<Emphasis Type="Italic">Pantoea ananatis</Emphasis> strains are differentiated into three groups based on reactions of tobacco and welsh onion and on genetic characteristics

Ninety-six strains of Pantoea ananatis were isolated from 14 plant species including melon, rice, tea and other crops of economic importance. They were classified into three groups (group I, II, III) based on a welsh onion stabbing assay, tobacco infiltration test, and polymerase chain reaction to detect indole acetic acid (IAA) biosynthesis genes (iaaM and iaaH) and a cytokinin biosynthesis gene (etz). Group Ι strains were characterized as causing significant blight symptom on welsh onion and inducing a hypersensitive response (HR)-like reaction on tobacco leaves after 36–48 h and encompassed 20 isolates from foxtail millet, hydrangea, pineapple, river water and rice. These 20 isolates did not possess iaaM, iaaH, or etz genes. Group II, consisting of 34 melon isolates, harbored iaaM, iaaH and etz genes, but did not cause either blight on welsh onion or HR-like reaction on tobacco. Group III strains did not have the iaaM, iaaH, and etz genes, nor did they cause any reaction on welsh onion or tobacco. The 42 strains in group III were isolated from bamboo grass, Chinese silver grass, citrus, dogwood, melon, mugwort, silk tree, sweet corn, tea and welsh onion. Representative strains of the three groups were tested for pathogenicity on melon and rice. Group Ι strains caused palea browning on rice but not internal fruit rot on melon. On the contrary, group II strains did not cause disease on rice but caused internal fruit rot on melon. Group III strains were not pathogenic on rice or melon. These results suggested that the host range of P. ananatis may be predicted by the reactions of welsh onion and tobacco and detection of iaaM, iaaH and etz genes. These tools may serve as rapid tests to identify the pathogenicity groups of P. ananatis.     

<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.     

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.     

Relationships of <Emphasis Type="Italic">Barley yellow dwarf virus</Emphasis>-PAV and <Emphasis Type="Italic">Cereal yellow dwarf virus</Emphasis>-RPV from Iran with viruses of the family <Emphasis Type="Italic">Luteoviridae</Emphasis>

Barley yellow dwarf disease is one of the most important problems confronting cereal production in Iran. Barley yellow dwarf virus-PAV (BYDV-PAV) and Cereal yellow dwarf virus-RPV (CYDV-RPV) are the predominant viruses associated with the disease. One isolate of BYDV-PAV from wheat (PAV-IR) and one isolate of CYDV-RPV from barley (RPV-IR) were selected for molecular characterisations. A genome segment of each isolate was amplified by PCR. The PAV-IR fragment (1264 nt) covered a region containing partial genes for coat protein (CP), read through protein (RTP) and movement protein (MP). PAV-IR showed a high sequence identity to PAV isolates from USA, France and Japan (96–97%). In a phylogenetic analysis it was placed into PAV group I together with PAV isolates from barley and oats. The fragment of RPV-IR (719 nt) contained partial genes for CP, RTP and MP. The sequence information confirmed its identity as CYDV. However, RPV-IR showed 90–91% identity with both RPV and Cereal yellow dwarf virus-RPS (CYDV-RPS). Phylogenetic analyses suggested that it was more closely related to RPS. These data comprise the first attempt to characterise BYD-causing viruses in Iran and southwest Asia. The nucleotide sequence data reported appear in the EMBL, GenBank and DDBJ Nucleotide Sequence Databases under the accession numbers AY450425 and AY450454     

Relationship between the incidence of latent infections caused by <Emphasis Type="Italic">Monilinia</Emphasis> spp<Emphasis Type="Italic">.</Emphasis> and the incidence of brown rot of peach fruit: factors affecting latent infection

