Differentiation of pathogenic and nonpathogenic isolates of <Emphasis Type="Italic">Geotrichum candidum</Emphasis> sensu Suprapta et al. (1995) on citrus fruit based on PCR-RFLP analysis of rDNA ITS and PCR using specific primers designed in polygalacturonase genes

Pathogenic and nonpathogenic isolates of Geotrichum candidum sensu Suprapta et al. (1995) that affect citrus fruit are indistinguishable morphologically. In this work, differentiation of the two pathogenicity types based on the internal transcribed spacer regions of ribosomal DNA (rDNA-ITS) and polygalacturonase (PG) genes was attempted. Polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) of rDNA ITS and PCR using specific primers to PG genes from each type could clearly differentiate the two pathogenicity types, and the profiles by PCR-RFLP of rDNA ITS genes corresponded with those by type-specific PCR of PG genes. These results indicate that the two pathogenic types can be differentiated at a molecular level and that PG genes are alternatively useful for distinguishing between the two types.     

Morphology,Physiology, and Virulence of <Emphasis Type="Italic">Bipolaris sorokiniana</Emphasis> Isolates

Bipolaris sorokiniana is a phytopathogenic fungus that causes diseases in cereal crops. The high morphological, physiological, and genetic variability makes the control of this fungus a difficult task. The aim of this work was to study the virulence, morphological, and physiological variability of B. sorokiniana isolates. For this, 35 B. sorokiniana isolates from different geographic regions in Brazil and other countries were used. The isolates were evaluated for their morphological variability, considering mycelium color, sector formation, and growth rate. Based on these morphological characteristics, the isolates were grouped in five different morphological groups. Extracellular enzymes activity in solid medium, virulence in wheat seeds and seedlings, and analysis of total proteins by SDS-PAGE were evaluated for all isolates. Variations among the isolates were found for enzymatic activity, and esterase was the enzyme that showed the highest activity indices. The results obtained from infection of seeds and seedlings showed that isolates from the same geographical region and morphological group had different degrees of virulence. The total protein profile shown by the isolates varied in the number of bands and intensity, where some of them may be used to characterize the specie.     

Vegetative Incompatibility Among Monoconidial Isolates of <Emphasis Type="Italic">Bipolaris sorokiniana</Emphasis>

Bipolaris sorokiniana is a phytopathogenic fungus that causes diseases of cereal crops, such as leaf-spot disease, common root rot, and black point of grain. Because of its great morphological, physiological, and genetic variability, this fungus is difficult to control. The aim of this investigation was to study the variability of isolates of B. sorokiniana by means of vegetative incompatibility. Thirty-five isolates of B. sorokiniana from different geographical regions in Brazil and other countries were used. The vegetative incompatibility between the isolates and the influences of different culture media on these reactions were evaluated. The total protein profile of the isolates was analyzed when the isolates were cultured separately, and in cultures of compatibility and incompatibility reactions. Eighteen of 31 confrontations showed vegetative incompatibility. The results obtained with different culture media for the vegetative compatibility/incompatibility genotypes suggested that the type of substratum influences these reactions. No differences in protein profiles among the isolates were observed. This result suggests that there is no induction of expression of different proteins in vegetative incompatibility reactions.     

Intra-isolate heterogeneity of the ITS region of rDNA in Pythium helicoides

Heterogeneity of the rDNA ITS region in Pythium helicoides and the phylogenetic relationship between P. helicoides and closely related species were investigated. In PCR-RFLP analysis of the rDNA ITS region of six P. helicoides isolates investigated, including the type culture, intraspecific variation was found at the HhaI site. The total length of fragments was longer than before cutting, indicating sequence heterogeneity within isolates. Digestion of the cloned rDNA ITS region derived from seven isolates with HhaI revealed polymorphisms among and within single zoospore isolates, and variability of the region was also present among the clones derived from the same isolate. To test whether the rDNA ITS region of closely related species and other regions in the genome of P. helicoides are also variable, the rDNA ITS region of P. ultimum and the cytochrome oxydase II (cox II) gene encoded in mitochondria were sequenced. P. ultimum had little variation in the rDNA ITS region. The cox II gene sequences of both species revealed only a low intraspecific variability and no intra-isolate variation. In the phylogenic tree based on the rDNA ITS sequences, all clones of P. helicoides formed one large clade that was distinct from the clades comprising morphologically similar species, such as P. oedochilum and P. ostracodes, and was closely related to P. chamaehyphon rather than the other species.     

