宇佐見 俊行

Based on the gene-for-gene model, research on the function of avirulence genes and resistance genes has had an unparalleled impact on breeding for resistance in most crops against individual pathogens. The soilborne fungal pathogen, Verticillium dahliae , is a major pathogen on many economically important crops.

Anaerobic soil disinfestation (ASD) consists of the application of labile organic materials to soil, flooding, and covering the soil surface with plastic film. Anaerobic soil disinfestation is a widely used ecofriendly alternative to chemical fumigation for eliminating soil-borne plant pathogens. However, the exact mode of action of ASD has not been elucidated. In particular, the potential role of anaerobic soil bacteria in disinfestation is unclear. In this study, we isolated a predominant bacterium designated as strain E801 from cocopeat medium after laboratory-scale ASD with ethanol as the carbon source. The strain was closely related with Clostridium kluyveri, and fermentatively produced butyrate and caproate from ethanol and acetate. Interestingly, the culture supernatant of strain E801 strongly suppressed the growth of Fusarium oxysporum f. sp. lycopersici (Fol) in a pH-dependent manner. Among the volatile fatty acids produced by E801, only caproate showed significant growth suppression at pHs below 5.5. In addition, caproate eliminated Fol conidia completely at pHs 5.5 and 5.0 and suppressed Fol growth even at a low temperature (15 °C). Furthermore, cocopeat medium amended with caproate eliminated Fol conidia completely within 6 days. These results suggest that caproate is one of the key disinfestation factors in ethanol-based ASD and that the direct application of caproate to soil could be a promising strategy for rapid and stable soil disinfestation.

Deciphering the gene-for-gene relationships during host-pathogen interactions is the basis of modern plant resistance breeding. In the <named-content content-type="genus-species">Verticillium dahliae</named-content> -tomato pathosystem, two races (races 1 and 2) and their corresponding avirulence ( <italic>Avr</italic> ) genes have been identified, but strains that lacked these two <italic>Avr</italic> genes exist in nature.

In August 2016, lettuce plants (Lactuca sativa) cultivated in Gunma Prefecture became stunted and often wilted. Dark brown to black lesions were observed on roots of diseased plants. Most small feeder roots were lost. Fungi isolated from lesions on roots were identified as Berkeleyomyces rouxiae based on their morphological and genetic characteristics. Pathogenicity of this fungus on lettuce was confirmed by inoculation and reisolation. This report is the first of black root rot caused by B. rouxiae on lettuce in Japan.

To investigate the effect of decontamination treatment on the nutritional attributes of fresh-cut produce, fresh-cut slices of immature bell peppers were soaked in sodium hypochlorite solutions or slightly acidic electrolyzed water with varying concentrations of effective free chlorine. Changes in the residual ratios of the water-soluble nutrients, vitamin C (L-ascorbic acid, L-AsA) and potassium, were measured, as well as aerobic plate counts (APCs), after decontamination. The L-AsA ratios of the samples that were decontaminated with the sodium hypochlorite solutions exhibited a gradual decrease when higher concentrations of detergent and longer soaking times were employed. In contrast, the potassium ratio remained mostly constant around 50% after 1 min of soaking. A decrease in the L-AsA ratio to 80% was observed within 5 min of a soak in deionized water (a 0 ppm solution). Soakings in the slightly acidic electrolyzed water also resulted in a decrease in L-AsA ratios, yet the trend was not similar to that associated with the sodium hypochlorite solutions. These results indicate that water-soluble nutrient contents of fresh-cut produce decrease during a decontamination process that uses chlorine-based solutions according to the free chlorine concentration and the soaking time. Moreover, the reduction in nutrient content varied depending on the disinfectant used, even if the free chlorine concentration was the same for the different disinfectants.

Plectosphaerella rot affects hydroponically grown lettuce, especially those grown using deep flow technique. Plectosphaerella species such as P. pauciseptata and P. cucumerina are reported as causal agents of this disease. However, the relation between fungal lineage and pathogenicity on lettuce has been unclear. From inoculation tests, we discovered that various lineages of Plectosphaerella can infect lettuce tissues. Even strains isolated from non-lettuce plants were pathogenic on lettuce. Furthermore, various lettuce cultivars were equally susceptible to a particular strain. These results indicate that strains from a wide lineage of Plectosphaerella can be pathogenic on various lettuce cultivars.

