Polymorphism in the internal transcribed spacer ( ITS ) of the ribosomal DNA of 26 isolates of ectomycorrhizal fungi

Interand intraspecific variation among 26 isolates of ectomycorrhizal fungi belonging to 8 genera and 19 species were evaluated by analysis of the internal transcribed sequence (ITS) of the rDNA region using restriction fragment length polymorphism (RFLP). The ITS region was first amplified by polymerase chain reaction (PCR) with specific primers and then cleaved with different restriction enzymes. Amplification products, which ranged between 560 and 750 base pairs (bp), were obtained for all the isolates analyzed. The degree of polymorphism observed did not allow proper identification of most of the isolates. Cleavage of amplified fragments with the restriction enzymes Alu I, Hae III, Hinf I, and Hpa II revealed extensive polymorphism. All eight genera and most species presented specific restriction patterns. Species not identifiable by a specific pattern belonged to two genera: Rhizopogon (R. nigrescens, R. reaii, R. roseolus, R. rubescens and Rhizopogon sp.), and Laccaria (L. bicolor and L. amethystea). Our data confirm the potential of ITS region PCR-RFLP for the molecular characterization of ectomycorrhizal fungi and their identification and monitoring in artificial inoculation programs.


Introduction
Ectomycorrhizal fungi have an important role in forest ecosystems because they mutualistically associate with different plant species, thereby increasing nutrient absorption while protecting the host plant against pathogens and abiotic stresses (Smith and Read, 1997;Allen, 1991).Taxonomically diverse, these fungi include between 5,000 and 6,000 species, mainly basidiomycetes (Molina et al., 1992).
Phenotypic differences between two isolates of the same species of an ectomycorrhizal fungus may be as pronounced as the differences between two distinct species, and for this reason identification of these fungi is not clear-cut (De la Bastide et al., 1995).Identification of ectomycorrhizal fungi is mainly based on the analysis of sporocarps whose presence is triggered by specific environmental conditions (Egger, 1995;Gardes and Bruns, 1996).Usually the identification of the mycosymbiont by morphological analyses of the mycorrhiza is more feasible than sporocarp analyses, al-though special skills are required to directly analyze a root-attached symbiont (Karén et al., 1997).
DNA-based molecular techniques combining the polymerase chain reaction (PCR) with analysis of restriction fragment length polymorphisms (RFLP) represent new tools to aid in properly identifying ectomycorrhizal fungi.The PCR-RFLP technique, coupling two known procedures to detect polymorphisms in DNA regions which have been amplified by specific oligonucleotide primers and restricted with different endonucleases, has been successfully used to analyze regions of ribosomal DNA of ectomycorrhizal fungi (Gardes et al., 1991;Erland et al., 1994;Henrion et al., 1994;Farmer and Sylvia, 1998;Gomes et al., 1999;Glen et al., 2001a).The internal transcribed spacer (ITS) region separating genes 17S and 25S, can be amplified by specific primers anchored in these two units.Since the ITS region is highly conserved intraspecifically but variable between different species it is often used in taxonomy (Bruns et al., 1991;Hillis and Dixon, 1991), but ITS region polymorphism for identifying ectomycorrhizal fungi species has been determined for only a restricted number of species, leaving its full potential as a taxonomic tool as yet unexplored (Karén et al., 1997).
In the work published in this paper, 26 isolates of ectomycorrhizal fungi belonging to 8 genera and 19 different species were analyzed using the PCR-RFLP technique as applied to the ITS regions of these fungi.These analyses aimed to confirm the classification of these fungi and also to find markers able to facilitate the identification of specific isolates for breeding purposes.

Fungal isolates and culture conditions
Ectomycorrhizal fungi isolates (Table I) were obtained from the fungal collection maintained at the Laboratory of Mycorrhizal Associations, Department of Microbiology, Federal University of Viçosa, Minas Gerais, Brazil.Cultures were maintained in Petri dishes containing modified Melin-Norkrans agar medium (MMN) (Marx, 1969) for 25 days at 28 °C.Mycelia for DNA extraction were obtained by inoculating MMN broth with agar plugs containing mycelia collected from actively growing colony margins, followed by 25 days static incubation at 28 °C.Ectomycorrhizae were obtained as described by Junghans et al. (1998).

DNA extraction
DNA was extracted from 0.5 to 1.0 g of fresh mycelium or ectomycorrhizae, according to the method of Schäfer and Wöstemeyer (1992) as modified by Junghans et al. (1998).DNA concentration was estimated by comparison with known standards in 1% (w/v) agarose gels stained with ethidium bromide.

