<Emphasis Type="Italic">Pseudomonas syringae</Emphasis> pv. <Emphasis Type="Italic">cerasicola</Emphasis>, pv. nov., the Causal Agent of Bacterial Gall of Cherry Tree

Bacterial gall on trunks and twigs of cherry trees (Prunus × yedoens, Someiyoshino) was found in Miyazaki and Saga prefectures, Japan. The surface of young galls are relatively smooth and light brown, but they become rough and dark brown. Characteristics of the bacterium isolated from galls on trunks or twigs are similar to those of Pseudomonas syringae pathovars, i.e., pv. actinidiae, pv. daphniphylli, pv. dendropanacis, pv. Morsprunorum, pv. myricae, pv. rhaphiolepidis, pv. syringae and pv. tremae. This bacterium produced galls on cherry and apricot, but not on 66 other species of plants belonging to 39 families. From these results, this bacterium was classified as a new pathovar of Pseudomonas syringae, and the name Pseudomonas syringae pv. cerasicola, pv. nov., is proposed. Strain M9501(ICMP 13926) was designated as the pathotype strain. Received 10 September 1999/ Accepted in revised form 24 December 1999     

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.     

Leaf Smut of <Emphasis Type="Italic">Sagittaria latifolia </Emphasis>Caused by <Emphasis Type="Italic">Doassansia horiana</Emphasis>

A smut-like disease was found on the leaves of Sagittaria latifolia in Japan. Spore balls collected from the leaves of S. latifolia and S. trifolia var. edulis were used to cross-inoculate leaves of pathogen-free plants of the two species to identify the pathogen. Spots and swellings formed on leaves of the two species 10 days after inoculation. These symptoms were quite similar to those of the leaf smut disease of S. trifolia var. edulis caused by Doassansia horiana, and the spore balls were characteristic of the fungus. Therefore, the authors conclude that D. horiana caused leaf smut disease on S. latifolia. Received 18 January 2000/ Accepted in revised form 14 May 2000     

<Emphasis Type="Italic">Citrus exocortis viroid</Emphasis> transmission through commercially-distributed seeds of <Emphasis Type="Italic">Impatiens</Emphasis> and <Emphasis Type="Italic">Verbena</Emphasis> plants

Surveys of Impatiens and Verbena species in local nurseries in Fredericton, Canada and Verbena species in New Delhi, India showed widespread infection of Citrus exocortis viroid (CEVd) in vegetatively-propagated and seed-grown plants. To determine viroid seed transmission, samples of eight varieties of Impatiens and 11 varieties of Verbena were obtained from four commercial sources. All 19 samples collected contained viroid infection irrespective of variety. The presence of viroid in non-germinated seed was 21%, while the transmission rate in seedlings was 66% in Impatiens walleriana in 2006. Following 2 years of seed storage, the respective figures were 6% and 26%. Similarly, in Verbena x hybrida the presence of viroid in seed was 13% in 2006 with a seed-transmission rate in seedlings of 28%, while the respective figures after 2 years of storage were 5% and 45%.     

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.     

Epidemiology of <Emphasis Type="Italic">Pseudomonas syringae</Emphasis> pv. <Emphasis Type="Italic">syringae</Emphasis> on tomato

Leaf spot of tomato, incited by Pseudomonas syringae pv. syringae, has been reported recently in Italy on grafted and non-grafted tomato plants (scion Cuore di Bue, rootstock Solanum lycopersicum x Solanum hirsutum cv. Beaufort). In some greenhouses, more than 80% of plants were affected, with a marked reduction in yield. This work was undertaken in order to understand the effect of the number of hours of incubation at high relative humidity (r.h.) and temperature as well as the effect of the presence of wounds at infection time on the development of leaf spot. A difference in sensitivity to leaf spot was observed in the various cultivars tested, in terms of severity of P. syringae pv. syringae, with “Cuore di Bue” being the most susceptible of these cultivars. The development of leaf spot is mostly favored by the presence of wounds, at temperatures between 15 and 20°C. The severity of the disease is lower at 10 and 25°C and very low at 30°C. Under the most favorable temperature conditions, the presence of wounds is sufficient to allow the development of the pathogen immediately upon incubation at high r.h. The effect of wounds and the relatively low requirement of hours of incubation at high r.h. suggest the need for careful management and handling of plants when temperatures range between 15 and 25°C, and particularly within 15 and 20°C. All operations carried out, particularly at transplant and immediately after, should avoid the creation of wounds.     

