Correct identification of wood-inhabiting fungi by ITS analysis
By - 12/17/2008
Annette Naumann #, Mónica Navarro-González # , Olivia Sánchez-Hernández # , Patrik J. Hoegger # and Ursula Kües #*
Georg-August-University Göttingen Büsgen-Institute Section Molecular Wood Biotechnology and Technical Mycology Büsgenweg 2, D-37077 Göttingen.
These authers contributed equally to the study#
*For correspondence : ukuees @ gwdg.de
Current Trends in Biotechnology and Pharmacy, Vol.1 (1) 41-61 (2007) ISSN :0973 - 8916
Abstract
In this study, we established ITS sequences from 20 strong wood degraders from different fungal genera and from 15 strains of the coprini which are occasionally found on wood. These se- quences were used in blast searches to evaluate their species identity. ITS sequences confirmed for 25 strains previous morphological species de- termination. ITS sequences from Coprinopsis scobicola were determined for the first time from DNA of two different strains. Finally, eight strains were found assigned to a wrong species or assignments were uncertain. We present prob- lems encountered in fungal identification by mo- lecular data, such as difficulties in previous mor- phological identification, occurrence of contami- nations or mixing-up of strains in fungal culture collections, and wrong entries in the NCBI GenBank sequence dataset. A long-term target of ITS sequencing of wood-inhabiting fungi is to establish a specific database that can be either used for blast searches or in ITS barcoding for species identification. We present first studies in ITS barcoding of coprini.
Key words
Wood degraders, Coprinoid mushrooms, misidentifications, phylogenetic analysis, barcoding
Introduction
Wood is threatened by various types of fungal damage. Blue stain fungi grow within parenchy- matic cells in wood and live from storage prod- ucts within these cells. They do not influence dimensional properties and stability of the wood. However, infestation by blue stain fungi makes wood unattractive for use because of melanin in the fungal cell walls that gleams in dirty blue- greyish colours through the wood. Furthermore, moulds might grow on the surface of wood spoil- ing its looks as well as being a hygienic problem by abundant production of asexual spores. Other fungi attack the wood structure by degrading cell wall compounds. Three major kinds of fungal decay are distinguished. White rot fungi degrade the lignin in the plant cell walls while brown rots and soft rots preferentially degrade cellulose and hemicellulose. As a result of such decay, stabil- ity of the wood is affected in early stages until the decay is completed leaving white strings of cellulosic materials in case of white rot, brown cubicles of lignin in case of brown rot and the middle lamellae in case of soft rot (1). Under certain environmental conditions such as higher levels of humidity and when in contact with soil, wood in service is in danger to be in- fested by destructive fungi. Early detection of fungi can be crucial, e.g. for rescuing houses in case of a dry rot infestation in a wooden construc- tion or for avoiding health problems such as spore allergies in case of moulds on wood. In living trees, early detection can help to decide upon measures to be taken against fungal pathogens. Early detection of harmful wood-inhabiting fungi implies in the best possible case also species identification [discussed further in references (2), (3) and (4)]. Traditionally, species identification has been done in late stages of infestation when sexual fruiting bodies occur or asexual spores are produced on the surface of infested wood (5). This, however, does not exclude that there are other species present in the wood not producing any obvious morphological structures.
Such fungi might be isolated from wood on suitable artificial medium for further evaluation of myc- elial cultures. Despite that not all fungi might be growing in culture or species might be overgrown by others during the isolation process (6), iden- tification by mycelial growth characters is very difficult or often impossible to perform even for a trained mycologist (7). Modern molecular bi- ology offers more accurate ways of identifica- tion. Very common is to apply sequencing of PCR (polymerase chain reaction)-amplified ITS (internal transcribed spacer) fragments, non-cod- ing regions localized in between ribosomal RNA genes (rDNA). rRNA gene clusters are conserved within species, but differ in sequence between species with the degree of differences reflecting distance of relation between organisms [(1), (4), (Fig. 1)]. PCR-amplification of ITS sequences can be applied on isolated fungal cultures but also on DNA obtained from wood samples without prior fungal strain isolation (8)-(12), our unpublished results]. In such a way, any bias by loss of species due to non- or unequal growth on artificial medium during fungal isolation is overcome.
