Occurrence of rocket larkspur witches’ broom caused by “<Emphasis Type="Italic">Candidatus</Emphasis> Phytoplasma asteris” in Japan

In October 2001, a disease of rocket larkspur (Cosolida ambigua (L.) P. W. Ball et Heyw), characterized by witches’ broom, yellows and virescence of flowers, was found in Yakage Town in Okayama Prefecture. Electron microscopy revealed the presence of phytoplasma-like bodies in the phloem of diseased plants. The causal phytoplasma was identified as “Candidatus Phytoplasma asteris” based on 16S rDNA sequence analysis, and demonstrated to be acquired by the leafhopper Macrosteles striifrons. The nucleotide sequence data reported are available in the DDBJ/EMBL/GenBank databases under the accession number AB258330.     

Timing and extent of hypersensitive response are critical to restrict local and systemic spread of Pepper mild mottle virus in pepper containing the L 3 gene

Amino acid changes in Pepper mild mottle virus (PMMoV) coat protein (CP) that enhance, decrease, or nullify the resistance-inducing activity in Capsicum plants carrying the L ³ gene have been identified. In this study, molecular events underlying the L ³ -gene-mediated resistance were analyzed through the expression of hypersensitive response (HR)-related genes, HSR203J-Cc and HIN1-Cc, and defense-related genes, PR1-Cc and PR4b-Cc, upon infection with PMMoV CP mutants. The expression kinetics of the genes correlated with the degree of restriction of virus distribution in the inoculated leaves. The results suggest that the timing and extent of HR are critical factors to restrict virus spread both locally and systemically in L ³ -gene-mediated resistance.The nucleotide sequence data reported are available in the DDBJ/GenBank/EMBL databases under accession numbers AB162220 (HSR203J-Cc), AB162221 (HIN1-Cc), AB162222 (PR1-Cc), and AB162223 (PR4b-Cc)     

Phylogenetic analysis of elongation factor Tu gene of phytoplasmas from Japan

The elongation factor Tu (tuf) gene from nine Japan phytoplasma isolates was amplified with the polymerase chain reaction, and the DNA sequences of the tuf gene were determined. The tuf gene from 14 phytoplasma isolates, including reference isolates and other bacteria, were phylogenetically analyzed. A nucleotide sequence of the tuf gene among seven aster yellows group (16Sr I-B and I-D) phytoplasmas had 97%–100% similarity, and the tuf gene of two phytoplasmas of the X-disease group (16Sr III-B) had 99% similarity. The tuf genes had lower homology than did the 16S rRNA gene in the phytoplasma groups. A phylogenetic tree of amino acid sequences of the tuf gene was nearly equal to that of the 16S rRNA gene but differed somewhat from the tree based on the 16S rRNA gene in that paulownia witches broom (PaW: 16Sr I-D) and American aster yellows (AAY: 16Sr I-B) were in a subclade.The nucleotide sequence data reported are available in the DDBJ/EMBL/GenBank databases under the accession numbers AB095495, AB095667, AB095668, AB095669, AB095670, AB095671, AB095672, AB095673 and AB095674     

Grapevine crown gall caused by <Emphasis Type="Italic">Rhizobium radiobacter</Emphasis> (Ti) in Japan

In 2005, characteristic symptoms of crown gall on grapevines (Vitis vinifera L. cv. Muscat of Alexandria, and cv. Seto Giants) were observed in a commercial greenhouse-orchard in Okayama Prefecture, Japan. Isolations from diseased tissues consistently yielded bacterial colonies that were white, glistening, and produced abundant polysaccharide on potato semi-synthetic agar (PSA) medium. Ten representative isolates were chosen for further characterization. A multiplex polymerase chain reaction (PCR) assay showed these strains were not Rhizobium vitis but did possess a Ti plasmid. The bacteriological characteristics of the isolates corresponded well with R. radiobacter. The almost complete 16S ribosomal DNA sequences of isolates AT06-1 and AT06-2, selected from 10 grapevine isolates, were determined and corresponded to sequences of R. radiobacter. The pathogenicity of the isolates was tested on young grapevine and tomato (Lycopersicon esculentum Mill.) plants. Gall symptoms developed on both plant species after inoculation, and bacteria with the same colony morphology as those inoculated were reisolated. Based on these results, the isolates were identified as R. radiobacter (Ti). This report is the first of the occurrence of R. radiobacter (Ti) on grapevine in Japan. Phylogenetic analyses using the partial nucleotide sequences of virC operon located on a Ti plasmid showed that the isolate of R. radiobacter (Ti) isolated from grapevine and some strains of R. vitis (Ti) belonged to the same monophyletic group, which differed from the groups of R. radiobacter (Ti) isolated from plants other than grapevine and of the majority of R. vitis (Ti) strains isolated from grapevine. The nucleotide sequence data reported are available in the DDBJ/EMBL/GenBank databases under accessions AB306890, AB306891, and AB465432–AB465459.     

