Biofilm formation and resistance to bactericides of <Emphasis Type="Italic">Pseudomonas syringae</Emphasis> pv. <Emphasis Type="Italic">theae</Emphasis>

Biofilm-grown cells of Pseudomonas syringae pv. theae (P.s.theae) wild-type strain K9301 on abiotic surface had remarkable resistance to kasugamycin in comparison to planktonically grown cells; however, the biofilm-grown cells of K9301 had the same sensitivity to copper sulfate. Because both the lesser biofilm-forming strain K9301S3 and enhanced biofilm-forming strain K9301-6 also had remarkable biofilm resistance to kasugamycin just as K9301 did and because epigallocatechin gallate, which enhanced biofilm formation of P.s.theae, had no effect on biofilm resistance to kasugamaycin, the degree of biofilm formation was not correlated with the antibiotic susceptibilities. In addition, K9301 and K9301S3 had less sensitivity to kasugamycin but had high sensitivity to copper sulfate on nonwounded leaf surfaces. These results indicate a possibility that the mechanism of P.s.theae biofilm resistance to bactericide functions on both abiotic and nonwounded leaf surfaces.     

Epigallocatechin gallate,a major tea catechin,induces biofilm formation of <Emphasis Type="Italic">Pseudomonas syringae</Emphasis> pv. <Emphasis Type="Italic">theae</Emphasis>

Plants constitutively produce a variety of secondary metabolites that have antimicrobial activities against phytopathogens; however, interactions between these performed antimicrobial compounds and phytopathogens were poorly understood. In this study, interactions between epigallocatechin gallate (EGCg), which was a major tea catechin that had antimicrobial activities against varieties of bacteria, and Pseudomonas syringae pv. theae (P.s. theae), the causal of bacterial shoot blight of tea, were investigated. EGCg had less antimicrobial activity against P.s. theae; however, subinhibitory concentrations of EGCg induced biofilm formation. Because biofilms are induced in the presence of sucrose in the culture medium but not by P.s. theae strains deficient in exopolysaccharide levan production, biofilm induction by EGCg and levan production are closely related. EGCg increased survival of P.s. theae under dry conditions on nonwounded leaf surfaces in the presence of sucrose. These data indicate the possibility that tea catechins affect the survival of P.s. theae on the phyllosphere. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Subcellular localization of the protein encoded by virulence gene <Emphasis Type="Italic">psvA</Emphasis> of <Emphasis Type="Italic">Pseudomonas syringae</Emphasis> pv. <Emphasis Type="Italic">eriobotryae</Emphasis>

The plasmid-encoded virulence gene psvA was previously isolated from Pseudomonas syringae pv. eriobotryae and sequenced. The deduced protein of the psvA gene had no significant similarity to any other protein sequences in the database. To gain a better understanding of the function of the PsvA protein its subcellular localization was examined. To localize the PsvA protein within the bacteria, the cells were fractionated into cytoplasmic, inner membrane, and outer membrane components. The cell fractions and culture supernatant were analyzed by immunoblotting. The PsvA protein was predominantly detected in the outer membrane fraction. Immunoelectron microscopy also showed that the PsvA protein was located in the outer membrane.     

Genetic relationships among strains of <Emphasis Type="Italic">Xanthomonas campestris</Emphasis>pv. <Emphasis Type="Italic">campestris</Emphasis>revealed by novel rep-PCR primers

Novel primers for rep-PCR were developed with the original software and based on `ancient diverged periodical sequences'. Rep-PCR with these primers was applied to study genetic relationships among 51 Xanthomonas campestris strains. The strains were collected from different countries including Russia, Japan, UK, Germany and Hungary. Reference strains of three X. campestrispathovars and five other Xanthomonas species were included. Based on qualitative differences in amplification profiles, the strains were divided into four major groups. Two subgroups recognised within X. campestrispopulation were similar to RFLP haplotypes. The third subgroup included strains of two other pathovariants and Japanese isolates of X. campestris pv. campestriswhile the fourth group comprised the other species of Xanthomonas. The analysis of the diversity within X. campestris resulted in the conclusion that isolates belong to distinct clonal populations (subgroups). The differences between the subgroups of X. campestris were only slightly smaller than between species of Xanthomonas. A PCR fragment about 600 bp amplified by primer KRPN2 was found in nearly all tested strains of X. campestris.SCAR primers designed for this marker produced a single specific band for strains of X. campestris, but not for other Xanthomonas, Pseudomonas and Erwiniastrains tested. Application of the new primer set for rep-PCR offers a rapid, simple and reproducible method for identification of bacterial strains. The X. campestris-specific SCAR primers may be used in diagnostics of this important plant pathogen.     