Although wood-inhabiting fungi are often del- eterious for wood as such, there are various bio- technological applications for such fungi [(1), (13)]. A variety of species are used in mushroom production on lignocellulosic waste materials from agriculture and forestry (14). Particularly the white rot fungi are sources of enzymes such as cellulases, hemicellulases, peroxidases, laccases, and others used in various types of in- dustry. Furthermore, such fungi are appointed in bioremediation of contaminated soils, purifica- tion of industrial effluents, etc. [(15), (16)]. Also in biotechnology, it is of interest to unequivo- cally identify the species for an exact definition of the fungal material applied in a process. Whilst analysis of ITS sequences is technically easy, there are, however, also problems related to this molecular approach. Established se- quences are usually submitted to public databases such as the NCBI GenBank (http:// www.ncbi.nlm.nih.gov/ ) or the EMBL Nucle- otide Sequence Database (http://www.ebi.ac.uk/embl/). These databases are open to submissions by everybody, with the consequence that up to 20% of sequence entries within a group of fungi may be faulty [(4), (17)]. Reasons for this can be that a species name has been assigned by ITS sequence identity to a formerly wrongly assigned species, that a parallel mycological identifica- tion by morphological characters was wrong, that another mycelium has been isolated unnoticed from a morphologically assigned fungal struc- ture taken non-sterile from nature, that unseen contaminations occurred in isolates in culture, or that fungal cultures were accidentally mixed up in a laboratory. Searchable experts´ sequence databases with controlled data entries are there- fore required [(1), (4)]. In this paper, we present case studies on ITS se- quences obtained from fungal cultures for the long-term goal to establish a large sequence da- tabase on wood-inhabiting fungi.
Material and Methods
Fungal strains and culture conditions: Fungi were routinely grown at 25°C or 28°C on 2% malt extract (agar) with the exception of Coprinopsis cinerea strains that were cultured at 37°C. Aerial mycelia were scraped from the surface of the agar for chromosomal DNA isola- tion by established methods [(18), 19)]. Origins of fungal strains used in this study for ITS sequencing are given in the footnotes of Table 1 and 2. Coprinoid mushrooms collected from the wild were morphologically identified according to the keys by Uljé (20) and Orton and Watling (21). Inner tissues from mushroom stipes were used to establish further fungal cultures for DNA iso- lation and barcoding by ITS sequence amplifi- cation. Coprinus comatus strains 1-2004, 2-2004, MN-23, MN-24 and MN-25 were mycelial iso- lates obtained from stipe tissues from independ- ent mushrooms collected in 2004 and 2006 on the grounds of the North Campus of the Georg- August-University Göttingen. Coprinellus micaceus strain MN-7 was isolated from a colony of fruiting bodies found in year 2006 on the grounds of the Georg-August-University Hos- pital in the bifurcation of a forked Robinia pseudoacacia tree breaching through its bark.
