<Emphasis Type="Italic">Physalis ixocarpa</Emphasis> and <Emphasis Type="Italic">P. peruviana</Emphasis>, new natural hosts of <Emphasis Type="Italic">Tomato chlorosis virus</Emphasis>

Tomato chlorosis virus causes yellow leaf disorder epidemics in many countries worldwide. Plants of Physalis ixocarpa showing abnormal interveinal yellowing and plants of Physalis peruviana showing mild yellowing collected in the vicinity of tomato crops in Portugal were found naturally infected with ToCV. Physalis ixocarpa and P. peruviana were tested for susceptibility to ToCV by inoculation with Bemisia tabaci, Q biotype. Results confirmed that ToCV is readily transmissible to both species. The infection was expressed in P. ixocarpa by conspicuous interveinal yellow areas on leaves that developed into red or brown necrotic flecks, while P. peruviana test plants remained asymptomatic. Infected plants of both P. ixocarpa and P. peruviana served as ToCV sources for tomato infection via B. tabaci transmission. This is the first report of P. ixocarpa and P. peruviana as natural hosts of ToCV.     

Isolation of pathogenicity- and gregatin-deficient mutants of <Emphasis Type="Italic">Phialophora gregata</Emphasis> f. sp. <Emphasis Type="Italic">adzukicola</Emphasis> through <Emphasis Type="Italic">Agrobacterium tumefaciens</Emphasis>-mediated transformation

Phialophora gregata f. sp. adzukicola, a causal agent of brown stem rot in adzuki beans, produces phytotoxic compounds: gregatins A, B, C, D, and E. Gregatins A, C, and D cause wilting and vascular browning in adzuki beans, which resemble the disease symptoms. Thus, gregatins are considered to be involved in pathogenicity. However, molecular analyses have not been conducted, and little is known about other pathogenic factors. We sought to isolate nonpathogenic and gregatin-deficient mutants through Agrobacterium tumefaciens-mediated transformation (ATMT) for cloning of pathogenicity-related genes. The co-cultivation of P. gregata and A. tumefaciens for 48 h at 20°C with 200 μM acetosyringone resulted in approximately 80 transformants per 10⁶ conidia. The presence of acetosyringone in the A. tumefaciens pre-cultivation period led to an increase in T-DNA copy number per genome. Of 420 and 110 transformants tested for their pathogenicity and productivity of gregatins, one nonpathogenic and three gregatin-deficient mutants were obtained, respectively. The nonpathogenic mutant produced gregatins, whereas the gregatin-deficient mutants had pathogenicity comparable to the wild-type strain. This is the first report of ATMT of P. gregata. Further analysis of these mutants will help reveal the nature of the pathogenicity of this fungus including the role of gregatin in pathogenesis.     

<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%).     

Susceptibility and resistance of <Emphasis Type="Italic">Beta vulgaris</Emphasis> subsp. <Emphasis Type="Italic">maritima</Emphasis> to foliar rub-inoculation with <Emphasis Type="Italic">Beet necrotic yellow vein virus</Emphasis>

Four lines (designated MR0, MR1, MR2, and M8) from 13 accessions of Beta vulgaris subsp. maritima were selected on the basis of phenotypes produced after foliar rub-inoculation with Beet necrotic yellow vein virus (BNYVV). The susceptible phenotype developed bright yellow local lesions, whereas the resistant phenotype had symptoms ranging from no visible lesions to necrotic lesions at the inoculation site. MR1 and MR2 lines had a resistant phenotype depending on the isolate and the MR0 line was susceptible to all isolates of BNYVV tested. The M8 line was highly susceptible; the virus spread systemically and caused severe stunting. These plant lines will be useful for distinguishing BNYVV isolates having different pathogenicities, especially those controlled by RNA3 and/or RNA5.     

Incidence of viruses in <Emphasis Type="Italic">Alstroemeria</Emphasis> plants cultivated in Japan and characterization of <Emphasis Type="Italic">Broad bean wilt virus-2, Cucumber mosaic virus</Emphasis> and <Emphasis Type="Italic">Youcai mosaic virus</Emphasis>

Alstroemeria plants were surveyed for viruses in Japan from 2002 to 2004. Seventy-two Alstroemeria plants were collected from Aichi, Nagano, and Hokkaido prefectures and 54.2% were infected with some species of virus. The predominant virus was Alstroemeria mosaic virus, followed by Tomato spotted wilt virus, Youcai mosaic virus (YoMV), Cucumber mosaic virus (CMV), Alstroemeria virus X and Broad bean wilt virus-2 (BBWV-2). On the basis of nucleotide sequence of the coat protein genes, all four CMV isolates belong to subgroup IA. CMV isolates induced mosaic and/or necrosis on Alstroemeria. YoMV and BBWV-2 were newly identified by traits such as host range, particle morphology, and nucleotide sequence as viruses infecting Alstroemeria. A BBWV-2 isolate also induced mosaic symptoms on Alstroemeria seedlings.     