PCR amplification and RFLP analysis
The primer pairs used to amplify the rDNA ITS region (ITS1 and ITS4) have been described by White et al. (1990).The cycling parameters were: 40 cycles, each cycle 478 Gomes et al.Samples were kept at -20 °C for 2 h and centrifuged at 12,000 x g for 30 min.The pellet was washed with 70% (v/v) ethanol, re-suspended in 10 µL of water and digested with different restriction enzymes.The DNA fragments were size-fractionated in 2% (w/v) agarose gel, stained with ethidium bromide (0.5 µg/mL) and photographed under UV light, either with a Polaroid camera or the images were captured and stored using the Eagle Eye II photodocumentation system (Stratagene, La Jolla, CA, USA).

Data analysis
After restriction with endonucleases the amplification products were scored as 1 (presence) or 0 (absence) of a restriction site and used for determining genetic distances between the isolates (Nei and Li, 1979).These distances were used to cluster the isolates by the unweighted pair group method with averages (UPGMA) method using the Statistica program (version 4.5 for Windows, StatSoft, Inc. 1993, Tulsa, OK, USA).

Results
The internal transcribed spacer (ITS) of the rDNA region of 26 isolates belonging to 8 genera of ectomycorrhizal fungi showed extensive length polymorphism (Figure 1), PCR amplification with specific primers for the ITS region generated bands ranging from 560 to 750 bp. Figure 1 shows that all but two species (Suillus brevipes and Hebeloma cylindrosporum) presented only one amplification product.No difference was detected between the 11 Rhizopogon isolates analyzed, while the amplification patterns for all three Suillus species were quite distinct.No polymorphism was observed within the three Laccaria species nor between the two Cenococcum geophilum isolates, although polymorphism was observed between the two Scleroderma isolates.
To detect a wider range of polymorphisms, the PCR products were cleaved with the restriction enzymes Alu I, Hae III, Hinf I, and Hpa II (Table II). Figure 2 shows the restriction patterns obtained with Alu I.However, restriction patterns with all these enzymes could not distinguish between the nine Rhizopogon isolates and for this reason the PCR products from these isolates were further cleaved with three other enzymes (Ava II, Mbo I, and Taq I) but still no polymorphism was detected.Only two of these isolates, belonging to the species R. subcaerulescens (RS1USA) and R. vinicolor (A 153), showed different patterns compared to the others.R. subcaerulescens showed a distinct pattern with all three enzymes and R. vinicolor, with enzymes Alu I and Ava II.All other species, except L. bicolor and L. amethystea, could be identified by at least one restriction enzyme pattern.
Restriction fragments obtained with all the endonucleases tested were used to determine genetic distances between the genotypes and cluster them into specific groups (Figure 3).In general, isolates grouped according to their genus, e.g.Rhizopogon, Laccaria, and Cenococcum isolates were clustered into three distinct groups.However, ITS polymorphism of ectomycorrhizal fungi 479   as shown in Figure 3, one Rhizopogon (RS1USA) and two Suillus (7598 and 9012) isolates were clustered in groups distinct from those specific for their genera and each of the two Scleroderma isolates formed a separate group.
The rDNA ITS region of ectomycorrhizae amplified successfully with the ITS1/ITS4 primer pair, the electrophoretic pattern of the amplification products showing two bands (Figure 4).