Sequence and RFLP analysis of the ITS2 ribosomal DNA in two Neotropical social bees, <Emphasis>Melipona beecheii</Emphasis> and <Emphasis>Melipona yucatanica</Emphasis> (Apidae,Meliponini)

Two stingless bees species of the genus Melipona, M. beecheii and M. yucatanica, are the only ones reported for the Yucatan Peninsula. The natural distribution of M. beecheii ranges from southern Mexico to Costa Rica, that of M. yucatanica from south Mexico to Guatemala. Colonies of both species occur in a variety of habitats and show adaptations to local conditions denoting the occurrence of ecotypes. The ITS2 of ribosomal DNA has been characterized in both species and its utility to discriminate among colonies has been investigated through RFLP experiments. The ITS2 region is unusually long, 1788 bp in M. beecheii and 1845 bp in M. yucatanica (including the 3′ end of the 5.8S gene and partial 5′ of the 28S gene). Mean nucleotide divergence between both ITS2 sequences is 16% (excluding sites with insertions/deletions) and 20% when the insertions/deletions are taken into account. The G+C content in both sequences is close to 53%. The PCR-RFLP assay was performed with 12 restriction enzymes on colonies of M. beecheii from Mexico (Yucatan, Campeche and Chiapas) Costa Rica, El Salvador and Guatemala, and of M. yucatanica from Mexico (Yucatan) and Guatemala. The restriction patterns obtained allow to discriminating colonies of both species with different origins. Both kinds of data are thus useful for assessing intra and interspecific genetic variability and for developing appropriate conservation strategies for these species. Received 20 June 2007; revised 31 August 2007; accepted 12 September 2007.     

PHYLOGENY AND GENETIC VARIANCE IN TERRESTRIAL MICROCOLEUS (CYANOPHYCEAE) SPECIES BASED ON SEQUENCE ANALYSIS OF THE 16S rRNA GENE AND ASSOCIATED 16S–23S ITS REGION1

Thirty‐one strains of Microcoleus were isolated from desert soils in the United States. Although all these taxa fit the broad definition of Microcoleus vaginatus (Vaucher) Gomont in common usage by soil algal researchers, sequence data for the 16S rRNA gene and 16S–23S internal transcribed spacer (ITS) region indicated that more than one species was represented. Combined sequence and morphological data revealed the presence of two morphologically similar taxa, M. vaginatus and Microcoleus steenstrupii Boye‐Petersen. The rRNA operons of these taxa were sufficiently dissimilar that we suspect the two taxa belong in separate genera. The M. vaginatus clade was most similar to published sequences from Trichodesmium and Arthrospira. When 16S sequences from the isolates we identified as M. steenstrupii were compared with published sequences, our strains grouped with M. chthonoplastes (Mertens) Zanardini ex Gomont and may have closest relatives among several genera in the Phormidiaceae. Organization within the 16S–23S ITS regions was variable between the two taxa. Microcoleus vaginatus had either two tRNA genes (tRNA^Ile and tRNA^Ala) or a fragment of the tRNA^Ile gene in its ITS regions, whereas M. steenstrupii had rRNA operons with either the tRNA^Ile gene or no tRNA genes in its ITS regions. Microcoleus vaginatus showed no subspecific variation within the combined morphological and molecular characterizations, with 16S similarities ranging from 97.1% to 99.9%. Microcoleus steenstrupii showed considerable genetic variability, with 16S similarities ranging from 91.5% to 99.4%. In phylogenetic analyses, we found that this variability was not congruent with geography, and we suspect that our M. steenstrupii strains represent several cryptic species.     

URP‐based DNA Fingerprinting of Bipolaris sorokiniana Isolates Causing Spot Blotch of Wheat