Genetic diversity of <Emphasis Type="Italic">Fusarium oxysporum</Emphasis> f. sp. <Emphasis Type="Italic">spinaciae</Emphasis> in Japan based on phylogenetic analyses of rDNA-IGS and <Emphasis Type="Italic">MAT1</Emphasis> sequences

Twenty-eight isolates of Fusarium oxysporum f. sp. spinaciae (FOS; the causal agent of spinach wilt) collected from Japan were assessed for mating type and subjected to phylogenetic analysis. Mating type analysis revealed all isolates to be MAT1-2, suggesting that there is no sexual recombination within the population. Phylogenetic analyses based on nucleotide sequences of the ribosomal DNA intergenic spacer (IGS) and the mating type locus (MAT1) suggested that FOS is polyphyletic. The cluster analysis based on IGS showed four phylogenetic groups (S1–S4) among the isolates. Two distinct lineages, S1 and S3, included FOS isolates both of the vegetative compatibility group (VCG) types, 0330 and 0331, demonstrating that VCG differentiation in FOS may not necessarily reflect the phylogenetic relationships based on IGS and MAT1-2-1.     

Virulence and diversity of <Emphasis Type="Italic">Blumeria graminis</Emphasis> f.sp. <Emphasis Type="Italic">hordei</Emphasis> in East China

Four hundred and sixty-one isolates of Blumeria graminis f.sp. hordei were obtained from eight populations occurring on cultivated barley (Hordeum vulgare) at four geographically distant locations in China during 2003 and 2004. Their virulence frequency was determined on 30 differential lines. No isolate was virulent on differential lines possessing the resistance genes Mla1, Mla3, Mla6, Mla7, Mla9, Mla12, Mla13, Mlat, Mlg, Mla10, Mla22, Mla23, Mlp1, Ml(N81) and Mlmw. Virulences to the first nine resistance genes are prevalent in Europe and constitute the main part of genetic distance between Chinese and European populations. Conversely, no isolate was avirulent on the differential lines possessing the genes Mla8 and Ml(Ch). The frequencies of isolates overcoming the genes Mla2, Mla11, Mlk1 and Mlk2 were .4–9.3%, and frequencies of isolates overcoming the genes Mlh, MlLa, Ml(Bw), Mlra, Ml(Ru2), mlw, MlGa, MlWo and Mlnn ranged from 18.2% to 98.7%. Based on reactions of differential lines possessing the genes Mlk1, Mlh, MlLa, Ml(Bw), Mlra and Ml(Ru2), pathotypes were identified and diversity parameters calculated. Eleven of 22 detected pathotypes were found in both years and comprised 94.6% of isolates. Generally, the populations from different locations in 1 year were more closely related than populations collected from the same locations in different years. Complete effectiveness of the resistance genes, for which no corresponding virulences were found, will allow Chinese breeders to access many modern European barley cultivars that are fully resistant to powdery mildew in China, including those possessing the non-host resistance gene mlo.     

Biofilm formation and resistance to bactericides of <Emphasis Type="Italic">Pseudomonas syringae</Emphasis> pv. <Emphasis Type="Italic">theae</Emphasis>

Biofilm-grown cells of Pseudomonas syringae pv. theae (P.s.theae) wild-type strain K9301 on abiotic surface had remarkable resistance to kasugamycin in comparison to planktonically grown cells; however, the biofilm-grown cells of K9301 had the same sensitivity to copper sulfate. Because both the lesser biofilm-forming strain K9301S3 and enhanced biofilm-forming strain K9301-6 also had remarkable biofilm resistance to kasugamycin just as K9301 did and because epigallocatechin gallate, which enhanced biofilm formation of P.s.theae, had no effect on biofilm resistance to kasugamaycin, the degree of biofilm formation was not correlated with the antibiotic susceptibilities. In addition, K9301 and K9301S3 had less sensitivity to kasugamycin but had high sensitivity to copper sulfate on nonwounded leaf surfaces. These results indicate a possibility that the mechanism of P.s.theae biofilm resistance to bactericide functions on both abiotic and nonwounded leaf surfaces.     

