cucumeris (Frank) Donk] is a common world-wide soil-borne fungus. Strains of ... A variety of molecular techniques based on genomic variation, such as DNA ...
Curr Genet (1996) 29: 174—181
( Springer-Verlag 1996
OR I G I N A L P AP E R
Marianne Boysen · Marise´ Borja Catalina del Moral · Oscar Salazar · Victor Rubio
Identification at strain level of Rhizoctonia solani AG4 isolates by direct sequence of asymmetric PCR products of the ITS regions
Received: 26 May/Accepted 20 July 1995
Abstract The relatedness of nine isolates of Rhizoctonia solani, belonging to anastomosis group (AG) 4, and one isolate of AG1 was determined by comparative sequence analysis based on direct sequencing of PCRamplified ribosomal DNA [the internal transcribed spacer (ITS) region and the 5.8 s ribosomal DNA]. The 5.8 s rDNA is completely conserved, but both ITS regions show variation among strains. AG1 was an outgroup based on anastomosis ability and RFLP analyses. Phylogenetic analyses based on the ITS sequences suggest that the analyzed AG4 strains can be divided into three groups that correlate with habitat and virulence. Key words Rhizoctonia solani · ITS sequences · Asymmetric PCR · Phylogenetic tree
Introduction Rhizoctonia solani Ku¨hn [teleomorph: ¹hanatephorus cucumeris (Frank) Donk] is a common world-wide soil-borne fungus. Strains of this fungus may grow as saprophytes or as mycorrhiza on orchids and other plants, and have been described as pathogens of at least 150 different plant species. R. solani is a complex species, and DNA homology between different members of this species can be as low as 14% (Ogoshi 1987). R. solani can be divided into 12 groups based on the ability of component strains to anastomose (Sneh et al.
M. Boysen1 · M. Borja · C. del Moral · O. Salazar · V. Rubio ( ) Centro Nacional de Biotecnologia, (CSIC-UAM), Campus de Cantoblanco, Universidad Autonoma de Madrid, E-28049 Madrid, Spain 1 Present address: Biotechnological Institute, Anker Engelundsvej 1, Building 227, DK-2800 Lyngby, Denmark Communicated by P.J.G.M. de Wit
1991; Carling et al. 1994). Some of these groups are specific to geographic locations and others are specific to particular plant species. For some of the anastomosis groups (AGs), subgroups have been defined in an attempt to clarify the system. Based on DNA homology, AG4 is divided to two sub-groups HG-I and HG-II (Kuninaga and Yokosawa 1984). Isolates of AG4 are known to cause damping-off and root-rot on a wide variety of hosts (Anderson 1982; Ogoshi 1987; Adams 1988). Biological control has been achieved using hypovirulent field isolates (Ichielevich-Auster et al. 1985). Rapid diagnostics are needed to identify the number and variability of R. solani strains in a given ecosystem and to develop efficacious control methods. What was originally defined as a single species actually appears to be a diverse group, as determined by molecular techniques. Some of the methods used to distinguish the members of this species complex included: reassociation kinetics (Vilgalys 1988), isozyme analyses (Liu et al. 1990), immunodetection (Thornton et al. 1994) and rDNA polymorphisms (Vilgalys and Gonzalez 1990; Cubeta et al. 1991; Liu and Sinclair 1993; Liu et al. 1993). A variety of molecular techniques based on genomic variation, such as DNA fingerprinting, restriction fragment length polymorphism (RFLP) or random amplified polymorphic DNA (RAPD) (Vilgalys and Gonzalez 1990; Liu et al. 1993; Blakemore et al. 1994; Muthumeenakshi et al. 1994) and DNA sequence analysis of various ribosomal spacer regions (Yao et al. 1992; Curran et al. 1994), have been used in fungi. In contrast with bacteria, in which the 16s region is variable between genus and species, the equivalent 18s region in fungi is highly conserved. Studies on the fungal ribosomal genes (5.8s, 18s and 28s) have shown that the genes are highly conserved at the genus level, or even higher, and have been used to determine the phylogenetic relationships between the Basidiomycetes, Ascomycetes and Chytridiomycetes (Bowman et al. 1992).