Verticillium dahliae infecting tomato can be differentiated into races 1 and 2 based on differential pathogenicity on tomato cultivars carrying resistance gene Ve1. Although no commercial cultivars resistant to race 2 are available, race 2-resistant rootstock cultivars Aibou and Ganbarune-Karis have been bred in Japan. Nevertheless, the resistance of these rootstocks appears to be unstable in commercial tomato fields. Pathogenicity assays conducted under controlled conditions revealed that these rootstock cultivars are resistant to some isolates of race 2; this resistance is controlled by a single dominant locus, denoted by V2, based on segregation of resistance in F2 populations from selfed rootstock cultivars. However, some other isolates of race 2 can overcome this resistance. Therefore it is proposed that the current race 2 of V. dahliae should be divided into two races, i.e. 'race 2' (nonpathogenic on Aibou) and 'race 3' (pathogenic on Aibou). The distribution of these races was surveyed in 70 commercial tomato fields in Hida, Gifu Prefecture, Japan. Race 3 was found in 45 fields, indicating that race 3 had already spread throughout the region. On the other hand, 25 fields had only race 2, and thus race 2-resistant rootstocks would be effective for disease management in these fields. Races 2 and 3 could not be identified by genomic Southern hybridization probed with a telomere sequence, nor with previously reported race-specific PCR assays. Elucidation of race-determining mechanisms and development of methods for quick race identification should be made in future studies.

Unknown spot diseases were observed on calla lily leaves and spathes in Japan. Fungi isolated from spots were identified as Plectosphaerella sp. based on morphological characteristics. Inoculation and reisolation confirmed the pathogenicity of the fungi on calla lily. The fungi were distinct from known species of Plectosphaerella in terms of rDNA nucleotide sequences, growth rate, and specific pathogenicity on Zantedeschia species. This report is the first in the world literature of a spot disease of calla lily caused by Plectosphaerella sp. Management of this pathogen is important for calla lily cultivation because it degrades spathes having high commercial value.

半身萎凋病菌（V. dahliae）は土壌伝染性の植物病原糸状菌で，幅広い双子葉植物に萎凋性病害をもたらす。本菌の菌株は複数種の植物に病原性を示すが，その宿主範囲は菌株ごとに様々である。また，トマトに病原性を示す系統では，真性抵抗性遺伝子Ve1を持つトマト品種に病原性を示すレース2と，示さないレース1に分化している。これらのレースは，各菌株が非病原力遺伝子VdAve1を持つか否かによって決定されている。本菌の完全世代はこれまで確認されていないが，交配型遺伝子の解析より，潜在的にはヘテロタリックな菌であると考えられる。しかし，交配型の分布が大きく偏っていることから，本菌が有性生殖を行っている可能性は低く，むしろ擬有性生殖により菌株間の遺伝的交雑が生じていると考えられる。一方，擬有性生殖を用いた病原性系統間の人為的な交雑により，トマトに対する病原性を決定する遺伝因子について情報が得られつつある。また，近年V. dahliaeによるレタスの病害が新たに発生した。さらに，レース2抵抗性のトマト台木品種を犯す新しいレースも報告されている。今後，本菌の病原性やレースが決定される遺伝的メカニズムを解明し，病害防除に役立てる必要がある。

We have produced a new UV-B lighting system comprising a UV-B florescent lamp, a reflective aluminum plate, a timer, and a control board. UV-B radiation induces resistance to strawberry against pathogens, especially powdery mildew (Sphaerotheca aphanis var. aphanis). We demonstrated control of powdery mildew with UV-B radiation in soil culture and in drip fertigation. The lighting system was suspended from the ceiling at intervals of 5 m in a line in the middle of a vinyl house. The lamp height was ca. 2 m above the strawberry plants. Strawberry plants were radiated using UV-B fluorescent lamps daily during 9:00-15:00. Plants received light energy of ca. 2-7 kJ m-2 d-1. The lighting system suppressed powdery mildew. Control was 100% at best. UV-B radiation induced transcription of strawberry genes associated with disease resistance, such as phenylalanine ammonia lyase (PAL), chalcone synthase (CHS), chalcone isomerase (CHI), β-1,3-glucanase, and osmotin-like protein. Furthermore, production of antifungal substances was induced in leaves. UV-B radiation also made strawberry fruits more red than the control, reflecting the accumulation of anthocyanin in fruits. Consequently, the new UV-B lighting system prevents powdery mildew of strawberry and improves fruit quality.

