Curr Genet (1996) 29: 174—181 ( Springer-Verlag 1996

ORIGINAL PAPER

Marianne Boysen · Marise BorjaCatalina del Moral · Oscar Salazar · Victor Rubio

Identification at strain level of Rhizoctonia solani AG4 isolates by directsequence of asymmetric PCR products of the ITS regions

Received: 26 May/Accepted 20 July 1995

Abstract The relatedness of nine isolates of Rhizo-ctonia solani, belonging to anastomosis group (AG) 4,and one isolate of AG1 was determined by comparativesequence analysis based on direct sequencing of PCR-amplified ribosomal DNA [the internal transcribedspacer (ITS) region and the 5.8 s ribosomal DNA]. The5.8 s rDNA is completely conserved, but both ITSregions show variation among strains. AG1 was anoutgroup based on anastomosis ability and RFLPanalyses. Phylogenetic analyses based on the ITS se-quences suggest that the analyzed AG4 strains can bedivided into three groups that correlate with habitatand virulence.

Key words Rhizoctonia solani · ITS sequences ·Asymmetric PCR · Phylogenetic tree

Introduction

Rhizoctonia solani Kuhn [teleomorph: ¹hanatephoruscucumeris (Frank) Donk] is a common world-widesoil-borne fungus. Strains of this fungus may grow assaprophytes or as mycorrhiza on orchids and otherplants, and have been described as pathogens of at least150 different plant species. R. solani is a complex spe-cies, and DNA homology between different members ofthis species can be as low as 14% (Ogoshi 1987).

R. solani can be divided into 12 groups based on theability 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 deCantoblanco, Universidad Autonoma de Madrid, E-28049Madrid, 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 arespecific to geographic locations and others are specific toparticular plant species. For some of the anastomosisgroups (AGs), subgroups have been defined in an attemptto clarify the system. Based on DNA homology, AG4 isdivided to two sub-groups HG-I and HG-II (Kuninagaand Yokosawa 1984). Isolates of AG4 are known to causedamping-off and root-rot on a wide variety of hosts(Anderson 1982; Ogoshi 1987; Adams 1988).

Biological control has been achieved using hypoviru-lent field isolates (Ichielevich-Auster et al. 1985). Rapiddiagnostics are needed to identify the number andvariability of R. solani strains in a given ecosystem andto develop efficacious control methods.

What was originally defined as a single species ac-tually appears to be a diverse group, as determined bymolecular techniques. Some of the methods used todistinguish the members of this species complex in-cluded: reassociation kinetics (Vilgalys 1988), isozymeanalyses (Liu et al. 1990), immunodetection (Thorntonet al. 1994) and rDNA polymorphisms (Vilgalys andGonzalez 1990; Cubeta et al. 1991; Liu and Sinclair1993; Liu et al. 1993).

A variety of molecular techniques based on genomicvariation, such as DNA fingerprinting, restrictionfragment length polymorphism (RFLP) or randomamplified polymorphic DNA (RAPD) (Vilgalys andGonzalez 1990; Liu et al. 1993; Blakemore et al. 1994;Muthumeenakshi et al. 1994) and DNA sequence anal-ysis of various ribosomal spacer regions (Yao et al.1992; Curran et al. 1994), have been used in fungi. Incontrast with bacteria, in which the 16s region is vari-able between genus and species, the equivalent 18sregion in fungi is highly conserved. Studies on thefungal ribosomal genes (5.8s, 18s and 28s) have shownthat the genes are highly conserved at the genus level,or even higher, and have been used to determinethe phylogenetic relationships between the Basidio-mycetes, Ascomycetes and Chytridiomycetes (Bowmanet al. 1992).