ITS sequence characterization: PCR was performed with ca 5 ng DNA of a fungal isolate in a total volume of 25 µl containing 10 mM Tris pH 8.8, 50 mM KCl, 0.1% Triton X-100, 1.5 mM MgCl 2, 0.2 mM dNTPs (Fermentas, St. Leon- Rot, Germany), each 0.4 µM of primer ITS1 [5'- TCCGTAGGTGAACCTGCGG-3' (22)] and primer ITS4 [5'-TCCTCCGCTTATTGATATGC -3', (22)], and 1 U of Taq DNA polymerase. PCR conditions were 2 min initial denaturation at 94 °C, followed by 35 cycles of 30 s at 94 °C, 30 s at 55 °C, and 30 s at 72 °C, and a final extension at 72 °C for 10 min. 20 µl per PCR reaction were precipitated with isopropanol and resuspended in 20 µl H 2O. Restriction enzyme digests and agarose gel electrophoresis of purified DNAs were performed by standard methods (23). For cycle sequencing reactions, 2 µl of the purified DNA were mixed with BigDye Terminator v3.1 (Applied Biosystems, Darmstadt, Germany) ap- plying a quarter of the reagents recommended by the manufacturer and either primer ITS1 or ITS4. After cycling following the manufacturer’s instructions, the reaction products were purified by sodium acetate/ethanol precipitation and washing with 70% ethanol and subsequently run on an ABI PRISM 3100 Genetic Analyzer (Ap- plied Biosystems). Obtained sequences were compared against the nucleotide entries in the NCBI GenBank by using search tool blastn (http://www.ncbi.nlm.nih.gov/). Identity values were taken either from the blastn results or from alignments performed by the tool for blasting two sequences bl2seq (http://www.ncbi.nlm.nih.gov/). For phylogenetic analysis, appropriate sequences were taken from the NCBI database and aligned with sequences established in this study using ClustalX (http://www-igbmc.u-strasbg.fr/ BioInfo/ClustalX/Top.html) and GeneDoc ver- sion 2.6.002 (http://www.psc.edu/biomed/ genedoc/). Phylogenetic trees of the nucleotide sequence alignments were calculated by the neighbour joining method of the programme MEGA version 3.1 (24).
Results and Discussion
Evaluating correct species assignment in a se- lection of wood-rotting fungi
ITS sequences were determined from 20 strains of strong fungal wood-decayers. The ITS se- quences of fifteen of them matched perfectly or nearly perfectly to sequences published in NCBI GenBank for strains registered under the same species names (Table 1). In the special cases of Polyporus brumalis FBI 5 and Trametes versi- color 051130.2, each two sequences were estab- lished that distinguished by a 1 bp insertion (Table 1). Such restricted nucleotide polymor- phism within one strain is not uncommon in higher basidiomycetes [(25)-(27), Hoegger et al. unpublished]. It is also quite often observed be- tween strains of a same species [(28)-(30)]. Spe- cies identification by ITS sequences is thus not always relying on 100% sequence identity but can include a certain degree of variation [(30), Table 1] that however might indicate ongoing specification in diverging morphotaxons [(31), (32)]. The five further cases of ITS sequences estab- lished in this study are discussed in the follow- ing: Pleurotus ostreatus var. florida N001 is a com- mercial mushroom production strain used in ge- netic characterisation of the genome of the spe- cies (33).
The ITS sequence of the strain is pre- sented for the first time. It is 100% identical to region 61-654 of sequence AY540322 from a fungus identified as Pleurotus floridanus. P. floridanus is considered a synonym for P. ostreatus (34). For Gloeophyllum odoratum, an ITS sequence was so far not present in the NCBI database. In blastn searches, the most similar sequence to the ITS sequence of strain G. odoratum 050601.3 was therefore from Gloeophyllum abietinum (AJ420947) with 86% identity. The ITS sequence from Trametes zonata 921011.17 was 99% identical to an ITS sequence from a strain called Trametes versicolor 3633 (AY840580) and 98% identical to an ITS se- quence from a strain called Trametes ochracea PRM 900601 (AY684177). T. ochracea is a syn- onym of T. zonata (35). Tomsovsky et al. (36) recently published a first extensive phylogenetic analysis of the genus Trametes. The ITS sequence of their T. versicolor voucher strain PRM 900594 (AY684179) showed 98% identity to our se- quence. Many of the GenBank entries involving the genus Trametes are mislabelled. There are several T. versicolor ITS sequences wrongly at- tributed to other fungal genera (4) but by the dif- ficult morphological definitions of Trametes fruiting bodies there are also various ITS se- quences assigned within the genus to a wrong species.