Five field experiments were performed in commercial orchards located in Lleida (Spain) over three growing seasons, 2000–2002, in order to estimate the relationship between the incidence of latent infection caused by Monilinia spp. in peaches and the incidence of post-harvest brown rot. No latent infection was recorded at popcorn and the maximum incidence occurred pre-harvest; in some orchards a second peak was detected during the pit hardening period. Monilinia laxa is the most prevalent species isolated from peaches with brown rot. There was a positive correlation between the incidence of latent infection and that of post-harvest brown rot. The average incidence of latent infection during the crop season explained 55% of the total variation in the incidence of post-harvest brown rot. The effect of temperature (T) and duration of wetness (W) on the incidence of latent infection in peach and nectarine orchards was analysed using multiple regression. The regression analysis indicated that T and W jointly explained 83% of the total variation in the incidence of latent infection. The model predicts no latent infections when T < 8°C, and >22 h wetness are required when T = 8°C but only 5 h at 25°C are necessary for latent infection to occur. The incidence of brown rot and latent infection of peaches caused by M. laxa under controlled experimental conditions were also affected by T and W, as well as by fruit maturity and inoculum concentration. Latent infections were produced in fruit when T was not suitable for the development of brown rot symptoms. In these experiments more than 4–5 h of daily wetness were required after embryo growth in fruit sprayed to run-off with an inoculum concentration higher than 10⁴ conidia ml⁻¹ of M. laxa for brown rot and latent infections to develop. The fitted model obtained from the field data was able to predict the observed data obtained under controlled environmental conditions.     

Genetic variation among <Emphasis Type="Italic">Fusarium</Emphasis> isolates from onion,and resistance to <Emphasis Type="Italic">Fusarium</Emphasis> basal rot in related <Emphasis Type="Italic">Allium</Emphasis> species

The aim of this research was to study levels of resistance to Fusarium basal rot in onion cultivars and related Allium species, by using genetically different Fusarium isolates. In order to select genetically different isolates for disease testing, a collection of 61 Fusarium isolates, 43 of them from onion (Allium cepa), was analysed using amplified fragment length polymorphism (AFLP) markers. Onion isolates were collected in The Netherlands (15 isolates) and Uruguay (9 isolates), and received from other countries and fungal collections (19 isolates). From these isolates, 29 were identified as F. oxysporum, 10 as F. proliferatum, whereas the remaining four isolates belonged to F. avenaceum and F. culmorum. The taxonomic status of the species was confirmed by morphological examination, by DNA sequencing of the elongation factor 1-α gene, and by the use of species-specific primers for Fusarium oxysporum, F. proliferatum, and F. culmorum. Within F. oxysporum, isolates clustered in two clades suggesting different origins of F. oxysporum forms pathogenic to onion. These clades were present in each sampled region. Onion and six related Allium species were screened for resistance to Fusarium basal rot using one F. oxysporum isolate from each clade, and one F. proliferatum isolate. High levels of resistance to each isolate were found in Allium fistulosum and A. schoenoprasum accessions, whereas A. pskemense, A. roylei and A. galanthum showed intermediate levels of resistance. Among five A. cepa cultivars, ‘Rossa Savonese’ was also intermediately resistant. Regarding the current feasibility for introgression, A. fistulosum, A. roylei and A. galanthum were identified as potential sources for the transfer of resistance to Fusarium into onion.     

<Emphasis Type="Italic">Turnip yellow mosaic virus</Emphasis> isolated from Chinese cabbage in Japan

A virus that caused a distinct yellow mosaic was isolated in Okayama, Japan from Chinese cabbage (Brassica rapa L., Pekinensis group). The virus, with spherical particles ca. 28 nm in diameter, was mechanically transmissible only to cruciferous species. From the host range, characteristic morphology of virus particles, serology and sequence analysis of coat protein gene, the causal virus was identified as Turnip yellow mosaic virus (TYMV). Seed transmission of TYMV at 0–2.2% in Chinese cabbage was confirmed. This report is the first of TYMV from Chinese cabbage and in Japan. The nucleotide sequence data reported are available in the DDBJ/EMBL/GenBank databases as accessions AB358971 and AB358972.     