茨城県では，土壌病害であるレタス根腐病とV. dahliaeによるレタスバーティシリウム萎凋病の発生が確認され，レタスの安定生産が脅かされつつある。レタス根腐病はレース1および2の発生が確認され，網羅的な現地調査によって発生状況が明らかになってきた。輪作や抵抗性品種の利用が進んできたが，課題も多い。レタスバーティシリウム萎凋病は初確認時以外では，被害が問題になっていないが，多犯性であり，他作物への影響も懸念される。遺伝的に異なる複数の菌が発生している状況も明らかにされている。今後もレタス栽培を継続していけるよう，本県の栽培体系に即した防除体系の構築が求められる。

Severe fruit rot of sweet pepper was found in Shimane, Hyogo, Chiba, Toyama, and Nagano prefectures, Japan from 2005 to 2011. Dark, sunken spots with concentric rings of orange conidial masses appeared on fruits. Pathogenic isolates from diseased fruits in the prefectures were identified as Colletotrichum scovillei. This species was added to the pathogens of sweet pepper anthracnose in Japan. The representative isolate was pathogenic to sweet pepper, tomato and chili pepper fruits, kidney bean pod, azuki bean, pea and strawberry leaves, but a caused no symptoms on cucumber or carrot in inoculation tests.

We developed a real-time PCR assay using a TaqMan probe (TM-qPCR) for specific detection and quantification of Phomopsis sclerotioides, causal agent of black root rot of cucurbit crops. The design of the primer sets and hybridization probe was based on the internal transcribed spacer region of the ribosomal DNA. The TM-qPCR assay was compared with a conventional, standard PCR (sPCR) assay and on a quantitative real-time PCR (SG-qPCR) assay based on SYBR Green I. The TM-qPCR assay had a detection limit of ca. 0.4 fg of P. sclerotioides DNA, which was approximately 100 times more sensitive than the sPCR assay and almost equivalent to the SG-qPCR assay. The TM-qPCR and SG-qPCR assays both were able to detect various quantities of P. sclerotioides DNA from diseased plants and infested soils, including DNA levels that were not detectable by the sPCR assay. However, the TM-qPCR was advantageous for samples containing PCR-inhibiting substances because its multiplex real-time PCR function allows the adjustment of cycle threshold values with an internal control. Based on the high specificity and sensitivity required for analyzing DNA in natural samples, the newly developed TM-qPCR assay was the most reliable tool for rapidly detecting and quantifying P. sclerotioides in plant and soil samples. © 2012 The Phytopathological Society of Japan and Springer Japan.

Fungal plant pathogens secrete effector molecules to establish disease on their hosts, and plants in turn use immune receptors to try to intercept these effectors. The tomato immune receptor Ve1 governs resistance to race 1 strains of the soil-borne vascular wilt fungi Verticillium dahliae and Verticillium albo-atrum, but the corresponding Verticillium effector remained unknown thus far. By high-throughput population genome sequencing, a single 50-Kb sequence stretch was identified that only occurs in race 1 strains, and subsequent transcriptome sequencing of Verticillium-infected Nicotiana benthamiana plants revealed only a single highly expressed ORF in this region, designated Ave1 (for Avirulence on Ve1 tomato). Functional analyses confirmed that Ave1 activates Ve1-mediated resistance and demonstrated that Ave1 markedly contributes to fungal virulence, not only on tomato but also on Arabidopsis. Interestingly, Ave1 is homologous to a widespread family of plant natriuretic peptides. Besides plants, homologous proteins were only found in the bacterial plant pathogen Xanthomonas axonopodis and the plant pathogenic fungi Colletotrichum higginsianum, Cercospora beticola, and Fusarium oxysporum f. sp. lycopersici. The distribution of Ave1 homologs, coincident with the presence of Ave1 within a flexible genomic region, strongly suggests that Verticillium acquired Ave1 from plants through horizontal gene transfer. Remarkably, by transient expression we show that also the Ave1 homologs from F. oxysporum and C. beticola can activate Ve1-mediated resistance. In line with this observation, Ve1 was found to mediate resistance toward F. oxysporum in tomato, showing that this immune receptor is involved in resistance against multiple fungal pathogens.