The high level of identity of ITS se- quences from different Trametes species makes species definition in this genus difficult. A thor- ough analysis of ITS sequences in larger num- bers of Trametes isolates is in progress (Hoegger et al. unpublished). The ITS sequence of a strain from the institute’s collection labeled Coniophora arida FBI 516 was 99% identical to region 30-677 of the ITS sequence from Coniophora puteana strain EBW 15 [(AJ344110), (37)]. Fig. 2 shows that in a phy- logenetic tree of C. arida and C. puteana ITS sequences taken from GenBank, the two species clearly distinguish from each other. The species concept within Coniophora is difficult since there are few and unstable morphological characteris- tics, especially for mycelia but also for fruiting bodies [(1), (7), (38)]. Present species identifi- cation is therefore based on molecular methods by comparing ITS sequences with those of stan- dard strains [(1), (32), (37)]. In conclusion, by the clustering of the strain from our institute’s collection with the C. puteana reference strains (Fig. 2), we suggest to rename our strain C. puteana. Regions 54-470 and 598-648 of the ITS sequence of our strain showed only 91% and 88% identity, respectively to regions 91-510 and 583- 630 of the reference strain MUCL 30844 of C. arida (AJ345007).
The most complicated case links to the strain FBI 52 from our institute’s collection labelled Trichaptum abietinum (Hirschioporus abietinus). Originally obtained as H. abietinus CBS 376.68 from the CBS culture collection (Centraalbureau voor Schimmelcultures), this strain has been stored for decades in our institute´s collection. In blastn searches of the ITS sequence of this strain, perfect or nearly perfect hits (98-100% identity) were obtained with a number of se- quences ; that should represent either Entrophospora ; sp., Ceratobasidium stevensii, Thanatephorus cucumeris (or its anamorph Rhizoctonia solani) or Bjerkandera strains. The one assignment (AY035664) of a soil fungus from a community of arbuscular mycorrhizal fungi to Entrophospora sp. (39) is easily identi- fied as being wrong by the fact that the genus Entrophospora belongs to the phylum Glomeromycota that is evolutionary distantly related to fungi from the phylum Basidiomycota in the subkingdom Dikarya (40). A number of NCBI GenBank submissions labelled T. cucumeris or with the anamorph name Rhizoctonia (AF455419, AF455435, AF455438, AF455459, AF455461, AF455463, AJ000198, AJ276054, AY443531, DQ117961, DQ278948, DQ426512, DQ426519, DQ426529) as well as one submission labelled Ceratobasidium stevensii (AJ427405) from the same fungal fam- ily Ceratobasidiaceae of the Basidiomycota have also to be considered as wrongly assigned.
This mislabelling likely goes back to sequence AJ000198 obtained from a strain T4 (IMI 360314, sequenced also in another laboratory: DQ278948) listed as Rhizoctonia solani isolated from rice from Ghana (41). Johanson et al. (41) noted at the time of first analysis that this se- quence was highly dissimilar to sequences from Rhizoctonia labelled strains from other sources. The other sequences were obtained later in his- tory from sequences amplified by PCR from air in hospital laboratory environment (DQ426512, DQ426519, DQ426529), nasal mucus (42) and leaf material from Ipomea asarifolia (43) and from fungi from root material of orchids (44). The ITS sequence from the single strain labelled C. stevensii came from a fungus that has been isolated from a twig of an apple tree (AJ427405). The final large group of about 20 sequences with high similarity to our ITS sequence represents the genus Bjerkandera. Among these are at least three sequences from independent isolates that were identified morphologically as Bjerkandera [(45), (46), DQ060096]. From strain Bjerkandera adusta VH57 (AB096737) in addition the se- quence from the 28S rDNA gene is known that matches with 98% identity that of strain B. adusta DAOM 215869 [(45), (47), (48)]. Mor- phological identification of at least four differ- ent strains argues strongly for correct assign- ments. These and some other Bjerkandera se- quences were extracted from the NCBI GenBank and used to construct the phylogenetic tree shown in Fig. 3. Two of the sequences had only 95-96% identity to the sequence of our strain and were obviously from the same species (Bjerkandera fumosa according to AJ006673) closely related to B. adusta (Fig. 3). The only entry labelled T. abietinum (U63474) showing a high degree iden- tity to our sequence (96%) was also included and two other T. abietinum labelled entries not hit in the blastn search by our ITS sequence.