Fig. 1 Schematic representationof the internal transcribedspacer (ITS) region of theribosomal DNA. The shadowedboxes represent the ribosomalsubunits. The arrows denote thepositions of the PCR andsequencing primers

Table 1 Anastomosis group,virulence, origin, source andclima of R. solani strains

R. solani strains AG! Virulence" Origin Source Clima

Me 8-2 4 5 Cucumis melo Almeria (Spain) Sub-aridMe 8-4 4 5 Cucumis melo Almeria (Spain) Sub-aridMe 8-7 4 5 Cucumis melo Almeria (Spain) Sub-aridPin JRS1 4 5 Pinus pinaster Jaen (Spain) ContinentalPin JRS3 4 5 Pinus pinaster Jaen (Spain) ContinentalRh2815 4 1 »icia faba Valencia (Spain) Sub-aridRh13 4 5 Soil Tel Aviv (Israel) Sub-arid521 4 1 Soil Tel Aviv (Israel) Sub-aridRSA 4 5 Phaseolus vulgaris Navarra (Spain) ContinentalAG1-331 1 5 INRA Collection Dijon (France)

! AG"Anastomosis group" 5 indicates that more than 80% of the plants were dead, 1 indicates that 1—10% of the plants were dead

Eucaryotic ribosomal genes (Fig. 1) are arranged ina tandem repeat with the 5.8s coding region flanked byinternal transcribed spacers (ITS) (White et al. 1990).The ITS regions are co-transcribed with the genesfor 18s, 5.8s and 28s rRNA, and in Saccharo-myces cerevisiae it has been shown that ITS regionsplay a major role in the production of the 18s and 28sRNAs (van der Sande et al. 1992; van Nues et al. 1994).

Two primer sets have been identified in the 18s andthe 28s regions (Gardes and Bruns 1993), one specificfor fungi in general and one specific for basidiomycetes.In other studies, the ITS regions have been used toconstruct phylogenetic trees in R. solani (Liu et al.1993).

In Penicillium, the phylogenetic tree reflects ecologi-cal groups of 28 different Penicillium species (Skouboeet al. 1995) In other cases the information fromspecies-specific sequences of the ITS regions has beenused to select species-specific primers (Johanson 1994;Boysen et al. 1995).

The variable genetics and pathology described with-in R. solani AG4 contrasted with the apparent lack ofvariation detected by rDNA RFLP analysis (Vilgalysand Gonzalez 1990). We considered that sequence anal-ysis could provide a more informative means of differ-entiating between AGs of R. solani. In this paper wepresent the use of the DNA sequences of the ITS regionto determine the relatedness between nine R. solaniAG4 isolates.

Materials and methods

Strains and pathogenicity test. The R. solani strains used in thisstudy, together with their characteristics, are listed in Table 1. Theyare all on deposit in either the ATCC or in the Coleccion Espan8 olade Cultivos Tipo (CECT), with the following numbers — 521: ATCC64643, Me82: CECT 20171, Me84: CECT 20172, Me87: CECT17320, PinJRS1: CECT 20174, PinJRS3: CECT 20175, Rh13: ATCC64662, Rh2815: CECT 2815 and Rsa: CECT 20176. The virulence ofeach strain used in this work was tested by a plate assay (Castanhoand Butler 1978) in five different hosts. The hosts used in these testswere 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 assaywas tested and confirmed in greenhouse tests and in field experi-ments (Ichielevich-Auster et al. 1985; Sneh et al. 1986). One-weekpost-inoculation, virulence was determined based on the number ofseedlings that were killed, showed lesions, or that remained healthy.An arbitrary scale from 1 to 5 reflects the degree of virulence (Snehet al. 1966).

Isolation of genomic DNA. R. solani strains were grown for 3—5 dayson 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) andincubated for 4—5 days at 28 °C with agitation (250 rpm). Myceliawere harvested by filtration, washed in sterile water, and stored at!80 °C until further use. General DNA extraction and manipula-tion protocols follow those of Sambrook et al. (1989). Specifically,approximately 2.0 g of mycelia was crushed in liquid nitrogen andtransferred to a 40 ml centrifuge tube. The tube was filled withextraction buffer [50 mM EDTA (pH 8.5), 0.2% SDS"dodecylsulphate sodium salt], incubated for 30 min. at 65 °C, and cooled to

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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. at17 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, andincubated on ice for at least 1 h. The supernatant was transferred toa new tube after centrifugation for 15 min at 27 000 g, and thenextracted twice with an equal volume of phenol/chloroform/isoamylalcohol (49/49/2). The DNA was precipitated by adding an equalvolume of isopropanol. After a 30-min incubation at room temper-ature, and a 15 min. centrifugation at 17 500 g, the recovered pelletwas washed with 80% cold ethanol, dried at 37 °C, and resuspendedin 2 ml of 1x TE-buffer [10 mM Tris-Cl (pH 7.4), 1 mM EDTA(pH 8.0)].