175 Fig. 1 Schematic representation of the internal transcribed spacer (ITS) region of the ribosomal DNA. The shadowed boxes represent the ribosomal subunits. The arrows denote the positions of the PCR and sequencing primers
Eucaryotic ribosomal genes (Fig. 1) are arranged in a tandem repeat with the 5.8s coding region flanked by internal transcribed spacers (ITS) (White et al. 1990). The ITS regions are co-transcribed with the genes for 18s, 5.8s and 28s rRNA, and in Saccharomyces cerevisiae it has been shown that ITS regions play a major role in the production of the 18s and 28s RNAs (van der Sande et al. 1992; van Nues et al. 1994). Two primer sets have been identified in the 18s and the 28s regions (Gardes and Bruns 1993), one specific for fungi in general and one specific for basidiomycetes. In other studies, the ITS regions have been used to construct phylogenetic trees in R. solani (Liu et al. 1993). In Penicillium, the phylogenetic tree reflects ecological groups of 28 different Penicillium species (Skouboe et al. 1995) In other cases the information from species-specific sequences of the ITS regions has been used to select species-specific primers (Johanson 1994; Boysen et al. 1995). The variable genetics and pathology described within R. solani AG4 contrasted with the apparent lack of variation detected by rDNA RFLP analysis (Vilgalys and Gonzalez 1990). We considered that sequence analysis could provide a more informative means of differentiating between AGs of R. solani. In this paper we present the use of the DNA sequences of the ITS region to determine the relatedness between nine R. solani AG4 isolates.
Table 1 Anastomosis group, virulence, origin, source and clima of R. solani strains
Materials and methods Strains and pathogenicity test. The R. solani strains used in this study, together with their characteristics, are listed in Table 1. They are all on deposit in either the ATCC or in the Coleccio´n Espan8 ola de Cultivos Tipo (CECT), with the following numbers — 521: ATCC 64643, Me82: CECT 20171, Me84: CECT 20172, Me87: CECT 17320, PinJRS1: CECT 20174, PinJRS3: CECT 20175, Rh13: ATCC 64662, Rh2815: CECT 2815 and Rsa: CECT 20176. The virulence of each strain used in this work was tested by a plate assay (Castanho and Butler 1978) in five different hosts. The hosts used in these tests were radish (Raphanus sativus L.), cotton (Gossypium hirsutum L.), onion (Allium cepa L.), bean (Phaseolus vulgaris L.) and carrot (Daucus carota L. var. sativa DC.). The reliability of the plate assay was tested and confirmed in greenhouse tests and in field experiments (Ichielevich-Auster et al. 1985; Sneh et al. 1986). One-week post-inoculation, virulence was determined based on the number of seedlings that were killed, showed lesions, or that remained healthy. An arbitrary scale from 1 to 5 reflects the degree of virulence (Sneh et al. 1966). Isolation of genomic DNA. R. solani strains were grown for 3—5 days on Bacto Potato Dextrose Agar (Difco Labs., Detroit, Michigan) plates at 28 °C. Mycelia were transferred to liquid complete media (CM media: malt extract, yeast extract and glucose, 5.0 g/l each) and incubated for 4—5 days at 28 °C with agitation (250 rpm). Mycelia were harvested by filtration, washed in sterile water, and stored at !80 °C until further use. General DNA extraction and manipulation protocols follow those of Sambrook et al. (1989). Specifically, approximately 2.0 g of mycelia was crushed in liquid nitrogen and transferred to a 40 ml centrifuge tube. The tube was filled with extraction buffer [50 mM EDTA (pH 8.5), 0.2% SDS"dodecyl sulphate sodium salt], incubated for 30 min. at 65 °C, and cooled to
R. solani strains
AG!