First report of blueberry red ringspot disease caused by <Emphasis Type="Italic">Blueberry</Emphasis><Emphasis Type="Italic">red</Emphasis><Emphasis Type="Italic">ringspot</Emphasis><Emphasis Type="Italic">virus</Emphasis> in Japan

Virus-like symptoms—red ringspots on stems and leaves, circular blotches or pale spots on fruit—were found on commercial highbush blueberry (Vaccinium corymbosum) cultivars Blueray, Weymouth, Duke and Sierra in Japan. In PCR testing, single DNA fragments were amplified from total nucleic acid samples of the diseased blueberry bushes using primers specific to Blueberry red ringspot virus (BRRV). Sequencing analysis of the amplified products revealed 95.7–97.7% nucleotide sequence identity with the BRRV genome. This paper is the first report of blueberry red ringspot disease caused by BRRV in Japan. The nucleotide sequence data reported in this paper are available in the GenBank/EMBL/DDBJ database as accessions AB469884 to AB469893 for BRRV isolates from Japan.     

Vascular blackening of wasabi rhizomes caused by <Emphasis Type="Italic">Pectobacterium carotovorum</Emphasis> subsp. <Emphasis Type="Italic">carotovorum</Emphasis>

Wasabi (Wasabia japonica) is grown for its highly-valued rhizome which is used as a condiment in Japanese food. Symptoms of vascular blackening in the rhizome were first observed in 2005 in plants grown in British Columbia, Canada. Microscopic observations and microbial isolation from infected tissues revealed that most of the xylem tracheid cells were blackened and bacteria were consistently associated with symptomatic plants. The bacterium most frequently recovered was identified as Pectobacterium carotovorum subsp. carotovorum (Pcc) using BioLog™ and sequencing of a specific ~510 bp IGS region. Pathogen-free plants obtained using meristem-tip micropropagation were inoculated with a wasabi isolate of Pcc. Vascular blackening symptoms developed in the rhizome after 8 weeks when the rhizome was first wounded by stabbing or cutting, or if the roots were pre-inoculated with Pythium species isolated from rhizome epidermal tissues, followed by inoculation with Pcc at 1 × 10⁸ cells ml⁻¹. Xylem tracheid cells were blackened and Pcc was reisolated from all diseased tissues. The highest frequency of rhizome vascular blackening occurred at 22°C and 27°C and these tissues occasionally succumbed to soft rot at higher temperatures, but not when inoculated tissues were incubated at 10°C. The rooting medium used by growers for vegetative propagation of wasabi was shown to contain Pcc but the pathogen was not recovered from the irrigation water. Entry of Pcc through wounds on wasabi rhizomes and the host tissue response result in symptoms of vascular blackening.     

White rust of <Emphasis Type="Italic">Ipomoea</Emphasis> caused by <Emphasis Type="Italic">Albugo ipomoeae-panduratae</Emphasis> and <Emphasis Type="Italic">A. ipomoeae-hardwickii</Emphasis> and their host specificity

In some areas of Japan, yellow spots with white pustules on leaves, stems, petioles, peduncles and calyces were found on Ipomoea nil, I. triloba, I. lacunosa and I. hederacea var. integriuscula. We demonstrated that the diseases on I. nil, I. triloba and I. lacunosa were caused by host-specific strains of Albugo ipomoeae-panduratae and defined three forma speciales of the fungus, respectively, for the three Ipomoea species: “f. sp. nile”, “f. sp. trilobae” and “f. sp. lacunosae”. Because the diseases were new to Japan, we coined the Japanese name “shirosabi-byo”, which means white rust. We also showed that the disease on I. hederacea var. integriuscula was caused by A. ipomoeae-hardwickii. We named this new disease “white rust (shirosabi-byo in Japanese)”.     

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.     

Pathogenicity,colony morphology and diversity of isolates of <Emphasis Type="Italic">Guignardia citricarpa</Emphasis> and <Emphasis Type="Italic">G. mangiferae</Emphasis> isolated from <Emphasis Type="Italic">Citrus</Emphasis> spp.