Identification and Discrimination of Pseudomonas syringae Isolates from Wild Cherry in England

A survey of wild cherry (Prunus avium) woodland plantations and nurseries was carried out in 2000/01. Trees with symptoms of bacterial canker were found in 20 of the 24 plantations visited and in three of seven nurseries. Fifty-four Pseudomonas syringae isolates from wild cherry together with 22 representative isolates from sweet cherry and 13 isolates from other Prunus spp., pear and lilac were characterised by physiological, biochemical, serological and pathogenicity tests. Isolates from wild cherry were predominantly P. syringae pv. syringae (Pss), but P. syringae pv. morsprunorum (Psm) races 1 and 2 were also found. Physiological and biochemical tests discriminated Psm races 1 and 2 from other P. syringae isolates. Agglutination and indirect-enzyme-linked immunosorbent assay tests with three different antisera showed that Psm race 1 and race 2 were very uniform and indicated high variability amongst other P. syringae isolates. However, pathogenic Pss isolates could not be distinguished from non-pathogenic isolates of P. syringae on the basis of physiological, biochemical or serological tests. Pathogenicity tests on rooted lilac plants and on micropropagated plantlets of lilac and two wild cherry clones differentiated Pss and Psm isolates and demonstrated a range of aggressiveness amongst Pss isolates. Serological tests could be used as an alternative to the classical physiological and biochemical tests to increase the speed of detection and discrimination of isolates, but pathogenicity tests are still necessary to discriminate the pathogenic Pss isolates.     

A diagnostic medium for the semi-selective isolation and enumeration of <Emphasis Type="Italic">Xanthomonas axonopodis</Emphasis>pv. <Emphasis Type="Italic">vignicola</Emphasis>

A semi-selective medium for isolation of Xanthomonas axonopodis pv. vignicola from cowpea (Vigna unguiculata) plant and soil samples was developed. Twelve carbon and five nitrogen sources were tested with four strains of X. axonopodispv.vignicola, and 25 antibiotics were screened against saprophytes. -cellobiose (10g) was selected as the optimal carbon source. Among the antibiotics, cefazoline inhibited growth of most of the saprophytes with little effect on strains of the pathogen. ,-methionine enhanced growth of X. axonopodispv.vignicola. Boric acid along with ammonium chloride suppressed growth of Pseudomonas fluorescens. The semi-selective medium designated as cefazoline-cellobiose-methionine (CCM) medium contained K₂HPO₄ 1.34g, KH₂PO₄ 0.4g, MgSO₄ 0.3g, H₃BO₃ 0.2g, NH₄Cl 1.0g, -cellobiose 10g, cycloheximide 0.2g, ,-methionine 1.0g, cefazoline 10mg and agar 14g per l of water (pH 7.2). Colonies of X. axonopodispv.vignicola on CCM medium were whitish, round, raised and 0.2–1.8mm in diameter 96h after incubation. CCM medium generally inhibited growth of Pantoea agglomerans, Bacillus subtilis and saprophytes isolated from cowpea leaves. Colonies of Pseudomonas fluorescens and a saprophytic bacterium, which were not completely suppressed by CCM, could be differentiated from X. axonopodispv.vignicola by their smaller size and different color. The CCM medium proved useful for isolation of X. axonopodispv.vignicola from cowpea plant and soil samples. This is the first report of a semi-selective medium developed for detection of X. axonopodispv.vignicola.     

Adsorption of OP<Subscript>1</Subscript>-related bacteriophages requires the gene encoding a TonB-dependent receptor-like protein in <Emphasis Type="Italic">Xanthomonas</Emphasis><Emphasis Type="Italic">oryzae</Emphasis> pv. <Emphasis Type="Italic">oryzae</Emphasis>

Xanthomonas oryzae pv. oryzae strain T7174R is lysed by bacteriophage OP_1h and OP_1h2. Three mutants tolerant to both OP_1h and OP_1h2 were isolated by transposon mutagenesis. The mutants had an insertion of the transposon in XOO1687, which is predicted to encode a TonB-dependent receptor gene. Plasmid pHMIroNB that contained XOO1687 of T7174R was constructed, and the mutant was transformed with the plasmid. The transformant recovered sensitivity to OP_1h and OP_1h2. Electron microscopic analysis demonstrated that OP_1h and OP_1h2 can adsorb to the wild type and the transformant, but they could not adsorb to the phage-tolerant mutant. These results suggest that the TonB-dependent receptor gene relates to adsorption and infection of T7174R by OP_1h2 and OP_1h. Y. Inoue and S. Tsuge have contributed equally to this work.     