Virulence"
Origin
Source
Clima
Me 8-2 Me 8-4 Me 8-7 Pin JRS1 Pin JRS3 Rh2815 Rh13 521 RSA AG1-331
4 4 4 4 4 4 4 4 4 1
5 5 5 5 5 1 5 1 5 5
Cucumis melo Cucumis melo Cucumis melo Pinus pinaster Pinus pinaster »icia faba Soil Soil Phaseolus vulgaris INRA Collection
Almeria (Spain) Almeria (Spain) Almeria (Spain) Jaen (Spain) Jaen (Spain) Valencia (Spain) Tel Aviv (Israel) Tel Aviv (Israel) Navarra (Spain) Dijon (France)
Sub-arid Sub-arid Sub-arid Continental Continental Sub-arid Sub-arid Sub-arid Continental
! AG"Anastomosis group " 5 indicates that more than 80% of the plants were dead, 1 indicates that 1—10% of the plants were dead
176 room temperature. After the addition of 3 ml of RNase (5 mg/ml), the mixture was incubated for 1 h at 37 °C, centrifuged for 15 min. at 17 500 g, and the supernatant transferred to a fresh tube on ice. A one-tenth volume of 5 M K acetate was added, then mixed, and incubated on ice for at least 1 h. The supernatant was transferred to a new tube after centrifugation for 15 min at 27 000 g, and then extracted twice with an equal volume of phenol/chloroform/isoamyl alcohol (49/49/2). The DNA was precipitated by adding an equal volume of isopropanol. After a 30-min incubation at room temperature, and a 15 min. centrifugation at 17 500 g, the recovered pellet was washed with 80% cold ethanol, dried at 37 °C, and resuspended in 2 ml of 1 x TE-buffer [10 mM Tris-Cl (pH 7.4), 1 mM EDTA (pH 8.0)]. Asymmetric PCR. The polymerase chain reaction was performed with some modifications from the original protocol (White et al. 1990) introduced by (Skouboe et al. 1995). Single-stranded DNA was prepared using primers in a ratio of 50:1 (Gyllensten and Erlich 1988). Primers used for amplification of the ITS region were ITS4 and ITS5 (White et al. 1990) and ITS1F and ITS4B as described by (Gardes and Bruns 1993) (Fig. 1). For each 50-ll reaction, a mixture was made containing 10 ng of genomic DNA, 10 mM Tris-Cl (pH 8.3), 50 mM KCl, 2.5 mM MgCl , 0.2 mM each of dATP, dCTP, dGTP and dTTP (Pharmacia, 2Sweden), 20 pmol of primer A, 0.4 pmol of primer B, 0.0025% Tween 20, 10% dimethyl-sulphoxide (DMSO) and 1.25 U of Taq DNA polymerase (USB, Cleveland Ohio). The mixture was covered with a drop of mineral oil. Amplification was performed using an automated thermal cycler (Perkin Elmer Cetus Corp. model 480, Norwalk, Conn.). The cycling parameters were an initial denaturation at 94 °C for 2.5 min., and then 40 cycles of: denaturation for 15 s at 94 °C, annealing at 53 °C (when ITS4/ITS5 were used as amplification primers) or 55 °C (when ITS1F/ITS4B were used as amplification primers) for 30 s, and extension at 72 °C for 1.5 min. The amplification was terminated by a final extension for 10 min at 72 °C. DNA cloning and sequencing. Prior to sequencing, excess primers and nucleotides were removed from the asymmetric amplification mixture by precipitation for 10 min at room temperature with an equal volume of 5 M NH Ac and 2.5 vol of 96% ethanol and 4 10 min at 9000 g. The PCR products pelleted by centrifugation for were washed with 80% cold ethanol, dried at 37 °C and finally resuspended in 17 ll of 1 ] TE. This DNA was used for sequencing without further purification. Sequencing was done by Sanger’s dideoxy method (Sanger et al. 1977) using the Sequenase Version 2.0 sequencing kit (USB, Cleveland Ohio), with single-stranded PCR products as templates and ITS1, ITS2, ITS3 and ITS4 as sequencing primers. Approximately 1 lg of ss-PCR product and 50 ng of primer were used for each reaction and labelling was done with (a-35S)-dATP (Amersham Int, Amersham, UK). The sequence fragments were separated in a denaturing 8.3 M urea/6% polyacrylamide gel. Sequencing analysis. Computer-aided alignments of the sequences of the ITS region were made using the CLUSTALV option of the IntelliGenetics software package, PC Gene Screen Device, Version 5.03 (IntelliGenetics, Inc., Mountain View, Calif.). Analysis of the nucleotide differences in the spacer and 5.8s regions were performed using the maximum-parsimony method with the branch-and-bound algorithm of the Phylogeny Using Parsimony Analysis (PAUP) program 3.1.1 (Swofford 1991), with gaps treated as missing data. Confidence intervals in tree topologies were estimated from bootstrap analyses (Felsenstein 1993) using the heuristic search option of PAUP with 500 replicates. The tree was confirmed using the neighbor-joining method (Saitou and Nei 1987) as implemented in the program neighbor from the PHYLIP package version 3.3 (Felsenstein 1993). The topological accuracy was again estimated by the bootstrap method with 500 replicates. The sequences obtained have been submitted to the Genbank database with the following acces-
sion numbers — 521: U19950, AG-1: U19551, Me82: U19952, Me82L: U19953, Me84: U19954, Me84L: U19955, Me87: U19956, Me87L: U19957, PinJRS1: U19958, PinJRS3: U19959, Rh13: U19960, Rh13L: U19961, Rh 2815: U19962, Rh 2815L: U19963 and Rsa: U19964.
