Bacterial Wilt of Russell Prairie Gentian Caused by <Emphasis Type="Italic">Burkholderia caryophylli</Emphasis>

A new bacterial disease of Russell prairie gentian (Eustoma grandiflorum) was found in Fukuoka Prefecture, Japan, in 1997. This disease was characterized by wilting and yellowing of the foliage. A cross section of the stem of a diseased plant revealed a tan to yellow-brown discoloration of the vascular tissue. A nonfluorescent, aerobic, Gram-negative bacterium was consistently isolated from infected plants. The bacteriological characteristics of 10 isolates of the bacterium coincided with those of the reference strains of Burkholderia caryophylli that were isolated from carnations. The bacterium, as well as the reference strains, attacked Russell prairie gentian and carnation after artificial inoculation and reproduced the symptoms similar to those after natural infections. On the basis of bacteriological characteristics and pathogenicities, the bacterium was identified as B. caryophylli. This is the first report of a disease caused by B. caryophylli on Russell prairie gentian ; therefore, bacterial wilt of Russell prairie gentian is proposed as the name of the disease. Received 5 April 2000/ Accepted in revised form 11 July 2000     

Molecular and biological characterization of <Emphasis Type="Italic">Chrysanthemum stem necrosis virus</Emphasis> isolates from distinct regions in Japan

Three isolates of Chrysanthemum stem necrosis virus (CSNV) were obtained from chrysanthemum plants in distinct regions of Japan in 2006 and 2007. All the original host plants showed severe necrotic symptoms on the leaves and stems. Amino acid sequence data of the nucleocapsid protein genes of the three isolates (CbCh07A, TcCh07A, and GnCh07S) showed high identities with those of two other CSNV isolates, HiCh06A L1 from Japan and Chry1 from Brazil. Furthermore, for the first time the complete nucleotide sequence of the S RNA was determined for CSNV (isolate HiCh06A). In phylogenetic analysis based on the non-structural protein genes from the genus Tospovirus, HiCh06A L1 was placed in the same genetic group as Tomato spotted wilt virus (TSWV) and Impatiens necrotic spot virus. Host range examination for isolates HiCh06A L1 and CbCh07A showed that green pepper (cv. ‘Kyoyutaka’, ‘Saitamawase’, ‘Tosakatsura’, ‘L3 sarara’ and ‘L3 miogi’) and tomato (cv. ‘Sekaiichitomato’) were systemically susceptible hosts, whereas TSWV-resistant Solanaceae species, Capsicum chinense, Lycopersicon peruvianum and a TSWV-resistant cultivar of green pepper (cv. TSR miogi), were resistant.     

Brown root rot of Russell prairie gentian caused by Subplenodomus drobnjacensis

Brown root rot of Russell prairie gentian was observed in the Aomori Prefecture, Japan in April 2011. The fungal isolate from the diseased root was identified as Subplenodomus drobnjacensis on the basis of its morphological characteristics and nucleotide sequences. The isolate induced similar root rot symptoms when inoculated in healthy Russell prairie gentian plants. We proposed the name “brown root rot” for this disease.     

Fusarium root rot of prairie gentian caused by a species belonging to the <Emphasis Type="Italic">Fusarium solani</Emphasis> species complex

Rotting of roots and stem bases and wilting of entire plants were found on a gentianaceous flowering plant, prairie gentian (Eustoma grandiflorum), grown in Kagawa Prefecture in the southwest region of Japan in April 2001. A mitosporic fungus, isolated repeatedly from the diseased plants, was identified as a species belonging to the clade 3 of Fusarium solani species complex based on the morphology and the sequence of the translation elongation factor gene. It was demonstrated to cause the disease by inoculating potted plants and reisolating the fungus from the diseased plants. We propose the name “Fusarium root rot of prairie gentian” for this disease.     