Spot blotch, caused by the pathogen Bipolaris sorokiniana is an important disease of wheat and is responsible for large economic losses world wide. In this study, molecular variability in B. sorokiniana isolates collected from different regions of India was investigated using URP‐PCR technique. All the 40 isolates used in the study were pathogenic when tested on susceptible host, Agra local, although they varied in pathogenicity. Isolate BS‐49 was least virulent showing 4.5 infection index while BS‐75 was the most virulent with 63.4 infection index. The universal rice primers (URPs’) are primers which have been derived from DNA repeat sequences in the rice genome. Out of the 12 URP markers used in the study, 10 markers were effective in producing polymorphic fingerprint patterns from DNA of B. sorokiniana isolates. The analysis of entire fingerprint profile using unweighted pair group method with arithmetic averages (UPGMA) differentiated B. sorokiniana isolates obtained from different geographic regions. One isolate BS‐53 from northern hill zone was different from rest of the isolates showing less than 50% similarity. Broadly, three major clusters were obtained using UPGMA method. One cluster consisted of isolates from North western plain zone; second cluster having isolates from North eastern plain zone and third cluster consisted of isolates from Peninsular zone showing more than 75% genetic similarity among them. One of the markers, URP‐2F (5′GTGTGCGATCAGTTGCTGGG3′) amplified three monomorphic bands of 0.60, 0.80 and 0.90 kb size which could be used as specific markers for identification of B. sorokiniana. Further, based on URP‐PCR analysis, the grouping of the isolates according to the geographic origin was possible. This analysis also provided important information on the degree of genetic variability and relationship between the isolates of B. sorokiniana.     

Variation in the nuclear ribosomal DNA internal transcribed spacer (ITS) region of Pinus rzedowskii revealed by PCR-RFLP

 In the genus Pinus the internal transcribed spacers (ITS1 and ITS2) and the 5.8s region of the nuclear ribosomal DNA are approximately 3000 bp in length. ITS1 is considerably longer than ITS2 and partial sequences of ITS1 indicate that this region is evolving rapidly and exhibits intraspecific variation. The ITS2 and 5.8s regions are relatively conserved. We surveyed restriction fragment length variability of PCR-amplified fragments (PCR-RFLP) of the ITS region in four populations (86 individuals) of Pinus rzedowskii, a pine endemic to western Michoacán, Mexico. Five of the restriction endonucleases assayed revealed variation, with a total of 13 variants, most of which were length mutations of 300–900 bp. A moderate degree of population differentiation was detected. The average diversity (Shannon’s index) of ITS fragment size patterns was 1.19, with 34% of the variation due to differences among populations and 66% due to differences among individuals within populations. The same individuals were assayed for nine polymorphic isozymes, which gave diversity measures similar to those of each restriction endonuclease. Received: 25 August 1997 / Accepted: 19 September 1997     

PCR-RFLP of the ribosomal DNA internal transcribed spacers (ITS) provides markers for the A and B genomes in<Emphasis Type="Italic"> Musa</Emphasis> L.

Musa acuminata Colla (AA genomes) and Musa balbisiana Colla (BB genomes) are the diploid ancestors of modern bananas that are mostly diploid or triploid cultivars with various combinations of the A and B genomes, including AA, AAA, BB, AAB and ABB. The objective of this study was to identify molecular markers that will facilitate discrimination of the A and B genomes, based on restriction-site variations in the internal transcribed spacers (ITS) of the nuclear ribosomal RNA genes. The ITS regions of seven M. acuminata and five M. balbisiana accessions were each amplified by PCR using specific primers. All accessions produced a 700-bp fragment that is equivalent in size to the ITS of most plants. This fragment was then digested with ten restriction enzymes (AluI, CfoI, DdeI, HaeIII, HinfI, HpaII, MspI, RsaI, Sau3AI and TaqI) and fractionated in 2% agarose gels, stained with ethidium bromide and visualized under UV light. The RsaI digest revealed a single 530-bp fragment unique to the A genome and two fragments of 350-bp and 180-bp that were specific to the B genome. A further 56 accessions representing AA, AAA, AAB, AB and ABB cultivars, and synthetic hybrids, were amplified and screened with RsaI. All accessions with an exclusively A genome showed only the 530-bp fragment, while accessions having only the B-genome lacked the 530-bp fragment but had the 350-bp and 180-bp fragments. Interspecific cultivars possessed all three fragments. The staining intensity of the B-genome markers increased with the number of B-genome complements. These markers can be used to determine the genome constitution of Musa accessions and hybrids at the nursery stage, and, therefore, greatly facilitate genome classification in Musa breeding.Communicated by H.F. Linskens     

Molecular characterization and phylogenetic inferences of Dermanyssus gallinae isolates in Italy within an European framework

In order to investigate the genetic relationships between Dermanyssus gallinae (Metastigmata: Dermanyssidae) (de Geer) isolates from poultry farms in Italy and other European countries, phylogenetic analysis was performed using a portion of the cytochrome c oxidase subunit 1 (cox1) gene of the mitochondrial DNA and the internal transcribed spacers (ITS1+5.8S+ITS2) of the ribosomal DNA. A total of 360 cox1 sequences and 360 ITS+ sequences were obtained from mites collected on 24 different poultry farms in 10 different regions of Northern and Southern Italy. Phylogenetic analysis of the cox1 sequences resulted in the clustering of two groups (A and B), whereas phylogenetic analysis of the ITS+ resulted in largely unresolved clusters. Knowledge of the genetic make‐up of mite populations within countries, together with comparative analyses of D. gallinae isolates from different countries, will provide better understanding of the population dynamics of D. gallinae. This will also allow the identification of genetic markers of emerging acaricide resistance and the development of alternative strategies for the prevention and treatment of infestations.     