Knowledge of pathogen biology and genetic diversity is a cornerstone of effective disease management, and accurate identification of the pathogen is a foundation of pathogen biology. Species names provide an ideal framework for storage and retrieval of relevant information, a system that is contingent on a clear understanding of species boundaries and consistent species identification. Verticillium, a genus of ascomycete fungi, contains important plant pathogens whose species boundaries have been ill defined. Using phylogenetic analyses, morphological investigations and comparisons to herbarium material and the literature, we established a taxonomic framework for Verticillium comprising ten species, five of which are new to science. We used a collection of 74 isolates representing much of the diversity of Verticillium, and phylogenetic analyses based on the ribosomal internal transcribed spacer region (ITS), partial sequences of the protein coding genes actin (ACT), elongation factor 1-alpha (EF), glyceraldehyde-3-phosphate dehydrogenase (GPD) and tryptophan synthase (TS). Combined analyses of the ACT, EF, GPD and TS datasets recognized two major groups within Verticillium, Clade Flavexudans and Clade Flavnonexudans, reflecting the respective production and absence of yellow hyphal pigments. Clade Flavexudans comprised V. albo-atrum and V. tricorpus as well as the new species V. zaregamsianum, V. isaacii and V. klebahnii, of which the latter two were morphologically indistinguishable from V. tricorpus but may differ in pathogenicity. Clade Flavnonexudans comprised V. nubilum, V. dahliae and V. longisporum, as well as the two new species V. alfalfae and V. nonalfalfae, which resembled the distantly related V. albo-atrum in morphology. Apart from the diploid hybrid V. longisporum, each of the ten species corresponded to a single clade in the phylogenetic tree comprising just one ex-type strain, thereby establishing a direct link to a name tied to a herbarium specimen. A morphology-based key is provided for identification to species or species groups.

The pea pathogen Fusarium oxysporum f. sp. pisi is able to detoxify pisatin produced as a defense response by pea, and the gene encoding this detoxification mechanism, FoPDA1, was 82% identical to the cytochrome P450 pisatin demethylase PDA1 gene in Nectria haematococca. A survey of E oxysporum f. sp. pisi isolates demonstrated that, as in N. haematococca, the PDA gene of F. oxysporum f. sp. pisi is generally located on a small chromosome. In N. haematococca, PDA1 is in a cluster of pea pathogenicity (PEP) genes. Homologs of these PEP genes also were found in the E oxysporum f. sp. pisi isolates, and PEP1 and PEPS were sometimes located on the same small chromosomes as the FoPDA1 homologs. Transforming FoPDA1 into a pda(-) E oxysporum f. sp. lini isolate conferred pda activity and promoted pathogenicity on pea to some transformants. Different hybridization patterns of FoPDA1 were found in E oxysporum f. sp. pisi but these did not correlate with the races of the fungus, suggesting that races within this forma specialis arose independently of FoPDA1. FoPDA1 also was present in the formae speciales lint, glycines, and dianthi of F. oxysporum but they had mutations resulting in nonfunctional proteins. However, an active FoPDA1 was present in F. oxysporum f. sp. phaseoli and it was virulent on pea. Despite their evolutionary distance, the amino acid sequences of FoPDA1 of F. oxysporum f. sp. pisi and E oxysporum f. sp. phaseoli revealed only six amino acid differences, consistent with a horizontal gene transfer event accounting for the origin of these genes.

In January 2002, Verticillium wilt of lettuce (Lactuca sativa L.) caused by Verticillium tricot-pus occurred in upland paddy fields in Hyogo Prefecture for the first time in Japan. This fungal species was first isolated from lettuce in California, USA. In the present study, the genetic relationships between the American and Japanese isolates of V. tricorpus from lettuce were analyzed to determine whether the pathogen could have migrated to Japan from the USA, the major lettuce-seed supplier for Japan. Nucleotide sequences of the rDNA internal transcribed spacer regions, as well as the genes coding for translation elongation factor 1-alpha and RNA polymerase II were compared among American and Japanese V. tricorpus isolates from lettuce. The Japanese isolates of V. tricomus were distinct from the American. Random amplified polymorphic DNA analyses also supported this conclusion. These results demonstrated that Verticillium wilt on lettuce caused by V. tricorpus in Japan was not related to the isolates causing the disease in California.