Powdery Mildew of Prairie Gentian: Characteristics,Molecular Phylogeny and Pathogenicity

In March 1999, we found prairie gentian (Eustoma grandiflorum) infected with powdery mildew in a greenhouse in Oita Prefecture, Japan. Morphological observation revealed that the causal fungus belongs to the mitosporic genus Oidium subgenus Pseudoidium [teleomorph: Erysiphe sensu Braun and Takamatsu (2000)]. Precise taxonomic position of the fungus, however, is uncertain due to lack of the perfect stage. We determined the nucleotide sequence of the rDNA ITS region of the fungus. Comparison of the sequence with those obtained from DNA databases of this fungal group revealed that the sequence is identical to those of powdery mildews from garden four-o'clock (Mirabilis jalapa) and broad bean (Vicia faba). Inoculation of an isolate from garden four-o'clock caused mildew on prairie gentian and broad bean, suggesting that the prairie gentian mildew originates from garden four-o'clock or broad bean. Molecular phylogenetic analysis indicated a close relationship of this fungus to Erysiphe baeumleri on Vicia spp. and E. trifolii on Trifolium pratense. From these results, we propose that prairie gentian mildew diverged from a Fabaceae-parasitic ancestor. Received 14 March 2002/ Accepted in revised form 28 May 2002     

Competence of <Emphasis Type="Italic">Frankliniella occidentalis</Emphasis> and <Emphasis Type="Italic">Frankliniella intonsa</Emphasis> strains as vectors for <Emphasis Type="Italic">Chrysanthemum stem necrosis virus</Emphasis>

The vector competence of Frankliniella occidentalis for Chrysanthemum stem necrosis virus (CSNV) was evaluated. Three vector strains with distinct competences for Tomato spotted wilt virus (TSWV) transmission were investigated, including an artificially selected strain (TsH) that has a particularly high competence (>90 %). Newly hatched larvae of F. occidentalis were given an acquisition access period of 5 days on CSNV-infected D. stramonium leaves, and reared to maturity. Their transmission efficiencies were examined using a leaf disk assay using Petunia x hybrida leaves. Following the leaf disk assay, the virus accumulation in the vectors was examined via a double antibody sandwich enzyme-linked immunosorbent assay (DAS-ELISA) of their bodies. The results showed that the CSNV acquisition and transmission efficiency of the TsH strain did not differ from those of the others, indicating that the competence of F. occidentalis as a vector for CSNV is not related to that for TSWV. The CSNV transmission and acquisition efficiencies of two F. intonsa strains (Hiroshima and Fukuoka) were also evaluated. In Hiroshima strain, 35 % of adults were viruliferous, but only two transmitters (3 %) were observed. In Fukuoka strain, 6 % were viruliferous, and no transmitters were observed. These results indicate that F. intonsa cannot be a major vector for CSNV. The accumulation of CSNV in the adults of F. occidentalis and F. intonsa evaluated using DAS-ELISA showed a significant difference in ELISA values among transmitter, viruliferous non-transmitter, and non-viruliferous individuals. These results clearly demonstrated that only transmitters that accumulated a threshold quantity of virus can transmit CSNV to plants.     

Genetic analysis of lethal tip necrosis induced by <Emphasis Type="Italic">Clover yellow vein virus</Emphasis> infection in pea

Clover yellow vein virus (ClYVV) elicits lethal tip necrosis in the pea line PI 118501. Pea line PI 118501 develops necrotic lesions and veinal necrosis on inoculated leaves, followed by systemic necrosis, leading to plant death. To understand the genetic basis of this lethal tip necrosis, we crossed lines PI 226564 and PI 250438, which develop mosaic symptoms in response to ClYVV inoculation. In reciprocal crosses of PI 118501 with PI 226564, all F₁ plants had mosaic symptoms with slight stem necrosis and early yellowing of upper leaves. Essentially the same symptom was manifested in PI 118501 × PI 250438 F₁ plants. In F₂ populations from the cross between PI 118501 and PI 226564, the observed ratios of necrosis, mosaic with slight stem necrosis, and mosaic fit the expected 1 : 2 : 1 ratio. These results indicate that a single incompletely dominant gene confers the induction of necrosis in PI 118501. This locus in pea, conferring necrosis induction to ClYVV infection, was designated Cyn1 (Clover yellow vein virus-induced necrosis). A linkage analysis using 100 recombinant inbred lines derived from a cross of PI 118501 and PI 226564 demonstrated that Cyn1 was located 7.5 cM from the SSR marker AD174 on linkage group III.     