When comparing each two sequences in the direct alignment, the ITS sequence of our strain matched only in each two short stretches to the sequences U63475 (190-353 with 96% identity and 540-572 with 96% identity) and AY781273 (190-353 with 98% identity and 510-533 with 95% identity). Finally, ITS sequences from Trametes versicolor ( AY354226), Ganoderma tsuga (DQ206985) and Polyporus squamosus (DQ267123) from the Polyporales were included in the phylogenetic analysis and the ITS sequence from Schizophyllum commune (AF249390) from the Agaricales was used as an outgroup (Fig. 2). The sequence submitted under U63474 clearly clustered with Bjerkandera sequences, although in an own short branch, whereas the sequences of the two other T. abietinum -labelled species grouped further away from the sequences of the three other genera of the Polyporales (Fig. 2). Grouping with B. adusta has been noted before and interpreted as likely misidentification or mis- labelling of the respective strain (49). The ITS sequence established in this study for strain FBI 52 implies that there has been also an error with this strain. T. abietinum CBS 376.68 is also known as DAOM 72245A. Ko Jung (50) analyzed the mitochondrial small subunit ribos- omal DNA of DAOM 72245A and found it to cluster with those of other T. abietinum strains. It is thus likely, that a mislabelling has happened at some point in the past in case of strain FBI 52. The ITS analysis implies that the strain in our collection should be relabelled as B. adusta.
Evaluating ITS sequences from coprini
Coprini present a group of approximately 200 different species that, with a few exceptions, are very difficult to distinguish from each other by morphological means. Coprini have been com- piled in one genus Coprinus until recently, when molecular data divided them into four new gen- era belonging to the Psathyrellaceae (Coprinopsis with an estimated 100 species, Coprinellus with more than 40 species and Parasola with currently 18 defined species) and the Agaricaceae (Coprinus with just three spe- cies), respectively (51). Most species grow on dung and plant litter in soil but about a third of the species has been observed growing also on (decaying) wood (Navarro-González, PhD the- sis in preparation). A collection of coprini ob- tained from different sources (Table 2) for test- ing of growth behaviour on wood (Navarro- González, PhD thesis in preparation) were sub- jected in this study to an analysis of ITS se- quences. ITS sequences verified species identity of two C. comatus strains, of a Coprinellus radians strain, 3 Coprinellus xanthothrix strains, and a Coprinopsis cinerea strain (Table 2). ITS se- quences for Coprinopsis scobicola were so far not available and we provide new ITS sequences from two monokaryotic strains well established in mating type analysis of C. scobicola [(52)- (53)]. Misidentified within the family of the Psathyrellaceae were obviously the strains la- belled as Coprinellus disseminatus C50, Parasola plicatilis C65, and Coprinopsis atramentaria C67. The first two should present two different further to be defined species from the genus Coprinellus, the third one a further to be defined species from the genus Coprinopsis (Table 2, Fig. 4). Mushrooms of the related species Coprinopsis lagopus and Coprinopsis lagopides are very dif- ficult to distinguish and misidentifications are easily possible (20). The ITS sequences of the Coprinopsis strains labelled C. lagopus C215 and C. lagopides C262 are both related to the NCBI sequence AF345815 assigned to C. lagopides and poorly to the NCBI sequence AF345813 assigned to C. lagopus . (Table 2, Fig. 4).
Since further background on the sequences AF345815 and AF345813 is not available, at the current state a complete species designation to the two tested strains is better left open until a more thorough analysis on the two species with several differ- ent isolates has been made. Finally, the snow mould used as Coprinopsis psychromorbida in microbiological practicals to demonstrate growth at low temperatures (2-4°C, optimum at 15°C) turned out to be an unknown basidiomycete with clamp cells at its hyphal septa. The only highly similar sequence (98 % identity) in the NCBI database is from an uncul- tured basidiomycete from forest soil (AY969953). The next similar sequence is from Athelia bombacina (ABU85795) (86% identity) belonging to the family of Atheliaceae in the Atheliales (Table 2, Fig. 4).