Structural conservation of the <Emphasis Type="Italic">hrp</Emphasis> gene cluster in <Emphasis Type="Italic">Xanthomons oryzae</Emphasis> pv. <Emphasis Type="Italic">oryzae</Emphasis>

The clustered hrp genes encoding the type III secretion system in the Japanese strains MAFF301237 and MAFF311018 of Xanthomonas oryzae pv. oryzae were sequenced and compared. The strains differ in their pathogenicity, location, and year of isolation. A 30-kbp sequence comprising 29 open reading frames (ORFs) was identical in its structural arrangement in both strains but differed from X. campestris pv. campestris, X. axonopodis pv. citri, and X. axonopodis pv. glycines in certain genes located between the hpaB-hrpF interspace region. The DNA sequence and the putative amino acid sequence in each ORF was also identical in both X. oryzae pv. oryzae strains as were the PIP boxes and the relative sequences. These facts clearly showed that the structure of the hrp gene cluster in X. oryzae pv. oryzae is unique.     

Notes on the Occurrence of Pathogenic Races of <Emphasis Type="Italic">Xanthomonas oryzae </Emphasis>pv. <Emphasis Type="Italic">oryzae </Emphasis>Found in Hiroshima Prefecture in 1999

The pathogenic race of 59 cultures of Xanthomonas oryzae pv. oryzae, a pathogen of bacterial leaf blight of rice, isolated from six locations in the inland mountainous area of Hiroshima Prefecture in 1999, were determined by a set of traditional differentials. Four races—I, II, V and VII—were found across the area; however, we noticed the composition of the races as well as the dominant race in each location different. All races were avirulent on differential cultivar Te-tep. Races V and VII were new to Hiroshima. The rice cultivars infected with bacterial leaf blight in Hiroshima are thought to be grouped into the Kinmaze group, which does not have any resistance genes. Apparently, a variety of races occurred unexpectedly on the cultivars contrary to stabilizing selection theory. Received 25 February 2000/ Accepted in revised form 13 July 2000     

Characterization of <Emphasis Type="Italic">Inago1</Emphasis> and <Emphasis Type="Italic">Inago2</Emphasis> retrotransposons in <Emphasis Type="Italic">Magnaporthe oryzae</Emphasis>

From the genome of a Japanese field isolate of the rice blast fungus, Magnaporthe oryzae, we newly identified Inago1 and Inago2 LTR retrotransposons. Both elements were found to be Ty3/gypsy-like elements whose copies were dispersed within the genome of Magnaporthe spp. isolates infecting rice and other monocot plants. Southern hybridization patterns of nine re-isolates derived from conidia of the strain Ina168 produced after a methyl viologen treatment were not changed, indicating that the insertion pattern of Inago elements is relatively stable.     

Pathogenicity of <Emphasis Type="Italic">Beauveria bassiana</Emphasis> and <Emphasis Type="Italic">Metarhizium anisopliae</Emphasis> against <Emphasis Type="Italic">Galleria mellonella</Emphasis>

The greater wax moth Galleria mellonella L. (Lepidoptera: Pyralidae) is occasionally found in beehives and is a major pest of stored wax. Entomopathogenic fungi have recently received attention as possible biocontrol elements for certain insect pests. In this study, 90 isolates of Beauveria bassiana and 15 isolates of Metarhizium anisopliae were screened for proteases and lipases production. The results showed significant variations in the enzymatic action between the isolates. In the bioassay, the selected isolates evinced high virulence against the 4th instar of the G. mellonella larvae. The isolates BbaAUMC3076, BbaAUMC3263 and ManAUMC3085 realized 100% mortality at concentrations of 5.5 × 10⁶ conidia ml⁻¹, 5.86 × 10⁵ conidia ml⁻¹, and 4.8 × 10⁶ conidia ml⁻¹, respectively. Strong enzymatic activities in vitro did not necessarily indicate high virulence against the tested insect pest. The cuticle of the infected larvae became dark and black-spotted, indicating direct attack of fungus on the defense system of the insects. The LC₅₀ values were 1.43 × 10³, 1.04 × 10⁵ and 5.06 × 10⁴ for Bba3263AUMC, Bba3076AUMC and Man3085AUMC, respectively, and their slopes were determined by computerized probit analysis program as 0.738 ± 0.008, 0.635 ± 0.007 and 1.120 ± 0.024, respectively.     