Molecular Characterization of <Emphasis Type="Italic">Bean yellow mosaic virus </Emphasis>from Vegetatively Propagated Dwarf <Emphasis Type="Italic">Gentiana </Emphasis>Plants

The causative virus (isolate No. 4) of gentian (Gentiana spp.) mosaic, which had been identified previously as Clover yellow vein virus (C1YVV) on the basis of host range and serological reactions, was re-identified as Bean yellow mosaic virus (BYMV) on the basis of the nucleotide sequences of the gene for the coat protein (CP) and the 3′-noncoding region, as well as the predicted amino acid sequence of CP. Received 16 April 2002/ Accepted in revised form 19 June 2002     

Stem and root rot of scarlet runner bean (Phaseolus coccineus) caused by Pythium myriotylum

A severe rot was found on the stems and roots of scarlet runner bean (Phaseolus coccineus) in Ibaraki Prefecture (Japan) in August 2004. The causal fungus was identified as Pythium myriotylum. We propose the name of stem and root rot of scarlet runner bean (“Kuki-negusare-byo” in Japanese) for this new disease.     

Foot Rot of Ulluco Caused by Pythium aphanidermatum

Severe rot of stem bases caused by Pythium aphanidermatum was found on ulluco (Ullucus tuberosus) grown in Kagawa Prefecture, Japan, in September 1999. The name “foot rot of ulluco” is proposed for this new disease. Received 14 November 2001/ Accepted in revised form 7 January 2002     

Establishment,purification, maintenance and serological diagnosis of <Emphasis Type="Italic">Sunflower necrosis virus</Emphasis> in callus

The calli induction from different Sunflower necrosis virus (SNV)-infected explants of sunflower was initiated on Murashige & Skoog medium amended with benzyladenine at 1 mg l ^-1 and indole acetic acid at 0.5 mg l ^-1. The virus was purified from the induced calli by sucrose density gradient centrifugation and the viral particles were confirmed by transmission electron microscopy. The SNV infection in uninfected calli was established by a soak prick method and the viral development was confirmed by a plant infectivity test. Serological analyses such as Ouchterlony double immnuodiffusion test, enzyme-linked immunosorbent assay and Western blot using anti-SNV antibodies yielded positive reactions with the purified SNV. These investigations have demonstrated that the SNV could be cultured in calli and maintained in vitro in pure form for a long period of time.     

Increase of tospoviral diversity in Brazil with the identification of two new tospovirus species, one from chrysanthemum and one from zucchini

ABSTRACT During a survey conducted in several different regions of Brazil, two unique tospoviruses were isolated and characterized, one from chrysanthemum and the other from zucchini. The chrysanthemum virus displayed a broad host range, whereas the virus from zucchini was restricted mainly to the family Cucurbitaceae. Double-antibody sandwich-enzyme-linked immunosorbent assay and western immunoblot analyses demonstrated that both viruses were serologically distinct from all reported tospovirus species including the recently proposed peanut yellow spot virus and iris yellow spot virus (IYSV) species. The nucleotide sequences of the nucleocapsid (N) genes of both viruses contain 780 nucleotides encoding for deduced proteins of 260 amino acids. The N proteins of these two viruses displayed amino acid sequence similarities with the previously described tospovirus species ranging from 20 to 75%, but they were more closely related to each other (80%). Based on the biological and molecular features, these viruses are proposed as two new tospovirus species, designated chrysanthemum stem necrosis virus (CSNV) and zucchini lethal chlorosis virus (ZLCV). With the identification of CSNV and ZLCV, in addition to tomato spotted wilt virus, groundnut ring spot virus, tomato chlorotic spot virus, and IYSV, Brazil harbors the broadest spectrum of tospovirus species reported.     