Analysis of sources of oxolinic acid-resistant field strains of <Emphasis Type="Italic">Burkholderia glumae</Emphasis> based on rep-PCR analysis and nucleotide sequences of <Emphasis Type="Italic">gyrB</Emphasis> and <Emphasis Type="Italic">rpoD</Emphasis>

Repetitive sequence-based polymerase chain reaction (rep-PCR) analysis using BOX and ERIC as primers showed a highly divergent phylogeny among field strains of Burkholderia glumae. To elucidate the sources of oxolinic acid (OA) resistance in field strains of B. glumae isolated from rice seedlings cultivated in Mie, Toyama, and Iwate prefectures, Japan, the amino acid at position 83 of GyrA (GyrA83), which is involved in OA resistance, and the DNA patterns from the rep-PCR and the partial nucleotide sequences of gyrB and rpoD from various strains were analyzed. The ten Mie strains, in which GyrA83 was isoleucine (Ile), were divided into two groups based on the band patterns in rep-PCR analysis, although the nucleotide sequences of gyrB and rpoD were identical among the strains. Based on the band patterns in the rep-PCR analysis and the gyrB and rpoD sequences, two highly OA-resistant Toyama strains, Pg-13 and Pg-14, for which GyrA83 was serine (Ser) and Ile, respectively, were in the same lineage. This suggests that the bacteria might acquire OA resistance faster than phylogenic diversity as determined with the repetitive sequences BOX and ERIC and with gyrB and rpoD. Furthermore, three Iwate strains (H95, H101, and H104), isolated from seedlings of different cultivars grown in different years and having Ile at GyrA83, are probably in the same lineage, suggesting that OA-resistant bacteria might be transferred among different cultivars.     

Characterization of biotin-autotrophic isolates derived from naturally occurring auxotrophic race 2 isolates of <Emphasis Type="Italic">Fusarium oxysporum</Emphasis> f. sp. <Emphasis Type="Italic">lactucae</Emphasis>

Race 2 isolates of Fusarium oxysporum f. sp. lactucae have been recognized as biotin auxotrophs and consequently have restricted growth on Puhalla's minimal medium (MM), which contains no biotin. Biotin-autotrophic isolates were raised from race 2 isolates through cultural mutation that grew as well on MM as they did on MM supplemented with biotin. These autotrophs were identical to the parental isolates in pathogenicity on race differential cultivars of lettuce (Patriot, Banchu Red Fire, and Costa Rica No. 4), and thus were designated as race 2. A vegetative compatibility test indicated that the autotrophic isolates fell into the same vegetative compatibility group as the parents. Culture filtrates of the autotrophs allowed abundant growth of the parental auxotroph on MM, and, through a competitive enzyme-binding assay, biotin was detected in the culture filtrates. These results suggest that biotin auxotrophy in the natural race 2 isolates has no direct relation to pathogenicity, qualitatively defined as physiological race, or to vegetative compatibility.     

Virulence and diversity of <Emphasis Type="Italic">Blumeria graminis</Emphasis> f.sp. <Emphasis Type="Italic">hordei</Emphasis> in East China

Four hundred and sixty-one isolates of Blumeria graminis f.sp. hordei were obtained from eight populations occurring on cultivated barley (Hordeum vulgare) at four geographically distant locations in China during 2003 and 2004. Their virulence frequency was determined on 30 differential lines. No isolate was virulent on differential lines possessing the resistance genes Mla1, Mla3, Mla6, Mla7, Mla9, Mla12, Mla13, Mlat, Mlg, Mla10, Mla22, Mla23, Mlp1, Ml(N81) and Mlmw. Virulences to the first nine resistance genes are prevalent in Europe and constitute the main part of genetic distance between Chinese and European populations. Conversely, no isolate was avirulent on the differential lines possessing the genes Mla8 and Ml(Ch). The frequencies of isolates overcoming the genes Mla2, Mla11, Mlk1 and Mlk2 were .4–9.3%, and frequencies of isolates overcoming the genes Mlh, MlLa, Ml(Bw), Mlra, Ml(Ru2), mlw, MlGa, MlWo and Mlnn ranged from 18.2% to 98.7%. Based on reactions of differential lines possessing the genes Mlk1, Mlh, MlLa, Ml(Bw), Mlra and Ml(Ru2), pathotypes were identified and diversity parameters calculated. Eleven of 22 detected pathotypes were found in both years and comprised 94.6% of isolates. Generally, the populations from different locations in 1 year were more closely related than populations collected from the same locations in different years. Complete effectiveness of the resistance genes, for which no corresponding virulences were found, will allow Chinese breeders to access many modern European barley cultivars that are fully resistant to powdery mildew in China, including those possessing the non-host resistance gene mlo.     