Asymmetric PCR. The polymerase chain reaction was performedwith some modifications from the original protocol (White et al.1990) introduced by (Skouboe et al. 1995). Single-stranded DNA wasprepared using primers in a ratio of 50:1 (Gyllensten and Erlich1988). Primers used for amplification of the ITS region were ITS4and ITS5 (White et al. 1990) and ITS1F and ITS4B as described by(Gardes and Bruns 1993) (Fig. 1). For each 50-ll reaction, a mixturewas made containing 10 ng of genomic DNA, 10 mM Tris-Cl (pH8.3), 50 mM KCl, 2.5 mM MgCl

2, 0.2 mM each of dATP, dCTP,

dGTP and dTTP (Pharmacia, Sweden), 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, ClevelandOhio). The mixture was covered with a drop of mineral oil. Amplifi-cation was performed using an automated thermal cycler (PerkinElmer Cetus Corp. model 480, Norwalk, Conn.). The cycling para-meters were an initial denaturation at 94 °C for 2.5 min., and then 40cycles of: denaturation for 15 s at 94 °C, annealing at 53 °C (whenITS4/ITS5 were used as amplification primers) or 55 °C (whenITS1F/ITS4B were used as amplification primers) for 30 s, andextension at 72 °C for 1.5 min. The amplification was terminated bya final extension for 10 min at 72 °C.

DNA cloning and sequencing. Prior to sequencing, excess primersand nucleotides were removed from the asymmetric amplificationmixture by precipitation for 10 min at room temperature with anequal volume of 5 M NH

4Ac and 2.5 vol of 96% ethanol and

pelleted by centrifugation for 10 min at 9000 g. The PCR productswere washed with 80% cold ethanol, dried at 37 °C and finallyresuspended in 17 ll of 1 ] TE. This DNA was used for sequencingwithout further purification. Sequencing was done by Sanger’sdideoxy method (Sanger et al. 1977) using the Sequenase Version 2.0sequencing kit (USB, Cleveland Ohio), with single-stranded PCRproducts as templates and ITS1, ITS2, ITS3 and ITS4 as sequencingprimers. Approximately 1 lg of ss-PCR product and 50 ng ofprimer were used for each reaction and labelling was done with(a-35S)-dATP (Amersham Int, Amersham, UK). The sequence frag-ments were separated in a denaturing 8.3 M urea/6% polyacryl-amide gel.

Sequencing analysis. Computer-aided alignments of the sequences ofthe ITS region were made using the CLUSTALV option of theIntelliGenetics software package, PC Gene Screen Device, Version5.03 (IntelliGenetics, Inc., Mountain View, Calif.). Analysis of thenucleotide differences in the spacer and 5.8s regions were performedusing the maximum-parsimony method with the branch-and-boundalgorithm 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 boot-strap analyses (Felsenstein 1993) using the heuristic search option ofPAUP with 500 replicates. The tree was confirmed using the neigh-bor-joining method (Saitou and Nei 1987) as implemented in theprogram neighbor from the PHYLIP package version 3.3 (Felsen-stein 1993). The topological accuracy was again estimated by thebootstrap method with 500 replicates. The sequences obtained havebeen 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 andRsa: 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 thearea was infection. Strains between these two valueswere not considered in this study.

PCR amplification of the ITS region

Fragments of approximately 700 bp were obtainedwhen using R. solani AG4 as a template for PCRamplification of the ITS region. Different combinationsof primers were tested for asymmetric amplification.

Asymmetric PCR products using ITS4/ITS5 in theratio 50:1 were found to give better results when se-quencing with ITS1 and ITS3, whereas the combina-tion of ITS1F/ITS4B (also in a 50 :1 ratio) was found togive better results when sequencing with ITS2 andITS4.

Figure 2 shows the products of asymmetric PCRamplification using either ITS4/ITS5 or ITS1F/ITS4B,

Fig. 2 PCR fragments produced by asymmetric PCR usingITS4/ITS5 or basidiomycete-specific ITS1F/ITS4B primers on thestrains indicated above each lane. The molecular-weight markersrepresent k cut with Bst1

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which yielded fragments of approximately 700 bp andapproximately 800 bp respectively. In both cases thehypovirulent strains (521 and Rh2815) gave rise frag-ments that were approximately 50 bp shorter than thevirulent strains.