A quantitative PCR assay for accurate <Emphasis Type="Italic">in planta</Emphasis> quantification of the necrotrophic pathogen <Emphasis Type="Italic">Phytophthora cinnamomi</Emphasis>

A reliable method for measuring disease progression is important when evaluating susceptibility in host—pathogen interactions. We describe a sensitive quantitative polymerase chain reaction (QPCR) assay that enables quantitative measurement of in planta DNA of the necrotrophic pathogen, Phytophthora cinnamomi, that avoids problems caused by variation in DNA extraction efficiency and degradation of host DNA during host tissue necrosis. Normalization of pathogen DNA to sample fresh weight or host DNA in samples with varying degrees of necrosis led to overestimation of pathogen biomass. Purified plasmid DNA, containing the pScFvB1 mouse gene, was added during DNA extraction and pathogen biomass was normalized based on plasmid DNA rather than host DNA or sample fresh weight. This method is robust and improves the accuracy of pathogen measurement in both resistant (non-host A. thaliana–P. cinnamomi) and susceptible (host Lupinus angustifolius–P. cinnamomi) interactions to allow accurate measurement of pathogen biomass even in the presence of substantial host cell necrosis.     

Spatial connectedness of plant species: potential links for apparent competition via plant diseases

This study evaluated the reactions of seven common C4 grasses of the tallgrass prairie of the USA Great Plains to the economically important wheat pathogens Pyrenophora tritici‐repentis and Gaeumannomyces graminis var. tritici (Ggt) isolated from wheat. The P. tritici‐repentis isolates (race 1) were pathogenic on all grasses tested, but symptom severity was markedly low. Three of the grass species inoculated with Ggt were highly susceptible, while four species exhibited no symptoms. Because measures of connectedness can provide a proxy for population processes, connectedness was evaluated within and among the seven grass species in representative tallgrass prairie environments for all potential pathogen‐sharing patterns. Andropogon gerardii was ubiquitous, so all plant species were well connected to it. Andropogon scoparius (= Schizachyrium scoparium), Sorghastrum nutans and Panicum virgatum were fairly common but specialized to particular environments. Bouteloua curtipendula was uncommon but occurred in all environments, while Buchloë dactyloides and Bouteloua gracilis were uncommon and only occurred in upland sites. Co‐occurrence of plant species was generally not reciprocal in that, for many species pairs, species A rarely occurred without potential exposure to inoculum from species B, while species B commonly occurred without species A. The three grass species susceptible to Ggt may act as sources of inoculum for each other within tallgrass prairie, with the potential to influence fitness, and tallgrass prairie and commercial wheat ecosystems in the Great Plains also have the potential to share both pathogens.     

Bacterial Wilt of China Aster Caused by Erwinia chrysanthemi

In July 2000, a bacterial wilt was found on China aster (Callistephus chinensis Nees) on Hokkaido, Japan. Bacteriological characteristics of the isolated bacteria were identical to Erwinia chrysanthemi. This is the first report on bacterial wilt of China aster caused by E. chrysanthemi. Received 4 June 2001/ Accepted in revised form 6 September 2001     

Fungicide resistance in Czech populations of cucurbit powdery mildews

A total of 108 isolates of cucurbit powdery mildew (CPM) fungi, 78Golovinomyces cichoracearum (Gc) and 30Podosphaera xanthii (Px), originating from nine Czech regions and 22 districts of the Czech Republic were collected in the years 2001–2004. These isolates were screened for tolerance and/or resistance to the three frequently used fungicides (fenarimol, dinocap, benomyl). Fungicide sensitivity was determined by a modified leaf-disc bioassay with five concentrations. Fungicide efficacy differed significantly: fenarimol was the most effective and all isolates of both CPM were controlled by the recommended concentration (36μg a.i. ml⁻¹). Some isolates expressed resistance (profuse sporulation) or tolerance (limited sporulation) to lower concentrations (9.6 and 18μg a.i. ml⁻¹). Specific temporal variation in tolerance/resistance was observed, with some isolates ofGc from 2002 evincing tolerant or resistant reactions to these low fenarimol concentrations, but isolates with similar reactions were not detected during the years 2003–2004. Dinocap showed decreasing efficacy during all 3 years. A shift to more tolerant reactions in the CPM populations was detected forGc in 2001–2002, and forPx in 2001 and 2004. Benomyl was found to be ineffective, because the majority of screened isolates (88%Gc and 97%Px) belonged to the highly resistant strains, with resistant reaction on the recommended concentration (250μg a.i. ml⁻¹) and tolerance or resistance on higher concentrations (500 and 1000μg a.i. ml⁻¹). Sensitivity differed between the CPM species. Whereas practically allPx isolates (except one from 2003) were resistant, 12% ofGc isolates from the years 2001–2003 showed sensitive and/or tolerant reactions. In 2004, only benomyl-resistantGc strains were detected. Variation in tolerance/resistance was detected to all screened fungicides during the course of this study at some repeatedly sampled locations. http://www.phytoparasitica.org posting June 12, 2008.     