In the present study, the pathogenicity of 36 isolates of Guignardia species isolated from asymptomatic ‘Tahiti’ acid lime fruit peels and leaves, ‘Pêra-Rio’ sweet orange leaves and fruit peel lesions, and a banana leaf were characterized. For pathogenicity testing, discs of citrus leaves colonized by Phyllosticta citricarpa under controlled laboratory conditions were kept in contact with the peels of fruit that were in susceptible states. In addition, pathogenicity was related to morphological characteristics of colonies on oatmeal (OA) and potato dextrose agar (PDA). This allowed the morphological differentiation between G. citricarpa and G. mangiferae. Polymerase chain reactions (PCRs) were also used to identify non-pathogenic isolates based on primers specific to G. citricarpa. A total of 14 pathogenic isolates were detected during pathogenicity tests. Five of these were obtained from leaf and fruit tissues of the ‘Tahiti’, which until this time had been considered resistant to the pathogen. Given that the G. citricarpa obtained from this host was pathogenic, it would be more appropriate to use the term insensitive rather than resistant to categorize G. citricarpa. A non-pathogenic isolate was obtained from lesions characteristic of citrus black spot (CBS), indicating that isolation of Guignardia spp. under these conditions does not necessarily imply isolation of pathogenic strains. This also applied to Guignardia spp. isolates from asymptomatic citrus tissues. Using fluorescent amplified fragment length polymorphism (fAFLP) markers, typically pathogenic isolates were shown to be more closely related to one another than to the non-pathogenic forms, indicating that the non-pathogenic isolates display higher levels of genetic diversity.     

Molecular characterization and variability analysis of <Emphasis Type="Italic">Apple scar skin viroid</Emphasis> in India

Apple scar skin viroid (ASSVd) infection is a major limitation to apple fruit quality and causes huge economic losses. In surveys of apple orchards in the northern Indian state of Himachal Pradesh, fruits with dappling symptoms were noticed. ASSVd was detected from these fruits and molecularly characterized. Ten clones from three isolates were sequenced, of which seven were new sequence variants of ASSVd. The clones had significant sequence variability (94–100%) with each other. Variability was more common in the pathogenic domain of the viroid genome. Four of the clones were 330 nucleotides (nt) long, and the other six had an additional nucleotide. Phylogenetic analysis showed close affinity of the present isolates with some Chinese and Korean isolates. The study reports seven new variants of ASSVd and also provides the first molecular evidence of viroid infection (ASSVd) in apple in India.     

Reaction of <Emphasis Type="Italic">Leersia</Emphasis> grasses to <Emphasis Type="Italic">Xanthomonas oryzae</Emphasis> pv. <Emphasis Type="Italic">oryzae</Emphasis> collected from Japan and Asian countries

The present study was conducted to determine if there is specificity in the host-pathogen relationship between the isolates of Xanthomonas oryzae pv. oryzae, the causal bacterium for rice blight and Leersia grasses, the alternative weed hosts of the disease. Plants of three species of Leersia, namely, L. sayanuka, L. oryzoides and L. japonica, were collected from various parts of Japan and were inoculated with the X. oryzae pv. oryzae isolates obtained from various locations in Japan and from 11 Asian countries. Four L. sayanuka plants were found susceptible to all Race II isolates and some Race I isolates, but were resistant to all Race III isolates. Race III is known to have a wider range pathogenicity to rice cultivar groups compared with Race I and II. Although the reactions of two L. oryzoides plants to Race I and II isolates were similar to that of L. sayanuka, the L. oryzoides plant collected from Niigata Prefecture showed a susceptible reaction to some Race III isolates. On the other hand, L. japonica plants gave reactions different those of L. sayanuka and L. oryzoides, with two plants of L. japonica found to be resistant to all test isolates collected from Japan. The Asian isolates exhibited a wide host range against the international differential rice cultivars, but almost all of them were avirulent to Leersia plants. These results indicate that the relationship between the pathogenicity of the causal bacterium and the resistance of host plants is very complex, and suggest that pathogenic diversity of X. oryzae pv. oryzae might be related to the resistance of Leersia spp.     