Sequencing PCR products

The ITS region, including the 5.8s rRNA coding region,is approximately 700 bp in AG1, approximately 620 bpin the hypovirulent strains 521 and Rh2815, and ap-proximately 680 bp for the remaining AG4s.

DNA sequencing revealed that some of the strainscontain 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, Me8-4 and 8-7, Rh 13 and Rh2815) out of ten isolates. Thesame sequence was obtained when the regions se-quenced were recovered from colonies that originatedfrom a single protoplast.

There was an insertion and a deletion in the Me 8-2and 8-7 strains, while in the Rh 13 strain there were twoinsertions. Sequences corresponding to the insertedversion were designated L. Sequences derived from thecloned ITS contained one of the two sequences foundin the direct PCR products. Individual clones weresequenced as a proof of the existence of two differentsequences in one strain.

Fig. 3 Sequencing gel of RH2815. The arrows indicate an insertion,which creates a ladder in the sequence (in sequences above theinsertion arrow), and a substitution, respectively

In the hypovirulent strains (521 and Rh2815) a fewbases were unidentifiable, as bands occurred in twosequence lanes. Further sequence analysis produced nobetter resolution. Since the anomalies appear in thesame positions regardless of which strand is sequenced,the errors are most likely the result of base-pair substi-tutions (Fig. 3). In strain 521 there are three C/T andthree A/G substitutions, while in Rh2815 there is oneA/T and two A/G substitutions. Only one of the A/Gsubstitutions is common between the two strains. Noindividual clones have been obtained to resolve thisphenomenon.

Alignments of the sequences

The results of the alignments of the sequenced strainsusing the CLUSTALV program are shown in Fig. 4.The 5.8s coding region is completely conserved andsimilar to that in other fungi (Nazar et al. 1987). WithinAG4 there is 0—40% variation in the ITS 1 region and0—18% variation in the ITS 2 region.

Phylogenetic analysis

One AG1 isolate of R. solani was included as a refer-ence for the AG4 isolates. As expected, RFLP analysiswith Hae III, Sau 3A I and Hind II digestions (data notshown) placed AG1 as an outgroup with the AG4strains closer to each other than to AG1. The most-parsimonious tree found by analysis of the alignedsequences was also the best tree obtained by the neigh-bor-joining method (Fig. 5). Phylogenetic relationshipsalso place the AG4 strains closer to each other than toAG1 and divide AG4 into three groups reflecting habi-tat and virulence. The two hypovirulent strains (521and Rh2815) grouped together, whereas the seven viru-lent strains were divided into two groups according tothe climate of the location where the isolate originatedfrom. Strains from one cluster (Rh 13, Me 8-2, Me 8-4and Me 8-7) were isolated from sub-arid areas in south-eastern Spain and Israel, and strains of the other cluster(RSA, Pin JRS1 and Pin JRS3) were isolated at highaltitude in continental and low-temperature areas inSpain. Even though Me 8-2 and Me 8-4 were isolatedfrom the same infected melon field they are furtherapart than Me 8-2 is from RH13, which was isolatedfrom Israel.

Discussion

Even though there are at least two hybridizationgroups present in AG4 (Vilgalys 1988), the variablegenetics and virulence described within AG4 contras-ted with the apparent lack of variation detected in this

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Fig. 4 Alignment of fungal ITSsequences. The R. solani strain isindicated. In the strains whereinsertions have occurred thelonger version has beendesignated L. R indicates A orG, ½ indicates C or T and¼ indicates A or T. Theasterisks indicate conservedpositions among all the strains.The arrows indicate the limits ofthe 5.8s rRNA gene

group by rDNA RFLP analysis (Vilgalys and Gonzalez1990). In the present study we sequenced the ITS regionof the rDNA from nine R. solani isolates belonging toAG4 and one belonging to AG1. This small group ofisolates can be distinguished according to virulence,based on differences in the size of the PCR product.