Characteristics of a <Emphasis Type="Italic">Plasmopara angustiterminalis</Emphasis> isolate from <Emphasis Type="Italic">Xanthium strumarium</Emphasis>

Leaves of Xanthium strumarium infected with downy mildew were collected in the vicinity of a sunflower field in southern Hungary in 2003. Based on phenotypic characteristics of sporangiophores, sporangia and oospores as well as host preference the pathogen was classified as Plasmopara angustiterminalis. Additional phenotypic characters were investigated such as the size of sporangia, the number of zoospores per sporangium and the time-course of their release. Infection studies revealed infectivity of the P. angustiterminalis isolate to both X. strumarium and Helianthus annuus. Inoculation of the sunflower inbred line, HA-335 with resistance to all known P. halstedii pathotypes, resulted in profuse sporulation on cotyledons and formation of oospores in the bases of hypocotyls. Infections of sunflower differential lines often led to damping-off. Molecular genetic analysis using simple sequence repeat primers and nuclear rDNA sequences revealed clear differences to Plasmopara halstedii, the downy mildew pathogen of sunflower.     

Behavior and mutation of <Emphasis Type="Italic">Ralstonia solanacearum</Emphasis> in <Emphasis Type="Italic">Solanum toxicarium</Emphasis> grown in aseptic culture

To study the behavior and mutation of Ralstonia solanacearum in Solanum toxicarium, which is resistant to bacterial wilt, S. toxicarium was grown in aseptic culture and inoculated with R. solanacearum. Although 60%–80% of the inoculated plants were wilting after 2 to 3 days, most wilted plants had recovered by 20 days after inoculation. The pathogen was reisolated from over 98% of inoculated plant stems, but the percentage of recovery decreased the closer the isolation sites were toward the upper stem sections. Three colony types, characterized as fluidal white, nonfluidal red, and a mixture of fluidal white and nonfluidal red, were reisolated from the stems. Nonfluidal red colonies were less virulent on tomato plants than fluidal white colonies.     

Genetics of host-pathogen interactions in the <Emphasis Type="Italic">Pyrenophora teres</Emphasis> f. <Emphasis Type="Italic">teres</Emphasis> (net form) – barley (<Emphasis Type="Italic">Hordeum vulgare)</Emphasis> pathosystem

The genetics of host-pathogen interactions in the Hordeum vulgare – P. teres f. teres pathosystem was studied in twelve resistant barley accessions, i.e. CI 9825, CI 9819, Diamond, CI 4922, CI 5401, Harbin, c-8755, c-21849, c-8721 c-23874, c-19979, c-15811. F₂ analyses of crosses with susceptible genotypes employing various isolates (from Europe, USA, Canada, and Australia) revealed that resistance is mostly isolate-specific and controlled by one or two genes. Segregation in ascospore progeny from two crosses between isolates of different origin revealed that avirulence in P. teres is also determined by one or two genes. An epistatic effect of suppressor genes on avirulence genes is proposed for the genetics of virulence to Diamond, Harbin, CI 5401 and c-8721 in the fungal crosses D (181-6 × A80) and F (H-22 × 92-178/9). Segregation in F₂ of crosses of three new sources of resistance (c-23874, c-19979, c-15811) to the susceptible cv. Pirkka was studied in laboratory and greenhouse tests by using seven P. teres isolates, i.e. 181-6, d8-3, d8-4, d9-1, d9-4, F4 and F74. In addition, virulence to these barley accessions of ascospore progeny from crosses of the same isolates was studied. Based on these studies it was concluded that depending on the isolate used, resistance of c-23874 is determined at least by two genes and in c-19979 and c-15811 by three genes. The results of this parallel analyses of genetics of resistance and genetics of virulence allows the postulation of a gene–for–gene interaction in the P. teres – H. vulgare pathosystem.     

Gummy stem blight of balsam pear caused by <Emphasis Type="Italic">Didymella bryoniae</Emphasis> and its anamorph <Emphasis Type="Italic">Phoma cucurbitacearum</Emphasis>

Gummy stem blight of balsam pear found in the Kanto district and in the Hokkaido Prefecture was demonstrated to be caused by Didymella bryoniae (Auerswald) Rehm based on inoculation experiments, molecular analysis, and morphological identification of the pathogenic fungus. This fungus was also pathogenic to related plants belonging to Cucurbitaceae. The imperfect stage of the fungus was identified as Phoma cucurbitacearum (Fr.: Fr.) Sacc. based on morphological similarities.     