Toxigenicity and pathogenicity of <Emphasis Type="Italic">Fusarium poae</Emphasis> and <Emphasis Type="Italic">Fusarium avenaceum</Emphasis> on wheat

In a field experiment between 2004 and 2006, 14 winter wheat varieties were inoculated with either a mixture of three isolates of F. poae or a mixture of three isolates of F. avenaceum. In a subsequent climate chamber experiment, the wheat variety Apogee was inoculated with individual single conidium isolates derived from the original poly conidium isolates used in the field. Disease symptoms on wheat heads were visually assessed, and the yield as well as the fungal incidence on harvested grains (field only) was determined. Furthermore, grains were analysed using LC-MS/MS to determine the content of Fusarium mycotoxins. In samples from field and climate chamber experiments, 60 to 4,860 μg kg⁻¹ nivalenol and 2,400 to 17,000 μg kg⁻¹ moniliformin were detected in grains infected with F. poae and F. avenaceum, respectively. Overall, isolate mixtures and individual isolates of F. avenaceum proved to be more pathogenic than those of F. poae, leading to a higher disease level, yield reductions up to 25%, and greater toxin contamination. For F. poae, all variables except for yield were strongly influenced by variety (field) and by isolate (climate chamber). For F. avenaceum, variety had a strong effect on all variables, but isolate effects on visual disease were not reflected in toxin production. Correlations between visual symptoms, fungal incidence, and toxin accumulation in grains are discussed.     

Gray mold of pearl lupine caused by <Emphasis Type="Italic">Botrytis cinerea</Emphasis>

Severe blight of stems, leaves and pods caused by Botrytis cinerea was found on pearl lupine (Lupinus mutabilis), a legume crop, grown in Kagawa Prefecture, Japan, in March–June 1996–2002. This disease was named “gray mold of pearl lupine” as a new disease. One of the fungal isolates obtained in this study was deposited in Genebank, National Institute of Agrobiological Sciences as accession MAFF238557.     

Anthracnose of <Emphasis Type="Italic">Enkianthus campanulatus</Emphasis> and <Emphasis Type="Italic">Rhynchosia acuminatifolia</Emphasis> caused by <Emphasis Type="Italic">Colletotrichum gloeosporioides</Emphasis> (new occurrence)

In 2003–2004, anthracnoses of Enkianthus campanulatus and Rhynchosia acuminatifolia were found for the first time in Kanagawa Prefecture and Tokyo in Japan. These pathogens were identified as Colletotrichum gloeosporioides based on their pathogenicity, morphology and ribosomal DNA spacer sequences. Results were presented at the annual meeting of The Phytopathological Society of Japan in 2004.     

PCR-based assays to detect and quantify <Emphasis Type="Italic">Phomopsis sclerotioides</Emphasis> in plants and soil

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 ≥ cucumber ≥ watermelon > pumpkin. These results suggest that Ph. sclerotioides infection is not species-specific but the rate of infection may differ among host species.     

<Emphasis Type="Italic">Pythium</Emphasis> and <Emphasis Type="Italic">Phytophthora</Emphasis> species associated with root and stem rots of kalanchoe

Pythium and Phytophthora species were isolated from kalanchoe plants with root and stem rots. Phytophthora isolates were identified as Phytophthora nicotianae on the basis of morphological characteristics and restriction fragment length polymorphism (RFLP) analysis of the rDNA-internal transcribed spacer regions. Similarly, the Pythium isolates were identified as Pythium myriotylum and Pythium helicoides. In pathogenicity tests, isolates of the three species caused root and stem rots. Disease severity caused by the Pythium spp. and Ph. nicotianae was the greatest at 35°–40°C and 30°–40°C, respectively. Ph. nicotianae induced stem rot at two different relative humidities (60% and >95%) at 30°C. P. myriotylum and P. helicoides caused root and stem rots at high humidity (>95%), but only root rot at low humidity (60%).