Results Pathogenicity test In this study, strains were designated hypovirulent, rated 1 in the test described in Materials and methods, and virulent, rated 5, which means that over 80% of the area was infection. Strains between these two values were not considered in this study. PCR amplification of the ITS region Fragments of approximately 700 bp were obtained when using R. solani AG4 as a template for PCR amplification of the ITS region. Different combinations of primers were tested for asymmetric amplification. Asymmetric PCR products using ITS4/ITS5 in the ratio 50:1 were found to give better results when sequencing with ITS1 and ITS3, whereas the combination of ITS1F/ITS4B (also in a 50 : 1 ratio) was found to give better results when sequencing with ITS2 and ITS4. Figure 2 shows the products of asymmetric PCR amplification using either ITS4/ITS5 or ITS1F/ITS4B,
Fig. 2 PCR fragments produced by asymmetric PCR using ITS4/ITS5 or basidiomycete-specific ITS1F/ITS4B primers on the strains indicated above each lane. The molecular-weight markers represent k cut with Bst1
177
which yielded fragments of approximately 700 bp and approximately 800 bp respectively. In both cases the hypovirulent strains (521 and Rh2815) gave rise fragments that were approximately 50 bp shorter than the virulent strains. Sequencing PCR products The ITS region, including the 5.8s rRNA coding region, is approximately 700 bp in AG1, approximately 620 bp in the hypovirulent strains 521 and Rh2815, and approximately 680 bp for the remaining AG4s. DNA sequencing revealed that some of the strains contain more than one sequence of the ITS 1 region. The insertion(s) created a ladder in the sequencing gel (Fig. 3). We observed this insertion in five (Me 8-2, Me 8-4 and 8-7, Rh 13 and Rh2815) out of ten isolates. The same sequence was obtained when the regions sequenced were recovered from colonies that originated from a single protoplast. There was an insertion and a deletion in the Me 8-2 and 8-7 strains, while in the Rh 13 strain there were two insertions. Sequences corresponding to the inserted version were designated L. Sequences derived from the cloned ITS contained one of the two sequences found in the direct PCR products. Individual clones were sequenced as a proof of the existence of two different sequences in one strain.
In the hypovirulent strains (521 and Rh2815) a few bases were unidentifiable, as bands occurred in two sequence lanes. Further sequence analysis produced no better resolution. Since the anomalies appear in the same positions regardless of which strand is sequenced, the errors are most likely the result of base-pair substitutions (Fig. 3). In strain 521 there are three C/T and three A/G substitutions, while in Rh2815 there is one A/T and two A/G substitutions. Only one of the A/G substitutions is common between the two strains. No individual clones have been obtained to resolve this phenomenon.
Alignments of the sequences The results of the alignments of the sequenced strains using the CLUSTALV program are shown in Fig. 4. The 5.8s coding region is completely conserved and similar to that in other fungi (Nazar et al. 1987). Within AG4 there is 0—40% variation in the ITS 1 region and 0—18% variation in the ITS 2 region.
Phylogenetic analysis One AG1 isolate of R. solani was included as a reference for the AG4 isolates. As expected, RFLP analysis with Hae III, Sau 3A I and Hind II digestions (data not shown) placed AG1 as an outgroup with the AG4 strains closer to each other than to AG1. The mostparsimonious tree found by analysis of the aligned sequences was also the best tree obtained by the neighbor-joining method (Fig. 5). Phylogenetic relationships also place the AG4 strains closer to each other than to AG1 and divide AG4 into three groups reflecting habitat and virulence. The two hypovirulent strains (521 and Rh2815) grouped together, whereas the seven virulent strains were divided into two groups according to the climate of the location where the isolate originated from. Strains from one cluster (Rh 13, Me 8-2, Me 8-4 and Me 8-7) were isolated from sub-arid areas in southeastern Spain and Israel, and strains of the other cluster (RSA, Pin JRS1 and Pin JRS3) were isolated at high altitude in continental and low-temperature areas in Spain. Even though Me 8-2 and Me 8-4 were isolated from the same infected melon field they are further apart than Me 8-2 is from RH13, which was isolated from Israel.