ITS in molecular barcoding of coprini
Once ITS sequences have been established and positions of recognition sites of restriction en- zymes deduced from these sequences, the infor- mation can be used as barcodes in species iden- tification (54), eliminating the need for further tedious sequencing and allowing also less equipped laboratories without modern sequencers an easy access to molecular species identifications. In the following, we describe ex- amples of barcoding of new isolates of C. comatus and Coprinellus micaceus. The edible C. comatus (Fig. 5 A to C) able to grow on wood (Navarro-González, PhD thesis in preparation) is not as easily mixed up with other coprini by the large size and the character- istic elongated-ovoid shape of the mushroom cap, the floccose, shaggy scales on the cap surface, a white annulus on the stipe upon opening the cap and an elastic cord suspended in the stipe that is not found in coprini belonging to the Psathyrellaceae (21). PCR-amplified ITS frag- ments of new isolates from C. comatus mush- rooms collected at different times and places on the grounds of the North Campus of the Georg- August-University Göttingen and a PCR-ampli- fied ITS fragment of the C. comatus strain C108 were digested with restriction enzymes HinfI and HhaI (Fig. 5 E and F). All showed the same band- ing patterns. Within the error range of the agarose gels, the size of the DNA fragments obtained cor- responded well with the expected sizes (Fig. 5 D). The ITS sequence of the closest related spe- cies, Coprinus sterquilinus (AF345821), is iden- tical in size to the ITS fragment of C. comatus strains. HinfI and HhaI digests can however clearly distinguish these two species by the fact that in the C. sterquilinus sequence the first HinfI site is missing (not shown).
Other genera of the coprini containing many more species than the newly defined genus Coprinus are more difficult to categorize. Fig. 6 shows an analysis of a strain isolated from easy to recognize fruiting bodies of the tree-patho- genic species C. micaceus. The HhaI restriction pattern of its ITS sequence is identical to that of strain C56 (Fig. 6C) that according to ITS sequencing (Table 2) likely presents a C. flocculosus strain. In contrast, the HinfI digests distinguish these two species (Fig. 6B). An evalu- ation of ITS sequences of other species of the genus showed that HhaI and HinfI digests are not always sufficient to distinguish C. flocculosus from other species such as Coprinellus verrucispermus and e.g. Coprinellus radians from species such as Coprinellus xanthothrix on agarose gels (Fig. 7). Whilst these four species divide into two groups due to a restriction site for HhaI shared only between C. flocculosus and C. verrucispermus, a third digest for example with enzyme DpnI is required to resolve the oth- erwise alike species (Fig. 7). In contrast, C. micaceus is easily recognized from the other species by a ; HinfI site shared only with C. disseminatus that in turn is characterized by multiple HhaI and HinfI sites (Fig. 7). Coprinellus curtus ( of which currently only a partial ITS sequence is available – AY461834) is distinct from all discussed species by lack of HinfI and/or HhaI sites (Fig. 7). Considering only 7 different species is a start of barcoding differ- ent members of the genus Coprinellus through restriction enzyme analysis of PCR-amplified ITS fragments. It is to be expected that in barcoding of the over 40 species of the genus, further enzymes will have to be incorporated in such ITS analysis.
Conclusion
In this study, we established the ITS sequences from collections of fungal strains of wood-in- habiting species and of a collection of coprini, of which several also occur on wood (Navarro- González, PhD thesis in preparation). To the best of our knowledge, in 71 % of the cases (25 of totally 35 sequences), ITS sequences confirmed species names assigned previously to the isolates. For one species (C. scobicola), ITS sequences were determined for the first time making use of different two strains. Two different strains (Bjerkandera sp. and an unidentified snow mould) were assigned to a wrong genus and in the six other cases left, strains were assigned to a wrong species within a genus (considering in case of Coprinellus sp. C65 the traditional ge- nus Coprinus) or assignments were uncertain. Our study documents the difficulty of correct identifications of fungal species by molecular means due to various types of errors that can oc- cur in the process (e.g. wrong morphological identification of a species, mixing up cultures in stock collections, assigning names to strains by wrong entries in the sequences databases). Once introduced into the databases, such mistakes are carried on and tend to multiply. To avoid mistakes in molecular identification, correct linkage between morphological charac- teristics and DNA sequences are required and in best cases, fungal voucher strains have been de- posited in a suitable strain collection allowing at any time a control (55). Since wrong entries in public databases cannot be avoided by the prin- ciple that submitting of sequences is open to eve- ryone, separate databases controlled by experts are required as already founded for mycorrhizal species and certain groups of food-inhabiting fungi (4). With enough numbers of species en- tries, such databases can then on the one hand be used for blast searching of sequences. On the other hand, such data are open to be used with suitable computer programs in the technically easier to perform barcoding of species.