<Emphasis Type="Italic">Physalis ixocarpa</Emphasis> and <Emphasis Type="Italic">P. peruviana</Emphasis>, new natural hosts of <Emphasis Type="Italic">Tomato chlorosis virus</Emphasis>

Tomato chlorosis virus causes yellow leaf disorder epidemics in many countries worldwide. Plants of Physalis ixocarpa showing abnormal interveinal yellowing and plants of Physalis peruviana showing mild yellowing collected in the vicinity of tomato crops in Portugal were found naturally infected with ToCV. Physalis ixocarpa and P. peruviana were tested for susceptibility to ToCV by inoculation with Bemisia tabaci, Q biotype. Results confirmed that ToCV is readily transmissible to both species. The infection was expressed in P. ixocarpa by conspicuous interveinal yellow areas on leaves that developed into red or brown necrotic flecks, while P. peruviana test plants remained asymptomatic. Infected plants of both P. ixocarpa and P. peruviana served as ToCV sources for tomato infection via B. tabaci transmission. This is the first report of P. ixocarpa and P. peruviana as natural hosts of ToCV.     

Susceptibility and resistance of <Emphasis Type="Italic">Beta vulgaris</Emphasis> subsp. <Emphasis Type="Italic">maritima</Emphasis> to foliar rub-inoculation with <Emphasis Type="Italic">Beet necrotic yellow vein virus</Emphasis>

Four lines (designated MR0, MR1, MR2, and M8) from 13 accessions of Beta vulgaris subsp. maritima were selected on the basis of phenotypes produced after foliar rub-inoculation with Beet necrotic yellow vein virus (BNYVV). The susceptible phenotype developed bright yellow local lesions, whereas the resistant phenotype had symptoms ranging from no visible lesions to necrotic lesions at the inoculation site. MR1 and MR2 lines had a resistant phenotype depending on the isolate and the MR0 line was susceptible to all isolates of BNYVV tested. The M8 line was highly susceptible; the virus spread systemically and caused severe stunting. These plant lines will be useful for distinguishing BNYVV isolates having different pathogenicities, especially those controlled by RNA3 and/or RNA5.     

Characteristics of a <Emphasis Type="Italic">Plasmopara angustiterminalis</Emphasis> isolate from <Emphasis Type="Italic">Xanthium strumarium</Emphasis>

Leaves of Xanthium strumarium infected with downy mildew were collected in the vicinity of a sunflower field in southern Hungary in 2003. Based on phenotypic characteristics of sporangiophores, sporangia and oospores as well as host preference the pathogen was classified as Plasmopara angustiterminalis. Additional phenotypic characters were investigated such as the size of sporangia, the number of zoospores per sporangium and the time-course of their release. Infection studies revealed infectivity of the P. angustiterminalis isolate to both X. strumarium and Helianthus annuus. Inoculation of the sunflower inbred line, HA-335 with resistance to all known P. halstedii pathotypes, resulted in profuse sporulation on cotyledons and formation of oospores in the bases of hypocotyls. Infections of sunflower differential lines often led to damping-off. Molecular genetic analysis using simple sequence repeat primers and nuclear rDNA sequences revealed clear differences to Plasmopara halstedii, the downy mildew pathogen of sunflower.     

