 and a second stand approximately 300 km away at Lammer Holz by hand for errors in the automated nucleotide detection. The (Braunschweig, Lower Saxony, Germany). The alder stand at corrected forward and reverse DNA sequences of each isolate Lake Belau with a size of ca. 1 ha is situated on the western shore. were aligned and the resulting consensus DNA sequences were Black alder [Alnus glutinosa (L.) Gaertn.] is the dominant tree further analyzed. The 5b and 3b positions of the ITS1, 5.8 S, ITS2 species (ca. 60 years old) growing intermixed with birch (Betula regions and the 5b start of the large subunit ribosomal DNA (Ta- pubescens Ehrh.) and, in elevated parts at the margin of the stand, ble 1) were set as in the sequence data published by Taylor and with oak (Quercus robur L.) and beech (Fagus sylvatica L.). Con- Bruns (1997). sidering the lake water table, the site may be divided into a gen- A sequence homology search in the GenBank Nucleotide da- erally wet and temporarily waterlogged part adjacent to the lake tabase of the National Center for Biotechnology Information (Lake Belau lakeside, BEl) and a somewhat dryer, elevated part (NCBI), Bethesda (http://www.ncbi.nlm.nih.gov/) was performed 0.5–1 m above the lake water level (Lake Belau hillside, BEh). using the program FastA 3.0 (Pearson and Lipman 1988). Se- The distance between the sampling sites BEh and BEl was ap- quences found during this search and which were included in this proximately 30 m. study for the purpose of sequence comparison to our data are The 2.5-ha stand at Lammer Holz (LH) is a drained alder for- listed in Table 1. In the case of sequence U65607 from GenBank est with a mix of other tree species forming an Alno-Fraxinetum. (Zhang et al. 1997), where the authors defined the span of the Samples at this stand were taken from locations within the forest ITS region slightly differently from Taylor and Bruns (1997), where black alders dominated. A more detailed description of spans and lengths given in Table 1 were adapted for comparison these locations is given by Dilly and Munch (1996) and Pritsch et on the basis of the Taylor and Bruns data. al. (1997a). Phylogenetic analysis Sampling and processing of root samples Two multiple alignments were created using ClustAl contained in Samples of black alder roots were collected from the litter layer ClustalW (Thompson et al. 1994). Gaps at the 5b and at the 3bend and kept at 4 7C during transport and storage. All samples from were excluded and gaps within alignments were treated as missing each location were pooled and cleaned by gentle shaking in tap data. Multiple alignments subsequently were corrected manually water within 24 h. Intact and turgescent-looking mycorrhizas with on a screen using LineUP. All programs are contained in the pro- a red-tipped root apex and mantle features characteristic of fully gram package for HUSAR version 3.0 DKFZ Heidelberg, Ger- active specimens of the morphotype “Alnirhiza cystidiobrunnea” many (http://www.dkfz-heidelberg.de). (Pritsch et al. 1997b) were sorted under a stereo microscope and Alignment 1 was created from all 19 alignable sequences of freed of all visible adherent soil particles. The color of mycorrhi- the mycorrhizal isolates of this study and related sequences from zas was recorded during superficial morphological examination. the ncbi database as indicated in Table 1. Alignment 1 was analyzed using distance-based methods under the Jukes-Cantor mod- el and phylogenetic trees were calculated based on these distant Isolation of fungal strains matrices by the neighbor-joining method (Saitou and Nei 1987). To ascertain relationships found by distance calculations, a 1000 Isolation from mycorrhizal tips followed a protocol modified replicate bootstrap analysis with the neighbor-joining method was from Erland and Söderström (1990). Ten mycorrhizal tips were performed with the program ClusTree, which is part of ClustalW transferred to a nylon 60-mm-mesh sachet and kept humid be- (Thompson et al. 1994) contained in the package for HUSAR tween wet filter papers soaked with a 0.2% solution of Tween 80 version 3.0. until sterilization in 30 ml 0.05% glutaraldehyde (v/v) in 0.05 M During sequence analyses, certain groups of isolates displayed Na-cacodylate buffer (pH 7) under continuous shaking for 3 min. minimal sequence deviation attributable to ambiguous bases and After rinsing three times in 300 ml of sterile distilled water, my- thus revealed almost complete sequence identity over the whole corrhizal tips were cut into pieces of approximately 4 mm length. alignable span. From these groups, with identical sequences, one The pieces of one mycorrhizal tip were plated together onto one representative sequence was submitted to the NCBI GenBank petri dish containing medium b (Kottke et al. 1987). To inhibit database. Accession numbers are given in Table 1. bacterial growth, 300 mg l–1 chlorotetracycline and 300 mg l–1 Based on the results of these first analyses, a second alignment streptomycin sulfate were added to medium b after autoclaving. (alignment 2, Table 1) was created including one representative Outgrowing mycelia were checked microscopically for the sequence as described above. The reduction of the number of se- presence of hyphae with clamps and, if clamps were present, rou- quences was a precondition to enable parsimony analysis. The tinely cultivated on medium b without antibiotics. Inoculation of phylogenetic relationships of sequences in alignment 2 (Table 1) isolates to sterile black alder seedlings according to the method of were analyzed using maximum parsimony-based calculations Wong and Fortin (1989) revealed Hartig net formation for all iso- without or with bootstrapping analyses from 1000 replicate sam- lates of the present study (data not shown). Isolates are main- ples. Parsimony-based analyses were performed under the branch tained in the culture collection of the Institute of Soil Ecology, and bound option using PAUP Portable version 4.0.0d55 for Unix GSF-National Center for Environment and Health, Neuherberg, contained in the program package of HUSAR version 3.0. Result- FRG under the names given in Table 1.

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