Molecular divergence of the soil yeasts <Emphasis Type="Italic">Williopsis</Emphasis> sensu stricto

Fifty-three strains of Saturn-spored yeasts were analyzed by means of restriction analysis of the amplified fragment of rDNA comprising the 5.8S rRNA gene and the internal transcribed spacers ITS1 and ITS2. The use of endonucleases HaeIII and MspI enabled clear differentiation of yeast species Williopsis mucosa, W. salicorniae, Zygowilliopsis californica, and Komagataea pratensis and the Williopsis sensu stricto complex. The minisatellite primer M13 was proposed for differentiation between sibling species of Williopsis sensu stricto, which have identical restriction profiles. PCR with primer M13 enabled reidentification of a number of collection strains, species identification of Saturn-spored isolates from the Far East, and detection of three strains affiliated to novel taxa. The latter have unique PCR profiles and differ in the nucleotide sequences of ITS1 and ITS2 fragments of rDNA. Possible variations in the results obtained with different molecular methods are discussed.Translated from Mikrobiologiya, Vol. 73, No. 6, 2004, pp. 768–776.Original Russian Text Copyright © 2004 by Naumova, Gazdiev, Naumov.     

Identification of Cyst Nematodes of Agronomic and Regulatory Concern with PCR-RFLP of ITS1

The first internally transcribed spacer region (ITS1) from cyst nematode species (Heteroderidae) was compared by nucleotide sequencing and PCR-RFLP. European, Asian, and North American isolates of five heterodefid species were examined to assess intraspecific variation. PCR-RFLP patterns of amplified ITS1 DNA from pea cyst nematode, Heterodera goettingiana, from Northern Ireland were identical with patterns from Washington State. Sequencing demonstrated that ITS1 heterogeneity existed within individuals and between isolates, but did not result in different restriction patterns. Three Indian and two U.S. isolates of the corn cyst nematode, Heterodera zeae, were compared. Sequencing detected variation among ITS1 clones from the same individual, between individuals, and between isolates. PCR-RFLP detected several restriction site differences between Indian and U.S. isolates. The basis for the restriction site differences between isolates from India and the U.S. appeared to be the result of additional, variant ITS1 regions amplified from the U.S. isolates, which were not found in the three India isolates. PCR-RFLP from individuals of the U.S. isolates created a composite pattern derived from several ITS1 types. A second primer set was specifically designed to permit discrimination between soybean (H. glycines) and sugar beet (H. schachtii) cyst nematodes. Fok I digestion of amplified product from soybean cyst nematode isolates displayed a uniform pattern, readily discernible from the pattern of sugar beet and clover cyst nematode (H. trifolii).     

Distinguishing homokaryons and heterokaryons in Phellinus sulphurascens using pairing tests and ITS polymorphisms

Phellinus sulphurascens Pilát causes laminated root rot of coniferous species in both western North America (WNA) and Asia. Accurate somatic incompatibility tests for mapping population structures have been difficult to conduct for P. sulphurascens because no single, unambiguous criterion has allowed differentiation of homokaryotic and heterokaryotic isolates. In a population study of P. sulphurascens in WNA, two types of ITS sequences were found in the single spore and vegetative isolates. All single spore isolates (SSIs) had either ITS type-1 or type-2 whereas some vegetative isolates had both ITS types. The segregation pattern for inheritance of ITS, which we observed in SSIs from eight basidiocarps, suggested that each ITS type occurred in a different nucleus and that each basidiospore inherited only one ITS type. In four SSIs from Russia and eight heterokaryotic isolates from Japan, nine different ITS types, referred to as type-3 to -11, were detected. A variety of pairing tests conducted between known Asian and WNA homokaryon and heterokaryon isolates did not always give consistent results with respect to fungal mat morphologies and formation of demarcation lines. However, the ITS types that occurred after pairing tests did follow consistent patterns. Thus, using ITS polymorphisms and pairing tests between Asian tester isolates and 49 vegetative isolates from WNA, we were able to accurately distinguish between homokaryotic and heterokaryotic isolates.     