Defective Long-distance Transport of <Emphasis Type="Italic">Cucumber mosaic virus </Emphasis>in Radish Is Efficiently Complemented by <Emphasis Type="Italic">Turnip mosaic virus</Emphasis>

A strain of Cucumber mosaic virus (CMV-D8) systemically infects Japanese radish (Raphanus sativus), but the Y strain of CMV (CMV-Y) only infects the inoculated leaves. Both of these strains cause severe systemic mosaic on the plants after dual infection with Turnip mosaic virus (TuMV). Synergistic interactions on long-distance transport of CMV-Y and CMV-D8 with TuMV were analyzed using an immunobinding assay. Direct tissue blots probed with either anti-CMV-Y or anti-TuMV antiserum clearly showed that CMV-Y efficiently spread and accumulated in the tissues of noninoculated upper leaves and roots when co-inoculated with TuMV, and that long-distance movement of CMV-D8 was enhanced by the presence of TuMV. Received 16 September 1999/ Accepted in revised form 5 February 2000     

Movement of <Emphasis Type="Italic">Cucumber Mosaic Virus</Emphasis> Is Restricted at the Interface between Mesophyll and Phloem Pathway in <Emphasis Type="Italic">Cucumis figarei</Emphasis>

Viral movement in the leaf tissues of a resistant host, Cucumis figarei, inoculated with the pepo strain of Cucumber mosaic virus (CMV) and incubated at 24°C or 36°C was investigated by fluorescence in situ hybridization (FISH), leaf-press blotting, tissue printing and immunogold-silver staining techniques. Observation by FISH revealed that at 24°C most infection sites with CMV at 0.01 mg/ml or 0.1 mg/ml were limited to a single cell during the incubation period, that the number of infection sites increased from 24hpi (hours post inoculation) to 80 hpi in the leaves inoculated with CMV at 0.5 mg/ml, and that the size as well as the number of infection sites rapidly increased with time in the leaves inoculated with CMV at 2.0 mg/ml. These results suggested that one factor for the resistance of C. figarei at 24°C might be an inhibition of viral movement in and out of the infection sites. Leaf-press blotting and tissue blotting indicated that CMV remained in the infection sites at 24°C, whereas it spread from the inoculated leaves to other parts of the plants through vascular systems at 36°C. Immunogold-silver staining demonstrated that at 24°C CMV infected bundle sheath (BS) cells in minor veins, whereas at 36°C it invaded not only BS cells, but also phloem parenchyma (PP)/ companion cell (CC) or PP/intermediary cell (IC) complexes in minor veins in the regions with chlorotic symptoms. These results indicated that at 24°C CMV had difficulty in passing through the interface between BS and PP/CC or PP/ IC complexes and that viral entry from mesophyll to the phloem pathway was inhibited in the inoculated leaves. Received 26 August 1999/ Accepted in revised form 14 December 1999     

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.     

<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.     

Characterization of <Emphasis Type="Italic">Pectobacterium carotovorum</Emphasis> subsp. <Emphasis Type="Italic">carotovorum</Emphasis> causing soft-rot disease on <Emphasis Type="Italic">Pinellia ternata</Emphasis> in China

Pinellia ternata is a traditional Chinese herb which has been used in China for over 1,000 years. A soft-rot disease characterized by water-soaked lesions and soft-rot symptoms with a stinking odour was commonly observed in cultivated fields of this plant, and Pectobacterium-like bacteria were consistently isolated from the infected tissues. Two typical strains (SXR1 and ZJR1), isolated from Shanxi and Zhejiang, respectively, were identified. Pathogenicity tests revealed that these strains were virulent to P. ternata and induced the same symptoms as observed in the field. Characterization involving fatty acid profile, metabolic and physiological properties, 16S rDNA sequence and PCR-RFLP identified both isolates as P. carotovorum subsp. carotovorum (Pcc). The 16S rDNA of both isolates shared 97–99% sequence similarity with that of Pcc strains. The phylogenetic trees showed that both isolates were clustered in the group of Pcc and P. carotovorum subsp. odorifera and both PCR-RFLP profiles were consistent with the pattern E produced by the minority of Pcc strains. Thus, isolates SXR1 and ZJR1 were characterized as Pcc in spite of some differences. This is the first report that Pcc has been proven as a causal agent of soft-rot disease on P. ternata.     

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.     

Genetic structure of <Emphasis Type="Italic">Pyrenophora teres</Emphasis> f. <Emphasis Type="Italic">teres</Emphasis> and <Emphasis Type="Italic">P. teres</Emphasis> f. <Emphasis Type="Italic">maculata</Emphasis> populations from western Canada