Isolation of pathogenicity- and gregatin-deficient mutants of <Emphasis Type="Italic">Phialophora gregata</Emphasis> f. sp. <Emphasis Type="Italic">adzukicola</Emphasis> through <Emphasis Type="Italic">Agrobacterium tumefaciens</Emphasis>-mediated transformation

Phialophora gregata f. sp. adzukicola, a causal agent of brown stem rot in adzuki beans, produces phytotoxic compounds: gregatins A, B, C, D, and E. Gregatins A, C, and D cause wilting and vascular browning in adzuki beans, which resemble the disease symptoms. Thus, gregatins are considered to be involved in pathogenicity. However, molecular analyses have not been conducted, and little is known about other pathogenic factors. We sought to isolate nonpathogenic and gregatin-deficient mutants through Agrobacterium tumefaciens-mediated transformation (ATMT) for cloning of pathogenicity-related genes. The co-cultivation of P. gregata and A. tumefaciens for 48 h at 20°C with 200 μM acetosyringone resulted in approximately 80 transformants per 10⁶ conidia. The presence of acetosyringone in the A. tumefaciens pre-cultivation period led to an increase in T-DNA copy number per genome. Of 420 and 110 transformants tested for their pathogenicity and productivity of gregatins, one nonpathogenic and three gregatin-deficient mutants were obtained, respectively. The nonpathogenic mutant produced gregatins, whereas the gregatin-deficient mutants had pathogenicity comparable to the wild-type strain. This is the first report of ATMT of P. gregata. Further analysis of these mutants will help reveal the nature of the pathogenicity of this fungus including the role of gregatin in pathogenesis.     

Tobacco cultivars vary in induction of systemic resistance against <Emphasis Type="Italic">Cucumber mosaic virus</Emphasis> and growth promotion by <Emphasis Type="Italic">Pseudomonas chlororaphis</Emphasis> O6 and its <Emphasis Type="Italic">gacS</Emphasis> mutant

The colonization of plant roots with certain rhizosphere bacteria promotes plant growth and induces long lasting systemic protection against a broad spectrum of plant pathogens. The role of the global regulator, GacS, in the rhizosphere colonist Pseudomonas chlororaphis O6 in stimulating growth promotion and induced resistance against Cucumber mosaic virus was examined in tobacco. Responses were compared in tobacco cvs Samsun and GX3. Root colonization of Samsun with wild-type O6 and the gacS complemented mutant-elicited reduced viral symptoms and viral titre. On GX3, there was little affect on symptoms when roots were colonized by the wild-type, gacS mutant or complemented mutant but colonization by both the wild-type and the gacS mutant lowered viral titre. Wild-type O6 and the gacS mutant caused plant growth to be maintained in both tobacco cultivars after viral infection, although the affect was stronger with GX3 than Samsun. In contrast, although a chemical inducer, benzothiadiazole, reduced symptoms and viral titre in both cultivars, plant growth was suppressed. Our results indicate rhizobacteria-elicited induced viral resistance without a negative impact on growth but there was a differential response between cultivars. Detailed knowledge regarding the mechanisms inherent to these differences between cultivars requires further investigation.     

Genetics of host-pathogen interactions in the <Emphasis Type="Italic">Pyrenophora teres</Emphasis> f. <Emphasis Type="Italic">teres</Emphasis> (net form) – barley (<Emphasis Type="Italic">Hordeum vulgare)</Emphasis> pathosystem

The genetics of host-pathogen interactions in the Hordeum vulgare – P. teres f. teres pathosystem was studied in twelve resistant barley accessions, i.e. CI 9825, CI 9819, Diamond, CI 4922, CI 5401, Harbin, c-8755, c-21849, c-8721 c-23874, c-19979, c-15811. F₂ analyses of crosses with susceptible genotypes employing various isolates (from Europe, USA, Canada, and Australia) revealed that resistance is mostly isolate-specific and controlled by one or two genes. Segregation in ascospore progeny from two crosses between isolates of different origin revealed that avirulence in P. teres is also determined by one or two genes. An epistatic effect of suppressor genes on avirulence genes is proposed for the genetics of virulence to Diamond, Harbin, CI 5401 and c-8721 in the fungal crosses D (181-6 × A80) and F (H-22 × 92-178/9). Segregation in F₂ of crosses of three new sources of resistance (c-23874, c-19979, c-15811) to the susceptible cv. Pirkka was studied in laboratory and greenhouse tests by using seven P. teres isolates, i.e. 181-6, d8-3, d8-4, d9-1, d9-4, F4 and F74. In addition, virulence to these barley accessions of ascospore progeny from crosses of the same isolates was studied. Based on these studies it was concluded that depending on the isolate used, resistance of c-23874 is determined at least by two genes and in c-19979 and c-15811 by three genes. The results of this parallel analyses of genetics of resistance and genetics of virulence allows the postulation of a gene–for–gene interaction in the P. teres – H. vulgare pathosystem.     