Molecular characterization of isolates of Beauveria bassiana obtained from overwintering and summer populations of Sunn Pest (Eurygaster integriceps)

Aim:  To examine whether isolates of the entomopathogenic fungus Beauveria bassiana are more closely associated to their summer hosts compared with overwintering hosts, with recently developed molecular tools based on mitochondrial regions. Methods and Results:  Primers for the traditional ITS1‐5·8S‐ITS2 region and two mitochondrial intergenic regions, namely, nad3‐atp9 and atp6‐rns, were used. All amplified products were sequenced, aligned and Neighbour‐Joining (NJ), parsimony and Bayesian phylogenetic inference analyses were performed. The isolates examined were grouped with very good support into three distinct groups, two of them showed geographical correlation, but no clear association to their host. Conclusions:  The mitochondrial intergenic regions used were more informative than the nuclear ITS1‐5·8S‐ITS2 sequences. The sequence variability observed, that allowed the phylogenetic placement of the isolates into distinct groups, depended on the geographical origin of the isolates and can be exploited for designing group‐specific and isolate‐specific primers for their genetic fingerprinting. No clear associations with summer Sunn Pest populations were observed. Significance and Impact of the Study:  Studies on the genetic variability of biocontrol agents like B. bassiana are indispensable for the development of molecular tools for their future monitoring.     

Ribosomal DNA-ITS sequence polymorphism in the sugarcane rust,Puccinia kuehnii

The two highly divergent ITS types previously observed in isolates ofPuccinia kuehnii were amplified from the same isolates through the use of ITS type-specific primers for PCR and are therefore considered as polymorphisms of ITS regions within the species. Although homology of sequences of one of the ITS types with otherPuccinia species was of expected levels, significantly high homology of sequences of the other type with those ofCronartium members indicates that abnormal genetic events led to the occurrence of these polymorphic regions. These results indicate that ITS gene tree phylogeny may not reflect true species phylogeny in this group of rust fungi. Rather, D1/D2 region tree phylogeny, which was concordant with the differences in morphology with and among related rusts, more correctly reflects phylogenetic relationships of sugarcane and related grass rusts. contribution No. 159, Laboratory of Plant Parasitic Mycology, Institute of Agriculture and Forestry, University of Tsukuba.     

Genetic Diversity Among Monoconidial and Polyconidial Isolates of Bipolaris sorokiniana

Spot blotch caused by Bipolaris sorokiniana is a destructive disease of wheat in warm and humid wheat-growing regions of the world. This fungus shows a high genetic diversity and morphological and physiologic variability. In this study, 19 polysporic and 57 monosporic isolates of B. sorokiniana were characterized using universal rice primers—URP-PCR. The results obtained when the dendrogram was constructed with all the data produced with the amplification products showed very distinct clusters. However, the similarity among the isolates was low where 37 and 26.3 % of the monosporic and polysporic isolates, respectively, showed similarity above 70 %. All primers amplified multiple DNA fragments of polysporic as well as the monosporic isolates. Isolates fingerprints were constructed based on binary characters revealed by the three primers. An amplified fragment of approximately 750 bp was observed among 40 % of the isolates, when primer URP-1F was used. When primers URP-4R and URP-2R were used, a fragment of 450 and 400 bp was present in 31.5 and 29 % of the isolates, respectively. It was expected a higher similarity among the isolates since the monosporic cultures were originated from the polysporic. The dendrogram did not enable the separation of B. sorokiniana isolates by their geographic origin. This low correlation suggests that gene transfer may have occurred by parasexual combination in this fungus population. However, in spite of the research efforts for that end, it has not been possible to establish patterns that characterize the profile of B. sorokiniana.     

Polymorphism in the internal transcribed spacer (ITS) region of the ribosomal DNA among different Fusarium species

Fusarium species causing wilt diseases in different plants were characterised by comparing nonpathogenic and different pathogenic species using rDNA RFLP analysis. The ITS (internal transcribed spacer) region of 12 isolates belonging to the section Elegans, Laseola, Mortiella, Discolor, Gibbosum, Lateritium and Sporotrichiella were amplified by universal ITS primers (ITS-1 and ITS-4) using polymerase chain reaction (PCR). Amplified products, which ranged from 522 to 565 bp were obtained from all 12 Fusarium isolates. The amplified products were digested with seven restriction enzymes, and restriction fragment length polymorphism (RFLP) patterns were analysed. A dendrogram derived from PCR-RFLP analysis of the rDNA region divided the Fusarium isolates into three major groups. Assessment of molecular variability based on rDNA RFLP clearly indicated that Fusarium species are heterogeneous and most of the forma speciales have close evolutionary relationships.     