Acknowledgements
We are very grateful to M.P. Challen, O. Holdenrieder, T.Y. James, S. Peddireddi, L. Ramírez, D. Rigling, K. Voigt, and H.A.B. Wösten for supplying fungal strains for our stud- ies. We thank Alexandra Dolynska for excellent technical work performed in frame of EFRE grant 2001.085 from the European Fund for Re- gional Development. AN holds a grant “Eigene Stelle” (NA 749/1-1) awarded by the DFG (Deutsche Forschungsgemeinschaft) for estab- lishing new methods for detection and identifi- cation of fungi in wood. MN was financially supported by a PhD scholarship (170607) awarded by CONACYT (Mexico). In frame of a grant (0330551) for analyzing fungal behaviour on beech and grand fir wood to UK, the BMBF (Bundesministerium für Bildung und Forschung) supports a long-term database project on fungal ITS sequences. The laboratory Molecular Wood Biotechnology was established by funds from the DBU (Deutsche Bundesstiftung Umwelt).
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Table 1 : Strains of wood-rotting fungi analyzed by ITS sequencing
Origins of strains:
#Origins of strains:1 isolated by O. Holdenrieder (Zurich);2 obtained from L. Ramírez
(Pamplona);3 monokaryons 4-39 and 4-40 were obtained from H. Wösten (Utrecht) and mated
by S. Peddireddi (Göttingen); 4 from the culture collection of the Institute of Forest Botany
(FBI) in Göttingen (now Büsgen-Institute); 5 obtained from K. Voigt (Pilz-Referenz-Zentrum
Jena),6 obtained from D. Rigling (WSL, Birmensdorf).
+Note that a few nucleotides at the ends of the ITS fragments can be missing due to poor
labelling in the sequencing reactions of PCR-amplified fragments.
*Species names were newly assigned according to the results from the comparison with ITS
sequences deposited in the NCBI GenBank.
Table 2 : Coprini analyzed by ITS sequencing
Origins of strains:
#Origins of strains:1 obtained from T. James (Duke University);2obtained from M. Challen
(Wellesbourne);3 from O. Holdenrieder via the fungal collection of the Institute for Microbi-
ology, ETH Zurich
+ Note that a few nucleotides at the ends of the ITS fragments can be missing due to poor
labelling in the sequencing reactions of PCR-amplified fragments.
* Species names were newly assigned according to the results from the comparison with ITS
sequences deposited in the NCBI GenBank.
Fig. 1: Sector from an rDNA cluster with a partial 18S rRNA gene, ITS 1, a 5.8S rRNA gene, ITS 2, and a partial 28S rRNA gene. The positions and directions of primers ITS1 and ITS4 are marked to indicate the region to be amplified in ITS analysis.
Fig. 2: Phylogenetic tree of ITS sequences from the genus Coniophora from the Boletales. Cp = ITS sequences from strains marked with Coniophora puteana, Ca = ITS sequences from strains marked as Coniophora arida. Codes indicate NCBI GenBank accession numbers. Stars mark the ITS sequences from reference strains of Schmidt et al. (37). Strain FBI 516 listed so far as C. arida (marked in grey) has now been renamed C. sp. (Csp FBI 516). The Schizophyllum commune se- quence (Sc AF249390) served in this analysis as outgroup. Bootstrap values (500 replications) above 50 are shown at tree branchings. The scale bar defines the number of nucleotide substitutions per site.