DNA sequence analysis of ribosomal ITS region 1 of Phytophthora vignae f. sp. adzukicola, the pathogen that causes stem rot of adzuki bean

Sequences of the internal transcribed spacer (ITS) region 1 were used to examine the phylogenetic relationships among races of 19 isolates of Phytophthora vignae f. sp. adzukicola and between this forma specialis and three isolates of the closely related P. vignae f. sp. vignae. The ITS 1 sequences were highly conserved (> 98.7% similarity) among representatives of both formae speciales groups. The results of this study indicate that P. vignae is a monophyletic group. The nucleotide sequence data reported are available in the DDBJ/EMBL/GenBank databases under the accession nos. AB120062–AB120080 and AB120122     

<Emphasis Type="Italic">Eremothecium coryli</Emphasis> and <Emphasis Type="Italic">E.</Emphasis><Emphasis Type="Italic">ashbyi</Emphasis> cause yeast spot of azuki bean

Yeast-like fungi were isolated from lesions on azuki bean (cv. Shin-Kyotodainagon) seeds that had been sucked by bean bugs in Kyoto Prefecture, Japan. On the basis of morphological and physiological characteristics and sequence data of the internal transcribed spacer (ITS) regions including the 5.8S rDNA, these yeasts were identified as Eremothecium coryli and E. ashbyi. Pathogenicity of those yeasts was confirmed by a reinoculation test. To our knowledge, this is the first report of the occurrence of yeast spot in azuki bean in Japan. The nucleotide sequence data reported are available in the GeneBank/EMBL/DDBJ database as accessions AB478291–AB478309 for E. coryli AZC1–19 and AB478310–AB478317 for E. ashbyi AZA1–8.     

The hrpZ and hrpA genes are variable, and useful for grouping Pseudomonas syringae bacteria

The hrpS to hrpB regions from strains of Pseudomonas syringae were amplified by polymerase chain reaction (PCR) and the DNA sequence determined. The order of hrpS, hrpA, hrpZ, and hrpB was consistent among P. syringae strains. The sequence of hrpS was highly conserved. In a cluster analysis with the hrpS sequence, P. syringae strains were divided into four groups (I, II, III, and IV) and one undetermined strain, in agreement with previous studies. In contrast, the hrpZ sequences contained insertions, deletions, and base substitutions followed by changes in amino acids. Based on cluster analysis of hrpA, hrpZ, and hrpB, P. syringae strains could be divided into five groups. One of the four groups (group I) in the cluster analysis of hrpS could be further divided into two subgroups (groups IA and IB). Groups II, III, and IV were the same in the two analyses. Group-specific primers were designed, based on the DNA sequences of hrpZ, that could differentiate the groups of P. syringae strains. The nucleotide sequence data reported are available in the DDBJ/EMBL/GenBank databases under the accession numbers AB112552 to AB112581     

Erwinia isolates from the bacterial shoot blight of pear in Japan are closely related to Erwinia pyrifoliae based on phylogenetic analyses of gyrB and rpoD genes

The phylogenetic relationships among Erwinia amylovora biovar 4 (the pathogen of bacterial shoot blight of pear in Japan), other biovars of E. amylovora, and Erwinia pyrifoliae were investigated using the sequences of 16S rRNA, gyrB, and rpoD genes. The tested isolates formed two distinct monophyletic groups in the phylogenetic trees constructed based on the gyrB gene, rpoD gene, or a combination of the three genes: group 1 contained E. amylovora biovars 1, 2, and 3; group 2 contained E. amylovora bv. 4 and E. pyrifoliae. This phylogenetic analysis showed that E. amylovora bv. 4 was more closely related to E. pyrifoliae than to other biovars of E. amylovora. The nucleotide sequence data reported are available in the DDBJ/EMBL/GenBank databases under the accession numbers AB242876 to AB242925.     