Discussion
Fig. 3 Sequencing gel of RH2815. The arrows indicate an insertion, which creates a ladder in the sequence (in sequences above the insertion arrow), and a substitution, respectively
Even though there are at least two hybridization groups present in AG4 (Vilgalys 1988), the variable genetics and virulence described within AG4 contrasted with the apparent lack of variation detected in this
178 Fig. 4 Alignment of fungal ITS sequences. The R. solani strain is indicated. In the strains where insertions have occurred the longer version has been designated L. R indicates A or G, ½ indicates C or T and ¼ indicates A or T. The asterisks indicate conserved positions among all the strains. The arrows indicate the limits of the 5.8s rRNA gene
group by rDNA RFLP analysis (Vilgalys and Gonzalez 1990). In the present study we sequenced the ITS region of the rDNA from nine R. solani isolates belonging to AG4 and one belonging to AG1. This small group of isolates can be distinguished according to virulence, based on differences in the size of the PCR product. Another interesting feature of these sequences is that variation within a single strain of R. solani can be detected. The heterogeneity caused by insertion or base substitution can be detected as the PCR used to amplify the ITS sequences amplifies the total rDNA popu-
lation present. In the sequences from cloned fragments, this phenomenon was not detected since sequencing of clones assesses only a single copy of the rDNAs. As R. solani is known to be multinucleate (Sneh et al. 1991), the heterogeneity could be due to chromosome variation present in different nuclei within the same strain. It is also possible that the insertion or substitution occurred in some of the approximately 200 copies (Bruns et al. 1991) of the ITS sequence on the same chromosome, or that the same nucleus has different rDNAs on different chromosomes. The variations do
179 Fig. 4 (continued)
not always occur in the same position; for example, of the six substitutions in strain 521 and the three substitutions and an insertion in strain Rh2815, only an A/G substitution in ITS2 is shared, while all of the other variants are unique. In the six strains with hetero-
geneous rDNA, the intensity of the bands in the sequencing gels are equivalent for a variants, so that the variants are presumed to be present in a similar number of copies. The secondary structure of the ITS sequences was predicted (data not shown) using the RNAFOLD option by the method of Zuker (Zuker and Stiegler 1981; Freier et al. 1986) in the GCG software package (Devereux et al. 1984). The structure indicated that the insertions and substitutions were placed in regions distant from the joining region of the 18s or 28s and the 5.8s rRNA and were, therefore, of minor importance for the processing of the precursor rRNA. Van Nues et al. b Fig. 5 Single most-parsimonious phylogenetic tree generated from a branch-and-bound algorithm in PAUP 3.1.1. The numbers indicate the percentage corresponding to the frequency with which a given branch appeared in a 500 bootstrap replications. Horizontal lengths represent genetic distances, vertical lengths are not meaningful. A similar phylogenetic tree was obtained by the neighbor-joining method. ¹wo asterisks and one asterisk denote branch points with confidence limits of '99% and '95%, respectively. AG1 was used as an outgroup
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(1994) showed that removal of a domain in the ITS 1 region distal to the joining of the 17s and 5.8s rRNA had no influence on the processing of rRNA in yeast. The presence of two (or more) different sequences in a single strain, despite high conservation of these regions, indicates that the ITS regions have been under less evolutionary pressure than the rDNA genes, presumably because the spacers are functionally less constrained than the rRNA coding region. Further experiments are needed to determine if there is any function for the insertions and substitutions in these strains. Both sequence analysis and RFLP polymorphisms placed the AG1 and AG4 isolates in separate clusters, which allowed us to use AG1 as an outgroup to root the phylogenetic trees. RFLP analysis of these AG4 strains grouped the isolates according to habitat, placing the hypovirulent strains (Rh2815 and 521) relatively closer to the high-altitude strains (Pin JRS 1 and 3 and RSA) with only two different restriction sites compared to 3-4 different ones in the sub-arid strains (Rh 13, Me 8-2, 8-4 and 8-7). In the parsimony analysis of the sequence data, the genetic distances between the hypovirulent and virulent strains and the AG1 outgroup are similar. Since more variation occurs in ITS1 than ITS2, it may be possible in future studies to consider only the sequence data from ITS1 to determine the relationships between different strains of the same AG group in R. solani. Nevertheless, whereas whole-cell fatty acid composition could only identify and differentiate between certain populations of AG-1 isolates (Johnk and Jones 1994), we have shown that rRNA ITS sequence analysis could be used to classify strains from AG4 according to virulence and habitat. With the information presented here, it will be possible to construct primers that are specific for a particular group which could then be used to specifically amplify sequences for diagnostic purposes. Using such primers a sufficiently large number of strains can be screened to obtain relevant population data. Acknowledgements This work was supported by Grant BIO 93 0923 C02 02 from the Comision Interministerial de Ciencia y Tecnologı´ a (CICYT), Spain and Grants BIOT/CT91030 and BIO2/CT94-3011 from the European Union. We thank Yigal Koltin, Javier Tello and Federico Uruburu for providing the strains for this study, Joaqun Dopazo for assistance with the phylogenetic analysis and for critically reading the manuscript. Lone Rossen, Jaap Keijer and Baruch Sneh gave valuable suggestions and criticisms to improve this work.
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