Fig. 3: Phylogenetic tree of species from the Polyporales. Ba = ITS sequences from strains marked with Bjerkandera adusta, Bf = ITS sequence from a strain marked with Bjerkandera fumosa, Bsp = ITS sequences from strains marked with Bjerkandera sp., Gt = ITS sequence from a strain marked with Ganoderma tsuga, Ps = ITS sequence from a strain marked with Polyporus squamosus, Ta = ITS sequences from strains marked with Trichaptum abietinum, Tv = ITS sequence from a strain marked with Trametes versicolor. Codes indicate NCBI GenBank accession numbers. Stars mark the ITS sequences from Bjerkandera strains that were morphologically defined. Strain FBI 52 listed so far as T. abietinum (marked in grey) has now been renamed B. sp. (Bsp FBI 52). The Schizophyllum commune sequence (Sc AF249390) served in this analysis as outgroup. Bootstrap values (500 replications) above 50 are shown at tree branchings. The scale bar defines the number of nucleotide substitutions per site.
Fig. 4: Phylogenetic tree of coprini. Codes indicate NCBI GenBank accession numbers. Strains marked in grey are those from this study that were assigned to a wrong species name. The Athelia bombacina sequence (ABU85795) served in this analysis as outgroup. Bootstrap values (500 repli- cations) above 50 are shown at tree branchings. The scale bar defines the number of nucleotide substitutions per site.
Fig. 5: Analysis of ITS sequences from Coprinus comatus: Fruiting bodies of C. comatus collected in year 2006 on the grounds of the North Campus of the Georg-August-University of Göttingen used to isolate strains MN-23 (A), MN-24 (B) and MN-25 (C) from stipe tissues. Physical map of the fragment with the internal transcribed spacer 1, the 5.8S rRNA gene, and the internal tran- scribed spacer 2 from C. comatus chromosomal DNA (AF345803) plus added ITS1 and ITS4 primer sequences (D). Sizes of fragments expected to be obtained in digests with restriction enzymes HinfI and HhaI are indicated. Restriction digests of PCR-amplified fragments with HinfI (E) and HhaI (F). Lane 1: size marker (1 kb ladder); lane 2 to 7: DNA amplified with primers ITS1 and ITS4 from genomic DNA of C. comatus C108, 1-2004, 2-2004, MN-23, MN-24, and MN-25, respectively.
Fig. 6: Analysis of ITS sequences from Coprinellus micaceus and Coprinellus flocculosus: A physical map of the DNA fragments with the internal transcribed spacer 1, the 5.8S rRNA gene, and the internal transcribed spacer 2 from C. micaceus and C. flocculosus chromosomal DNA plus added ITS1 and ITS4 primer sequences were constructed using the sequences deposited in NCBI GenBank under AF345808 and AF345818, respectively (A). A PCR-amplified ITS fragment from a mycelial culture of a strain isolated from C. micaceus mushrooms (shown in E) taken from a colony of fruiting bodies found in year 2006 on the grounds of the Georg-August-University Hospital breaching in the bifurcation of an infested forking Robinia pseudoacacia tree through its bark (D) were analyzed with restriction enzymes HinfI (B: lane 2) and HhaI (C: lane 2). Lanes 1 in the two agarose gels present the size marker (1 kb ladder), lanes 3 HinfI- and HhaI-digested PCR-amplified ITS se- quences from strain Coprinellus sp. C65 (EU168107).
Fig. 7: Physical maps of ITS fragments of Coprinellus micaceus (Cm, AF345808), Coprinellus flocculosus (Cf, AF345818), Coprinellus verrucispermus (Cv, AY521250), Coprinellus radians (Cr, AF345822), Coprinellus xanthothrix (Cx, AF361228) Coprinellus curtus (Cc, AY46181834; incomplete sequence!), and Coprinellus disseminatus (Cd, AF345809) showing positions of re- striction sites for enzymes HinfI, HhaI, and DpnI.