Infection by Pyrenophora teres f. teres (Ptt) or P. teres f. maculata (Ptm), the causal agents of the net and spot forms of net blotch of barley, respectively, can result in significant yield losses. The genetic structure of a collection of 128 Ptt and 92 Ptm isolates from the western Canadian provinces of Alberta (55 Ptt, 27 Ptm), Saskatchewan (58 Ptt, 46 Ptm) and Manitoba (15 Ptt, 19 Ptm) were analyzed by simple sequence repeat (SSR) marker analysis. Thirteen SSR loci were examined and found to be polymorphic within both Ptt and Ptm populations. In total, 110 distinct alleles were identified, with 19 of these shared between Ptt and Ptm, 75 specific to Ptt, and 16 specific to Ptm. Genotypic diversity was relatively high, with a clonal fraction of approximately 10 % within Ptt and Ptm populations. Significant genetic differentiation (PhiPT = 0.230, P = 0.001) was found among all populations; 77 % of genetic variation occurred within populations and 23 % between populations. Lower, but still significant genetic differentiation (PhiPT = 0.038, P = 0.001) was detected in Ptt, with 96 % of genetic variation occurring within populations. No significant genetic differentiation (PhiPT = 0.010, P = 0.177) was observed among Ptm populations. Isolates clustered in two distinct groups conforming to Ptt or Ptm, with no intermediate cluster. The high number of haplotypes observed, combined with an equal mating type ratio for both forms of the fungus, suggests that P. teres goes through regular cycles of sexual recombination in western Canada.     

Web Blight of Fan Columbine, <Emphasis Type="Italic">Aquilegia flabellata, </Emphasis>Caused by <Emphasis Type="Italic">Rhizoctonia solani </Emphasis>AG1-IB : A New Disease Record

A new disease, found on fan columbine in August of 1997, first appeared as necrotic spots on leaves and within a week caused wilting of all the leaves. Fungal mycelia bound aerial parts of the plants together, formed mats of mycelia and eventually killed the plants. The pathogen, isolated from the infected leaves and stalks, was identified as Rhizoctonia solani AG1-IB in respect to hyphal anastomosis and culture types. The common name of web blight is proposed for this new occurrence on fan columbine (Kumonosu-byo in Japanese). Received 24 November 1999/ Accepted in revised form 4 February 2000     

Role of secondary metabolites in the biocontrol activity of <Emphasis Type="Italic">Pseudomonas corrugata</Emphasis> and <Emphasis Type="Italic">Pseudomonas mediterranea</Emphasis>

Colletotrichum gloeosporioides is the causal agent of Camellia oleifera anthracnose, mainly infecting fruits and leaves. The fungus secretes degrading enzymes to destroy the cuticle of aerial plant parts and help infect the host successfully. To validate whether a cutinase gene (CglCUT1) was required for cutinase activity and pathogenicity of C. gloeosporioides, the CglCUT1 gene was cloned and analyzed. The characterization of CglCUT1 predicted protein suggests that the cloned DNA encoded a cutinase in C. gloeosporioides affecting C. oleifera. The CglCUT1 showed a high homology to those from C. gloeosporioides causing papaya anthracnose and C. capsici causing pepper anthracnose, as well as those of other ascomycetes. The whole CglCUT1 gene was knocked-out and the knockout mutant (?CglCUT39) was subsequently complemented using Agrobacterium tumefaciens mediated transformation. The knockout transformants exhibited significant decreases in cutinase activity and virulence compared with the wild-type strain. The complemented transformants of the disrupted transformant ?CglCUT39 showed a significant increase in cutinase activity and virulence compared with the disrupted transformant ?CglCUT39. This study suggests that the CglCUT1 gene has a positive effect on fungal virulence of the hemibiotrophic C. gloeosporioides on C. oleifera.     

Isolation of Molecular Markers Linked to the <Emphasis Type="Italic">Cry</Emphasis> Locus Conferring Resistance to <Emphasis Type="Italic">Cucumber mosaic cucumovirus </Emphasis>Infection in Cowpea

We previously demonstrated that cowpea [Vigna unguiculata (L.) Walp.] cultivar Kurodane-Sanjaku contains the Cry gene, which confers resistance against Cucumber mosaic cucumovirus infection. In this paper, randomly amplified polymorphic DNA (RAPD) analysis was carried out to tag the Cry locus. Bulked segregant analysis for RAPD resulted in many polymorphisms in amplified DNA patterns. Candidates were further screened using parental and/or F₂ cowpea DNAs. As a result, we obtained three RAPD markers, D13/E14-350, WA3-850 and OPE3-500, flanking the Cry locus. In addition, we amplified cowpea sequences coding for the putative nucleotide-binding site (NBS). Degenerate primers based on NBS sequences of tobacco N and Arabidopsis RPS2 disease resistance genes were used for polymerase chain reaction, and resultant products were cloned and sequenced. Among eight independent clones, cowpea resistance gene analog (CRGA) 5 showed a distinct polymorphism when used as a probe for restriction fragment length polymorphism analysis against the susceptible cowpea cultivar PI 189375 and a near-isogenic line for the Cry. Linkage analyses of these molecular markers showed that genetic distances of CRGA5, D13/E14-350, WA3-850 and OPE3-500 to the Cry locus were 0.7, 5.2, 11.5 and 24.5 cM, respectively. Received 16 December 1999/ Accepted in revised form 22 March 2000