Effects of temperature,free moisture duration and inoculum concentration on infection of sweet cherry by<Emphasis Type="Italic">Pseudomonas syringae</Emphasis> pv.<Emphasis Type="Italic">syringae</Emphasis>

The effects of temperature, free moisture duration and inoculum concentration on infection caused byPseudomonas syringae pv.syringae (Pss), on sweet cherry (Prunus avium) were investigated. Epiphytic populations ofPss are an important source of inoculum for bacterial canker and it has been demonstrated that a cyclic pattern exists during the year, from undetectable during the warm and dry periods to large populations following cool and wet periods. The effects of temperature and inoculum concentration on the infection caused byPss on immature fruits and 1-yr-old twigs were significant (P<0.001). Fruit and twig infection increased linearly in proportion to the logarithm ofPss when bacterial concentrations were higher than 10³ cfu ml⁻¹ and temperatures were between 5 and 20°C. Regardless of the inoculum concentration and the free moisture duration, fruit and twig infection was either absent or low at 5°C but it increased linearly as temperature increased from 5 to 20°C. Growth ratein vitro was very slow (0.03–0.04 cfu h⁻¹) at 5°C and fast (0.21–0.23 cfu h⁻¹) at 20°C. Therefore, it is possible that multiplication of the epiphytic populations may be significantly reduced in the field with air temperatures below 5°C. A significant (P<0.001) effect of free moisture was obtained only when a low inoculum concentration (10³ cfu ml⁻¹) was used, and a significant linear response between free moisture and disease incidence was obtained only at 10°C. An apparent threshold population ofPss higher than 10³ cfu ml⁻¹ was needed to infect immature fruits and 1-yr-old twigs of sweet cherry. http://www.phytoparasitica.org posting July 10, 2002.     

Incidence of viruses in <Emphasis Type="Italic">Alstroemeria</Emphasis> plants cultivated in Japan and characterization of <Emphasis Type="Italic">Broad bean wilt virus-2, Cucumber mosaic virus</Emphasis> and <Emphasis Type="Italic">Youcai mosaic virus</Emphasis>

Alstroemeria plants were surveyed for viruses in Japan from 2002 to 2004. Seventy-two Alstroemeria plants were collected from Aichi, Nagano, and Hokkaido prefectures and 54.2% were infected with some species of virus. The predominant virus was Alstroemeria mosaic virus, followed by Tomato spotted wilt virus, Youcai mosaic virus (YoMV), Cucumber mosaic virus (CMV), Alstroemeria virus X and Broad bean wilt virus-2 (BBWV-2). On the basis of nucleotide sequence of the coat protein genes, all four CMV isolates belong to subgroup IA. CMV isolates induced mosaic and/or necrosis on Alstroemeria. YoMV and BBWV-2 were newly identified by traits such as host range, particle morphology, and nucleotide sequence as viruses infecting Alstroemeria. A BBWV-2 isolate also induced mosaic symptoms on Alstroemeria seedlings.     

A new <Emphasis Type="Italic">Rhizoctonia</Emphasis> sp. closely related to <Emphasis Type="Italic">Waitea circinata</Emphasis> causes a new disease of creeping bentgrass

Isolates of an unidentified Rhizoctonia sp. (UR isolates) were obtained from creeping bentgrass and Kentucky bluegrass with reddish brown sheath and foliar rots. Because the UR isolates anastomosed with isolates of three varieties of Waitea circinata (var. oryzae, var. zeae, and var. circinata), colony morphology, hyphal growth rate at different temperatures, pathogenicity, sequence analysis of the internal transcribed spacers (ITS) region of ribosomal RNA genes (rDNA) were compared. The colony color of mature UR isolates was distinct from isolates of the other three varieties of W. circinata. In pathogenicity tests on creeping bentgrass, the severity of the disease caused by UR isolates was significantly higher than that caused by the three varieties of W. circinata. Sequence similarities of the rDNA-ITS region between UR isolates and between isolates within each variety were high (97–100%), but they were lower among isolates from UR and the varieties of W. circinata (88–94%). In a phylogenetic tree based on the rDNA-ITS sequences, UR isolates formed a cluster separate from each of the clusters formed by the three varieties of W. circinata. These results indicate that the UR isolates clearly differ from the three varieties of W. circinata. We therefore propose that the UR isolates be classified as new Rhizoctonia sp. that are closely related to W. circinata and that the disease on creeping bentgrass should be called Waitea reddish-brown patch disease (Sekikasshoku-hagusare-byo in Japanese).