We developed polymerase chain reaction (PCR) assays to detect and quantify Phomopsis sclerotioides, the causal agent of black root rot of cucurbits. We used internal transcribed spacers 1 and 2 of the ribosomal DNA (rDNA) from representative isolates to search for target sequences. Primer pairs were selected after testing against 40 fungal isolates including 13 Ph. sclerotioides isolates, 9 Phomopsis isolates other than Ph. sclerotioides, and 18 soilborne fungi that were either pathogenic or nonpathogenic to cucurbits. Conventional PCR assays with the primer pair of CPs-1 (forward) and CPs-2 (reverse) produced target DNA amplicons from all Ph. sclerotioides isolates but none of the other isolates tested. From soil and root samples collected from fields naturally infested with black root rot of cucumber and melon, the CPs-1/CPs-2 primer pair successfully amplified target DNA fragments in conventional PCR assays. Moreover, we applied the CPs-1/CPs-2 primer pair in a real-time PCR assay with SYBR Green I, and PCR-amplified products were successfully quantified by reference to a standard curve generated by adding known amounts of target DNA. Target Ph. sclerotioides DNA fragments were similarly detected in artificially inoculated roots of cucumber, melon, pumpkin, and watermelon, but quantities of Ph. sclerotioides DNA in their hypocotyls of the hosts varied as follows: melon a parts per thousand yen cucumber a parts per thousand yen watermelon &gt; pumpkin. These results suggest that Ph. sclerotioides infection is not species-specific but the rate of infection may differ among host species.

Verticillium dahliae, a soilborne plant pathogen, causes wilt disease in many important crops. We reported previously that the mating type gene MAT1-2-1 is spread to isolates of this asexual fungus. However, we did not determine whether V. dahliae is homothallic or heterothallic because the opposite mating type gene, MAT1-1-1, had not been identified. In the present study, we identified the MAT1-1-1 gene from an isolate lacking MAT1-2-1 and the mating type idiomorphs of V. dahliae. Each isolate we tested contained either the MAT1-1 or MAT1-2 idiomorph, indicating that the asexual fungus V. dahliae is potentially heterothallic.

We investigated the effect of a microbial suspension prepared from compost-amended soil on the developmentof bacterial wilt disease of tomato caused by Ralstonia solanacearum in a hydroponic system under greenhouseconditions. When the compost-amended soil suspension was applied to the root zone of tomato seedlings and to thenutrient solution before and after transplanting, respectively, the disease was slightly suppressed. The effect wasenhanced by using a bacterial group, which was incubated in bouillon medium after collection from a compostamendedsoil suspension before application. The population of R. solanacearum in the nutrient solution treatedwith the soil bacterial group remained lower than in the untreated control. In the rhizoplane of tomato plants at 4weeks after treatment with the bacterial group, the total microbial activity evaluated by fluorescein diacetatehydrolysis was higher than those in the control plants and PCR-DGGE analysis showed that a unique bacterialcommunity different from control plants was established. These results indicated that the bacterial group activatedand diversified by compost amendment led to the disease suppression and that these bacteria could be suitable forthe control of bacterial wilt of tomato in hydroponic systems.コンポストの施用効果の一つとして,土着微生物の活性化による発病抑制効果が挙げられる.本研究では,これらの活性化された微生物を養液栽培における作物の根部病害の防除に応用することを目的として,トマト青枯病(病原菌:Ralstonia solanacearum)を対象に発病抑制効果について検討した.土耕栽培ではコンポストの施用(5%,w/w)により顕著な発病抑制効果が確認された.そこで,同様に複数の組成から成る市販コンポスト添加4週間後の土壌懸濁液およびそれに含まれる細菌群を培養・増殖して得た懸濁液を定植前のトマト苗根部および定植後の培養液に処理した.その結果,いずれにおいても発病抑制効果が見られたが,培養細菌群を処理した方がその効果は高かった。培養細菌群を処理したトマトの根面では,FDA(fluorescein diacetate)分解活性を指標にした微生物活性が無処理区よりも有意に増加し,PCR-DGGE(denaturing gradient gel electrophoresis)解析によって細菌群集構造も変化していることがうかがわれた.以上の結果より,コンポスト添加により活性化した細菌群は発病抑制に関与することが示唆され,養液栽培におけるトマト青枯病の防除にも利用し得るものと考えられた.

Mating type genes of Verticillium dahliae, a wilt pathogen affecting many plant species, were identified to examine sexual recombination between Japanese pathotypes. We amplified a DNA sequence encoding high mobility group (HMG) box from V. dahliae using PCR. A cloned genomic DNA fragment included a sequence homologous to MAT1-2-1 gene. Despite that sequence&apos;s presence in all V. dahliae isolates we used, MAT1-1-1 (an opposite mating type gene) was never amplified. We concluded that V. dahliae is potentially heterothallic. Furthermore, sexual bias practically obviates sexual recombination between Japanese pathotypes. This report describes, for the first time, a mating type gene of phytopathogenic Verticillium. (C) 2009 Elsevier Ltd. All rights reserved.