Genetic relationship of Tricholoma matsutake and T. nauseosum from the Northern Hemisphere based on analyses of ribosomal DNA spacer regions

The genetic relationship among Tricholoma matsutake and T. nauseosum strains collected from various parts of the Northern Hemisphere was investigated using sequence analysis of the rDNA ITS region and PCR-RFLP analysis of the rDNA IGS-1 region. ITS sequence similarity between T. matsutake and T. nauseosum ranged between 98.1% and 100%. The strains of T. matsutake from coniferous forests and those from broad-leaved forests showed more than 99.8% similarity in their ITS sequences. Three distinct RFLP types were detected when IGS-1 regions were digested with Cfr13I. RFLP patterns showed no variability among the strains of T. nauseosum and those of T. matsutake from broad-leaved forests. This pattern corresponded to the dominant RFLP type in the Japanese population of T. matsutake. Thus, strains belonging to this RFLP type are widely distributed throughout East Asia and Europe and associated with many tree species of Pinaceae and Fagaceae. The result suggests that T. matsutake in coniferous and broad-leaved forests and T. nauseosum should be treated as the same species genetically.     

广东省水库微囊藻的产毒特征和ITS序列的遗传多样性分析

为了解广东省水库微囊藻的产毒特征和ITS 序列的遗传多样性,从广东省供水水库中分离得到28 株微囊藻(Microcystisspp.),对它们的产毒特征和15 株微囊藻的ITS 序列进行了分析.高效液相色谱(HPLC)和微囊藻毒素合成酶基因mcyE 的检测结果表明,广东省水库中的微囊藻以产毒藻株占优势,微囊藻毒素的主要类型为MC-RR.广东省15 株藻株的ITS 序列相似性大于93.2%,在用相邻法(NJ)构建的系统树上,不同形态的种和不同地理区域的藻株没有区分开,产毒和非产毒藻株没有形成独立分支.这说明微囊藻ITS 序列的遗传多样性较低,ITS 序列和mcyE 存在没有相关性,表型不能够反映藻株的进化关系.因此,有必要将藻类传统分类方法与分子方法结合起来对蓝藻进行重新分类.     

Evolutionary Diversification Indicated by Compensatory Base Changes in ITS2 Secondary Structures in a Complex Fungal Species, <Emphasis Type="Italic">Rhizoctonia solani</Emphasis>

The rRNA cistron (18S–ITS1–5.8S–ITS2–28S) is used widely for phylogenetic analyses. Recent studies show that compensatory base changes (CBC) in the secondary structure of ITS2 correlate with genetic incompatibility between organisms. Rhizoctonia solani consists of genetically incompatible strain groups (anastomosis groups, AG) distinguished by lack of anastomosis between hyphae of strains. Phylogenetic analysis of internal transcribed spacer (ITS) sequences shows a strong correlation with AG determination. In this study, ITS sequences were reannotated according to the flanking 5.8S and 28S regions which interact during ribogenesis. One or two CBCs were detected between the ITS2 secondary structure of AG-3 potato strains as compared to AG-3 tobacco strains, and between these two strains and all other AGs. When a binucleate Rhizoctonia species related to Ceratobasidiaceae was compared to the AGs of R. solani, which were multinucleate (3–21 nuclei per cell), 1–3 CBCs were detected. The CBCs in potato strains of AG-3 distinguish them from AG-3 tobacco strains and other AGs yielding further evidence that the potato strains of AG-3 originally described as R. solani are a species distinct from other AGs. The ITS1–5.8S–ITS2 sequences were analyzed by direct sequencing of PCR products from 497 strains of AG-3 isolated from potato. The same 10 and 4 positions in ITS1 and ITS2, respectively, contained variability in 425 strains (86%). Nine different unambiguous ITS sequences (haplotypes) could be detected in a single strain by sequencing cloned PCR products indicating that concerted evolution had not homogenized the rRNA cistrons in many AG-3 strains. Importantly, the sequence variability did not affect the secondary structure of ITS2 and CBCs in AG-3. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.