Eremothecium ashbyi causes soybean yeast-spot and is associated with stink bug, Riptortus clavatus

Many fusiform ascospores observed on soybean seeds with yeast spot disease symptoms differed significantly from those of Eremothecium coryli, the known causal agent of yeast spot disease in soybean. On the basis of morphological and physiological characteristics and sequence data of the internal transcribed spacer regions including the 5.8S rDNA and D1/D2 regions of 26S rDNA, this fungus was identified as E. ashbyi. Pathogenicity of E. ashbyi was confirmed by reinoculation test. This report is the report on E. ashbyi causing soybean yeast spot disease. In addition, this study showed that E. ashbyi was transmitted by the stink bug, Riptortus clavatus, as was E. coryli, the two Eremothecium yeasts may have been acquired when the stink bug fed on infected soybeans and overwintered in this insect species. The nucleotide sequence data reported are available in the GeneBank/EMBL/DDBJ database as accessions AB294407 to AB294412 for E. ashbyi EA1, EA7 and EA11.     

Fusarium rot of hyacinth caused by <Emphasis Type="Italic">Gibberella zeae</Emphasis> (anamorph: <Emphasis Type="Italic">Fusarium</Emphasis><Emphasis Type="Italic"> graminearum</Emphasis>)

Severe rot of leaves, peduncles and flowers caused by Gibberella zeae (anamorph: Fusarium graminearum) was found on potted plants of hyacinth (Hyacinthus orientalis), a liliaceous ornamental, in greenhouses in Kagawa Prefecture, Japan, in January 2001. This disease was named “Fusarium rot of hyacinth” as a new disease because only the anamorph, F. graminearum, was identified on the diseased host plant. The authors contributed equally to this work. The fungal isolate and its nucleotide sequence data obtained in this study were deposited in the Genebank, National Institute of Agrobiological Sciences and the DDBJ/EMBL/GenBank databases under the accession numbers MAFF239499 and AB366161, respectively.     

Identification and pathogenicity of Pythium species causing damping-off of kidney bean

Five Pythium species (Pythium irregulare, P. mamillatum, P. myriotylum, P. spinosum and P. ultimum var. ultimum) were isolated from the hypocotyls and roots of kidney bean plants with damping-off from a commercial field and from experimental plots that have undergone either continuous cropping with kidney bean or rotational cropping with arable crops. In inoculation tests, all five Pythium species were pathogenic to kidney bean. This is the first report of damping-off of kidney bean caused by Pythium species; we named this disease damping-off of kidney bean. The nucleotide sequence data reported are available in the DDBJ/EMBL/GenBank databases under accession numbers AB291811, AB291944 and AB291945.     

Sclerotinia rot of blueberry caused by <Emphasis Type="Italic">Sclerotinia sclerotiorum</Emphasis>

In 2002, rotted flower clusters and blighted shoot tips and leaves were observed on highbush blueberry (Vaccinium corymbosum L.) and rabbiteye blueberry (V. ashei Reade) plants in Chiba, Japan. The causal fungus isolated from the diseased plants was morphologically identified as Sclerotinia sclerotiorum (Libert) de Bary. The fungus reproduced natural symptoms after inoculation, then reisolated from the symptomatic parts. This is the first report of blueberry sclerotinia rot caused by S. sclerotiorum. The nucleotide sequence data reported are available in the DDBJ/EMBL/GenBank databases under accession numbers AB269903#(020501) and AB233346 (020505).     

Occurrence of chrysanthemum virescence caused by “<Emphasis Type="Italic">Candidatus</Emphasis> Phytoplasma aurantifolia” in Okinawa

In 1999, a disease of chrysanthemum [Dendranthema grandiflorum (Ramat.) Kitamura], characterized by virescence of flowers, occurred in Okinawa Prefecture. The causal agent was identified as “Candidatus Phytoplasma aurantifolia” based on 16S rDNA sequencing. The nucleotide sequence data reported are available in the DDBJ/EMBL/GenBank databases under accession number AB247462.     