Discussion
Detection of polymorphism using PCR-RFLP analyses of the rDNA ITS region has been successfully used for identifying several species of fungi (Amicucci et al., 1996).This simple technique requires only minute amounts of DNA and two specific primers flanking the ITS region.We found that the amplification products for the ITS region of 26 isolates of ectomycorrhizal fungi collected in Brazil, the United States and France ranged from 560 to 750 bp, coinciding with the sizes obtained for other ectomycorrhizal fungi (Gardes et al., 1991;Káren et al., 1997).Despite the length polymorphism observed for many of the isolates, ITS analysis alone was not able to separate all the genotypes, the exceptions being S. brevipes and H. cylindrosporum which presented two amplification products allowing their identification in relation to the other isolates (Figure 1).These two bands may be due to either polymorphism within the rDNA repetitive unit or the presence in the same isolate of more than one nucleus bearing different rDNA sequences (Sanders et al., 1996).
A low degree of ITS region intraspecific polymorphism in ectomycorrhizal fungi has been reported by other authors (Gardes et al., 1991;Erland et al., 1994).According to Karén et al. (1997) ITS region intraspecific polymorphism varies among different genera and species, although no general conclusion can be drawn due to the small number of genera and species analyzed.
On the other hand, length polymorphism analysis of the ITS region followed by endonuclease restriction was sufficient to categorize most of the 26 isolates studied by us and place them in their correct species (Figure 2).RFLP analysis of the ITS region has been suggested by several authors as a means for discriminating between ectomycorrhizal fungi at species level (Gardes et al., 1991;Bruns et al., 1991).At genus level, ITS region fragment length polymorphism separated the 26 isolates into their 8 genera with any of the enzymes used.
Cleavage of the ITS region with Alu I (Figure 2) allowed differentiation of 12 out of the 19 species studied.However, none of the restriction enzymes produced a distinct pattern for five Rhizopogon species (R. nigrescens, R. reaii, R. roseolus, R. rubescens, and Rhizopogon sp.) and two Laccaria species (L.bicolor and L. amethystea).Martin et al. (1998) analyzed the nuclear rDNA ITS region of Rhizopogon, in order to reexamine its present taxonomic classification in terms of the presence of specific DNA polymorphisms, and found that, based on both gel electrophoresis of digested PCR products and previous morphological observations, the five different species represent only one species, R. villosulus.
Cleavage of the ITS region of Pisolithus tinctorius isolate PT 145 resulted in the same DNA fragment pattern presented by other Pisolithus isolates collected in Brazil (Gomes et al., 1999).Indeed, cluster analysis based on RAPD-PCR analysis grouped the Brazilian isolates, while ITS polymorphism of ectomycorrhizal fungi 481  isolates collected in the USA and France have been shown to be quite distinct (Junghans et al., 1998).The two bands observed by us on the PCR amplified ITS region of ectomycorrhizae correspond to plant and fungi patterns, this result confirming that ITS1/ITS4 primers are not fungi specific (Paolocci et al., 1995 andAmicucci et al., 1996).However, in some cases, heterologous DNA from the plant material did not interfere with PCR amplification (Gardes et al., 1991;Henrion et al., 1992;Erland, 1995).Glen et al. (2001b) tested six primers pairs (targeting three nuclear and three mitochondrial regions) for specificity, sensitivity and species discrimination on identified collections of fungi.Two sets of these primers, one newly designed and targeting the ITS region and the other amplifying a ribosomal DNA fragment of the large mitochondrial subunit met the requirements of high specificity and sensitivity, amplifying DNA from a broad range of the larger basidiomycetes, with no amplification of plant, bacterial or ascomycete DNA.These specific primers discriminated fungi to species level for 91 fungal species from 28 families and are a potential practical PCR-RFLP tool for identifying basidiomycetes in plants from field samples.
The results outlined in this paper show that while interspecific variation of the ectomycorrhizal fungi ITS region is relatively high, intraspecific variability is very limited and that ITS restriction fragment analysis has potential for developing species-level markers for many, but not necessarily all, ectomycorrhizal fungi species.It appears that ITS-RFLP is a potent tool for the taxonomic study of ectomycorrhizal fungi, with the minute amounts of DNA required and the high reproducibility of this procedure making it an ideal method both for studying population heterogeneity in the field and the identification and monitoring of specific strains introduced into the soil in controlled mycorrhization programs.

Figure 1 -
Figure 1 -Gel electrophoresis of the amplified rDNA internal transcribed sequence (ITS) region of 26 ectomycorrhizal fungal isolates.Lanes are numbered according to the isolate identification scheme given in Table I.Size markers (pUC 19 digested with Hinf I and Rsa I) are indicated by M.

Figure 2 -
Figure 2 -Gel electrophoresis of the amplified rDNA internal transcribed sequence (ITS) region of 26 ectomycorrhizal fungal isolates digested with the Alu I restriction enzyme.Lanes are numbered according to the isolate identification scheme given in Table I.Size markers (pUC 19 digested with Hinf I and Rsa I) are indicated by M.

Figure 3 -
Figure 3 -Unweighted pair group method with averages (UPGMA) cluster diagram of the relationships between 26 ectomycorrhizal fungi isolates.The dendrogram was based on genetic similarity coefficients determined by the presence and absence of rDNA internal transcribed sequence (ITS) region restriction enzyme patterns.

Table I -
The ectomycorrhizal fungi studied and their origin and hosts. of a denaturation step at 94 °C for 1 min, an annealing step at 50 °C for 1 min and an extension step at 72 °C for 1 min 30 s.After the 40th cycle, a final extension step was performed at 72 °C for 7 min.Components for 25 µL PCR reactions were: 10 ng of DNA template, a The state of Minas Gerais.bThe state of Santa Catarina.*Usedfor inoculation of Eucalyptus grandis.consisting

Table II -
Restriction fragments (pair bases) of the internal transcribed spacer (ITS) rDNA amplified regions of ectomycorrhizal fungi isolates produced by different restriction enzymes.