The chromosome number and electrophoretic karyotype of Japanese isolates of Verticillium dahliae were investigated. In a genomic Southern blot analysis of seven isolates probed with a telomere consensus sequence (TTAGGG) 5, 12 or 14 bands were observed. Furthermore, pulsed-field gel electrophoresis (PFGE) of these isolates revealed five or six chromosomal bands. A band (approx. 3.5 Mbp) common to all isolates apparently contained more than two chromosomes. From these results, we concluded that each isolate&apos;s chromosome number is six (an eggplant pathotype isolate) or seven (all isolates of tomato and sweet pepper pathotypes). Although the chromosome sizes differed among isolates, karyotypes were similar within tomato and sweet pepper pathotypes. A small chromosome (approx. 1.8 Mbp) was observed only in the sweet pepper pathotype. Subsequent PFGE-Southern hybridization analyses revealed that the three DNA fragments specific to tomato pathotype are located on the same chromosome. These results suggest that the tomato-pathotype-specific DNA sequences might coexist on one chromosome.

Japanese isolates of Verticillium dahliae, a causal agent of wilt disease in many plants, are classifiable into pathotypes based on their pathogenicity. Because these pathotypes are morphologically indistinguishable, establishing a rapid identification method is very important for the control of this pathogen in Japan. For cloning DNA fragments that are useful for identification and specific detection of V. dahliae pathotypes, we performed random amplified polymorphic DNA (RAPD) analyses using various isolates. One polymerase chain reaction (PCR) product, E10-U48, was specific to isolates pathogenic to sweet pepper. The other product, B68-TV, was specific to race 1 of isolates pathogenic to tomato. The specificity of these sequences was confirmed by genomic Southern hybridization. Further analyses revealed that the region peripheral to B68-TV obtained from the genomic DNA library includes the sequence specific to all isolates pathogenic to tomato (races 1 and 2). Moreover, sequence tagged site (STS) primers designed from B68-TV and its peripheral region showed race-specific and pathotype-specific amplification in a PCR assay. The probes and primers obtained in this study are likely to be useful tools for the identification and specific detection of pathotypes and races of V. dahliae. © 2007 The Phytopathological Society of Japan and Springer-Verlag.

小麦フスマを用いた還元処理土壌より分離した細菌の中から,単独で処理した場合にもトマト萎凋病菌に対し通常の還元処理と同様の殺菌効果を示す3菌株を選抜した。これらの菌株は単独処理しても還元処理土壌に特有の腐敗臭が発生した。また,この腐敗臭を含む還元処理土壌からの気体には,トマト青枯病菌に対しては殺菌的な,トマト萎凋病菌には静菌的な作用が認められた。PCR-DGGE解析により,還元処理土壌中ではKlebsiella pneumoniaeやEnterobacter sp.のような細菌群が優占化していたが,単独で処理した場合に殺菌効果が得られた3菌株由来のバンドも還元処理土壌のDGGEに認められ,16SrDNA配列からこれらの菌株は全てClostridium sp.であることが示唆された。以上のことから,還元処理土壌中における殺菌機構として,トマト青枯病菌に対しては,土着の嫌気性細菌が産生する揮発性物質や有機酸による直接的な殺菌作用が,これに加えてトマト萎凋病菌の場合には,厚膜胞子の発芽や揮発性物質による静菌作用が効果的に作用していることが考えられた。

Although the causal agent of black root rot of Cucurbitaceae in Japan has been proposed as Phomopsis sclerotioides, the species identification of the pathogen has remained inconclusive because of a lack of spore formation. We confirmed that a Japanese isolate of Phomopsis sp. obtained from a diseased pumpkin root produced pycnidia containing α spores in sterilized bean pods. In phylogenetic analyses of rDNA-ITS regions, nine Japanese Phomopsis sp. isolates from melon, watermelon grafted onto bottle gourd, and pumpkin diagnosed with black root rot, formed a single clade with P. sclerotioides standard isolates. We identified the causal agent of the black root rot of melon, pumpkin, bottle gourd, and watermelon in Japan as P. sclerotioides and propose the Japanese name "Phomopsis-negusare-byo" for the disease. Patterns of random amplified polymorphic DNA (RAPD) of these Japanese isolates were also similar to those of P. sclerotioides, thus supporting the species identification. However, mycelial incompatibilities were found for many combinations among these P. sclerotioides isolates, suggesting some genotypic variations of this fungus in Japan at a level that the RAPD analyses cannot discriminate. © The Phytopathological Society of Japan and Springer-Verlag 2006.