Corynespora blight of sweet pepper (<Emphasis Type="Italic">Capsicum annuum</Emphasis>) caused by <Emphasis Type="Italic">Corynespora cassiicola</Emphasis> (Berk. &; Curt.) Wei

In 2004, Corynesopra cassiicola was isolated from dark brown spots on leaves and fruits and from black blights on stems of sweet pepper plants in Kochi Prefecture, Japan. The isolated fungus was then used to inoculate sweet pepper plants and subsequently reisolated from the plants with dark brown spots and black blights, showing that C. cassiicola is a new pathogen causing Corynespora blight on sweet pepper plants. The nucleotide sequence data reported are available in the DDBJ/EMBL/GenBank databases as accession numbers AB366649 (TS-C11), AB366650 (TS-C21), AB366651 (TI-C32) and AB366652 (TI-C51)     

DNA transposon fossils present on the conditionally dispensable chromosome controlling AF-toxin biosynthesis and pathogenicity of Alternaria alternata

The strawberry pathotype of Alternaria alternata produces host-specific AF-toxin and causes Alternaria black spot of strawberry. Previously, we isolated cosmid clones pcAFT-1 and pcAFT-2 from strain NAF8 of the strawberry pathotype that contain AF-toxin biosynthetic genes, named AFT genes. In a molecular characterization here of pcAFT-1 and pcAFT-2, 11 AFT genes and five transposon-like sequences, named TLS-S1 to TLS-S5, were detected. The nucleotide sequences of TLS-S1 and TLS-S4 share high homology, and their putative products have similarity to transposases of the hAT family transposons. Thus, TLS-S1 and TLS-S4 were renamed TLS-S1-1 and TLS-S1-2, respectively. Amino acid sequences deduced from TLS-S2, TLS-S3, and TLS-S5 have similarity to transposases of the Fot1/Pogo family transposons, but they are significantly different. All five sequences have incomplete open reading frames (ORFs) for transposases owing to deletions, termination codons, and/or frameshifts, indicating that they are inactivated elements. Analysis of genomic distribution of these sequences revealed that they are specifically distributed on a 1.05-Mb chromosome of NAF8, which has been identified as a conditionally dispensable (CD) chromosome encoding AFT genes. The presence of three, four, and three copies of TLS-S1, TLS-S2, and TLS-S3, respectively, and a single copy of TLS-S5 on the CD chromosome were estimated by DNA gel blot analysis. The remaining copy of TLS-S1 and the three copies of TLS-S2 were isolated and identified to also encode incomplete ORFs. Thus, it appears that all copies of the transposon-like sequences identified are inactivated elements (fossils) unique to the CD chromosome in the genome of the strawberry pathotype. The nucleotide sequence data reported are available in the DDBJ/EMBL/GenBank databases under the accession numbers AB236733 (TLS-S1-1), AB236734 (TLS-S1-2), AB236735 (TLS-S1-3), AB236736 (TLS-S2-1), AB236737 (TLS-S2-2), AB236738 (TLS-S3), and AB236739 (TLS-S5)     

The coat protein of <Emphasis Type="Italic">Tomato mosaic virus</Emphasis> L<Subscript>11</Subscript>Y is associated with virus-induced chlorosis on infected tobacco plants

The L₁₁Y strain of Tomato mosaic virus (ToMV) causes severe chlorosis on infected tobacco leaves. Sequencing analysis for the genome showed that L₁₁Y contained multiple nucleotide changes and that some led to amino acid substitutions, when compared with that of the common L strain of ToMV. The chimeric virus, which has the CP of L₁₁Y in the context of the L strain RNA genome, caused severe chlorosis on infected tobacco plants, suggesting that the CP of L₁₁Y containing three amino acid changes (E33S, A86T and E97K) was the determinant of the chlorosis. Two of these amino acid changes (A86T and E97K) were associated with the induction of chlorosis when present together in the CP. Severe destruction and deformation of chloroplasts and the formation of discrete dark-staining materials adjacent to chloroplasts were observed with electron microscopy in L₁₁Y-infected plants. Fewer virus particles accumulated in the cytoplasm of L₁₁Y-infected plant cells. The level of accumulation of CP subgenomic RNA and CP in the infected protoplasts was similar between L and L₁₁Y. Fewer virus particles accumulated in L₁₁Y-infected protoplasts, and many of them were shorter-than-full-length. The nucleotide sequence data reported is available in DDBJ/EMBL/GenBank databases as accession AB355139.     