Another interesting feature of these sequences is thatvariation within a single strain of R. solani can bedetected. The heterogeneity caused by insertion or basesubstitution can be detected as the PCR used to am-plify the ITS sequences amplifies the total rDNA popu-

lation present. In the sequences from cloned fragments,this phenomenon was not detected since sequencing ofclones assesses only a single copy of the rDNAs. AsR. solani is known to be multinucleate (Sneh et al.1991), the heterogeneity could be due to chromosomevariation present in different nuclei within the samestrain. It is also possible that the insertion or substitu-tion occurred in some of the approximately 200 copies(Bruns et al. 1991) of the ITS sequence on the samechromosome, or that the same nucleus has differentrDNAs on different chromosomes. The variations do

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Fig. 4 (continued)

not always occur in the same position; for example, ofthe six substitutions in strain 521 and the three substi-tutions and an insertion in strain Rh2815, only an A/Gsubstitution in ITS2 is shared, while all of the othervariants are unique. In the six strains with hetero-

geneous rDNA, the intensity of the bands in the se-quencing gels are equivalent for a variants, so that thevariants are presumed to be present in a similar num-ber of copies.

The secondary structure of the ITS sequences waspredicted (data not shown) using the RNAFOLD op-tion 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 theinsertions and substitutions were placed in regions dis-tant from the joining region of the 18s or 28s and the5.8s rRNA and were, therefore, of minor importance forthe processing of the precursor rRNA. Van Nues et al.

bFig. 5 Single most-parsimonious phylogenetic tree generated froma branch-and-bound algorithm in PAUP 3.1.1. The numbers indicatethe percentage corresponding to the frequency with which a givenbranch appeared in a 500 bootstrap replications. Horizontal lengthsrepresent genetic distances, vertical lengths are not meaningful.A similar phylogenetic tree was obtained by the neighbor-joiningmethod. ¹wo asterisks and one asterisk denote branch points withconfidence limits of '99% and '95%, respectively. AG1 wasused as an outgroup

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(1994) showed that removal of a domain in the ITS1 region distal to the joining of the 17s and 5.8s rRNAhad no influence on the processing of rRNA in yeast.The presence of two (or more) different sequences ina single strain, despite high conservation of theseregions, indicates that the ITS regions have been underless evolutionary pressure than the rDNA genes,presumably because the spacers are functionally lessconstrained than the rRNA coding region. Furtherexperiments are needed to determine if there is anyfunction for the insertions and substitutions in thesestrains.

Both sequence analysis and RFLP polymorphismsplaced the AG1 and AG4 isolates in separate clusters,which allowed us to use AG1 as an outgroup to rootthe phylogenetic trees. RFLP analysis of these AG4strains grouped the isolates according to habitat, plac-ing the hypovirulent strains (Rh2815 and 521) relativelycloser to the high-altitude strains (Pin JRS 1 and 3 andRSA) with only two different restriction sites comparedto 3-4 different ones in the sub-arid strains (Rh 13, Me8-2, 8-4 and 8-7). In the parsimony analysis of thesequence data, the genetic distances between the hy-povirulent and virulent strains and the AG1 outgroupare similar.

Since more variation occurs in ITS1 than ITS2, itmay be possible in future studies to consider only thesequence data from ITS1 to determine the relationshipsbetween different strains of the same AG group inR. solani. Nevertheless, whereas whole-cell fatty acidcomposition could only identify and differentiate be-tween certain populations of AG-1 isolates (Johnk andJones 1994), we have shown that rRNA ITS sequenceanalysis could be used to classify strains from AG4according to virulence and habitat.

With the information presented here, it will be pos-sible to construct primers that are specific for a particu-lar group which could then be used to specificallyamplify sequences for diagnostic purposes. Using suchprimers a sufficiently large number of strains can bescreened to obtain relevant population data.

Acknowledgements This work was supported by Grant BIO 93 0923C02 02 from the Comision Interministerial de Ciencia y Tecnologıa(CICYT), Spain and Grants BIOT/CT91030 and BIO2/CT94-3011from the European Union. We thank Yigal Koltin, Javier Telloand Federico Uruburu for providing the strains for this study,Joaqun Dopazo for assistance with the phylogenetic analysis and forcritically reading the manuscript. Lone Rossen, Jaap Keijer andBaruch Sneh gave valuable suggestions and criticisms to improvethis work.

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