Pathogenicity of <Emphasis Type="Italic">Mycochaetophora gentianae</Emphasis>, causal fungus of gentian brown leaf spot,as affected by host species,inoculum density,temperature, leaf wetness duration,and leaf position

When the influence of host species, inoculum density, temperature, leaf wetness duration, and leaf position on the incidence of gentian brown leaf spot caused by Mycochaetophora gentianae, was examined, the fungus severely infected all seven Gentiana triflora cultivars, but failed to infect two cultivars of G. scabra and an interspecific hybrid cultivar. Inoculum density correlated closely with disease incidence, and a minimum of 10² conidia/mL was enough to cause infection. In an analysis of variance, temperature and leaf wetness duration had a significant effect upon disease incidence, which increased with higher temperature (15–25°C) and longer duration of leaf wetness (36–72 h). No disease developed at temperatures lower than 10°C or when leaf wetness lasted <24 h. At 48-h leaf wetness, disease incidence was 0, 28, 77, and 85% at 10, 15, 20, and 25°C, respectively. Middle and lower leaves on the plant were more susceptible than upper leaves. In microscopic observations of inoculated leaves, >50% of conidia germinated at temperatures >15°C after 24-h leaf wetness. More appressoria formed at higher temperatures (15–25°C) with extended duration of leaf wetness (24–72 h). At 48-h leaf wetness, appressorium formation was 0, 8, 26, and 73% at 10, 15, 20, and 25°C, respectively. These results suggest that temperature and leaf wetness duration were important factors for infection of gentian leaves.     

The competence of four thrips species to transmit and replicate four tospoviruses

The tospoviruses Tomato spotted wilt virus (TSWV), Tomato chlorotic spot virus (TCSV), Groundnut ringspot virus (GRSV) and Chrysanthemum stem necrosis virus (CSNV) are well-known pathogens on tomato in Brazil. The thrips species Frankliniella occidentalis , F. schultzei , Thrips tabaci and T. palmi were studied for their competence to transmit these tospoviruses. Frankliniella occidentalis transmitted all four tospoviruses with different efficiencies. Frankliniella schultzei transmitted TCSV, GRSV and CSNV. Although F. schultzei has been reported as a vector of TSWV, the F. schultzei population in the present study did not transmit the TSWV isolate used. A population of T. tabaci known to transmit Iris yellow spot virus (onion isolate) did not transmit any of the studied tospoviruses, and nor did T. palmi . Replication of these tospoviruses could be demonstrated by ELISA, not only in the thrips species that could transmit them, but also in those that could not. The results strongly suggest that competence to transmit is regulated not only by the initial amount of virus acquired and replication, but also by possible barriers to virus circulation inside the thrip's body.     

Aster yellows subgroup (Candidatus Phytoplasma sp. AY 16S-group,AY-sg) phytoplasma associated with porcelain vine showing witches' broom symptoms in South Korea

 This is the first report of a phytoplasma in porcelain vine [Ampelopsis brevipedunculata var. heterophylla (Thunb.) Hara.] with severe witches' broom symptoms in Korea. On the basis of the polymerase chain reaction-amplified ribosomal DNA, the phytoplasma infecting porcelain vine was classified as a member of the aster yellows subgroup. Received: October 21, 2002 / Accepted: December 20, 2002