甘肃定西地区马铃薯线虫病病原的分离鉴定

为明确甘肃省定西市马铃薯线虫病的病原种群分类地位,采用形态学结合分子生物学的方法对该地区马铃薯上的4个线虫群体进行了鉴定,观察和测量其形态特征值,基于r DNA-ITS序列以UPGMA法构建线虫群体的系统发育树,并按照柯赫氏法则进行了致病性测定。结果表明,4个线虫群体在形态学上与马铃薯腐烂茎线虫Ditylenchus destructor一致,但群体DX27与群体DX11、DX16、DX19雌虫的体长、体长/食道长、体长/尾长值存在极显著差异。利用通用引物TW81/AB28扩增r DNA-ITS序列均获得长度为915 bp的片段;序列比对分析表明,群体DX27与其它3个群体相比在ITS1区的第96~255 bp片段内有25个碱基的差异;系统发育树显示,群体DX27与C型群体聚为1支,群体DX11、DX16、DX19与B型群体聚为1支。根据形态学特征及r DNA-ITS序列分析结果确定该病原线虫为马铃薯腐烂茎线虫,其中群体DX27属于C型,群体DX11、DX16、DX19属于B型。     

Occurrence of strawberry marginal chlorosis caused by “Candidatus Phlomobacter fragariae” in Japan

In February 2004, a disease of strawberry (Fragaria × ananassa Duch.), causing little-leaf, proliferation, malformation of fruits, and marginal chlorosis of leaves, occurred in Ehime Prefecture, Japan. The causal pathogen was identified as a phloem-restricted bacterium-like organism, “Candidatus Phlomobacter fragariae,” based on polymerase chain reaction (PCR) detection, electron microscopy, and sequence analysis of PCR products. This is the first report of strawberry marginal chlorosis in Asia. The nucleotide sequence data reported are available in the DDBJ/EMBL/GenBank databases under the accession number AB246669.     

Internal fruit rot of netted melon caused by <Emphasis Type="Italic">Pantoea ananatis</Emphasis> (=<Emphasis Type="Italic">Erwinia ananas</Emphasis>) in Japan

An internal fruit rot with a malodor was found in netted melons (Cucumis melo L.) in commercial greenhouses in Kochi Prefecture, Japan, in 1998, despite their healthy appearance and lack of water-soaking or brown spots on the surface. A yellow bacterium was consistently isolated from the affected fruits. To confirm the pathogenicity of eight representative isolates of the yellow bacterium, we stub-inoculated ovaries (immature-fruits) 5–7 days after artificial pollination, with a pin smeared with bacteria. After the melon fruits had grown for 60 more days, an internal fruit rot resembling the natural infection appeared, and the inoculated bacterium was reisolated. The melon isolates had properties identical with Pantoea ananatis, such as gram-negative staining, facultative anaerobic growth, indole production, phenylalanine deaminase absence, and acid production from melibiose, sorbitol, glycerol, and inositol. Phylogenetic analysis based on 16S rDNA sequences showed that the melon bacterium positioned closely with known P. ananatis strains. The melon bacterium had indole acetic acid (IAA) biosynthesis genes (iaaM and iaaH) and a cytokinin biosynthesis gene (etz). The bacterium could be distinguished from the other ‘Pantoea’ group strains by rep-PCR genomic fingerprinting. From these results, the causal agent of internal fruit rot was identified as a strain of P.ananatis [Serrano in (Philipp J Sci 36:271–305, 1928); Mergaert et al. in (Int J Syst Bacteriol 43:162–173, 1993)]. The nucleotide sequence data reported are available in the DDBJ database under accessions AB297969, AB373739, AB373740, AB373741, AB373742, AB373743 and AB373744.