Adsorption of OP<Subscript>1</Subscript>-related bacteriophages requires the gene encoding a TonB-dependent receptor-like protein in <Emphasis Type="Italic">Xanthomonas</Emphasis><Emphasis Type="Italic">oryzae</Emphasis> pv. <Emphasis Type="Italic">oryzae</Emphasis>

Xanthomonas oryzae pv. oryzae strain T7174R is lysed by bacteriophage OP_1h and OP_1h2. Three mutants tolerant to both OP_1h and OP_1h2 were isolated by transposon mutagenesis. The mutants had an insertion of the transposon in XOO1687, which is predicted to encode a TonB-dependent receptor gene. Plasmid pHMIroNB that contained XOO1687 of T7174R was constructed, and the mutant was transformed with the plasmid. The transformant recovered sensitivity to OP_1h and OP_1h2. Electron microscopic analysis demonstrated that OP_1h and OP_1h2 can adsorb to the wild type and the transformant, but they could not adsorb to the phage-tolerant mutant. These results suggest that the TonB-dependent receptor gene relates to adsorption and infection of T7174R by OP_1h2 and OP_1h. Y. Inoue and S. Tsuge have contributed equally to this work.     

A new <Emphasis Type="Italic">Rhizoctonia</Emphasis> sp. closely related to <Emphasis Type="Italic">Waitea circinata</Emphasis> causes a new disease of creeping bentgrass

Isolates of an unidentified Rhizoctonia sp. (UR isolates) were obtained from creeping bentgrass and Kentucky bluegrass with reddish brown sheath and foliar rots. Because the UR isolates anastomosed with isolates of three varieties of Waitea circinata (var. oryzae, var. zeae, and var. circinata), colony morphology, hyphal growth rate at different temperatures, pathogenicity, sequence analysis of the internal transcribed spacers (ITS) region of ribosomal RNA genes (rDNA) were compared. The colony color of mature UR isolates was distinct from isolates of the other three varieties of W. circinata. In pathogenicity tests on creeping bentgrass, the severity of the disease caused by UR isolates was significantly higher than that caused by the three varieties of W. circinata. Sequence similarities of the rDNA-ITS region between UR isolates and between isolates within each variety were high (97–100%), but they were lower among isolates from UR and the varieties of W. circinata (88–94%). In a phylogenetic tree based on the rDNA-ITS sequences, UR isolates formed a cluster separate from each of the clusters formed by the three varieties of W. circinata. These results indicate that the UR isolates clearly differ from the three varieties of W. circinata. We therefore propose that the UR isolates be classified as new Rhizoctonia sp. that are closely related to W. circinata and that the disease on creeping bentgrass should be called Waitea reddish-brown patch disease (Sekikasshoku-hagusare-byo in Japanese).     

Effect of seed treatment of<Emphasis Type="Italic">Arachis hypogaea</Emphasis> with<Emphasis Type="Italic">Bacillus subtilis</Emphasis> on nodulation in biocontrol of southern blight (<Emphasis Type="Italic">Sclerotium rolfsii</Emphasis>) disease

The employment of formulatedBacillus subtilis for peanut seeds (Arachis hypogaea cv. ‘Shulamit’) counteracted the destructive effects of the seedborne pathogenSclerotium (Athelia)rolfsii on the nodulation, leghemoglobin and nitrogenase activity of peanuts. Moreover, the changes in crop vigor index, total nitrogen content and survivability of bothRhizobium spp. andB. subtilis have been related to compatibility and even an occasional synergism between them. http://www.phytoparasitica.org posting Nov. 12, 2006.     

A diagnostic medium for the semi-selective isolation and enumeration of <Emphasis Type="Italic">Xanthomonas axonopodis</Emphasis>pv. <Emphasis Type="Italic">vignicola</Emphasis>

A semi-selective medium for isolation of Xanthomonas axonopodis pv. vignicola from cowpea (Vigna unguiculata) plant and soil samples was developed. Twelve carbon and five nitrogen sources were tested with four strains of X. axonopodispv.vignicola, and 25 antibiotics were screened against saprophytes. -cellobiose (10g) was selected as the optimal carbon source. Among the antibiotics, cefazoline inhibited growth of most of the saprophytes with little effect on strains of the pathogen. ,-methionine enhanced growth of X. axonopodispv.vignicola. Boric acid along with ammonium chloride suppressed growth of Pseudomonas fluorescens. The semi-selective medium designated as cefazoline-cellobiose-methionine (CCM) medium contained K₂HPO₄ 1.34g, KH₂PO₄ 0.4g, MgSO₄ 0.3g, H₃BO₃ 0.2g, NH₄Cl 1.0g, -cellobiose 10g, cycloheximide 0.2g, ,-methionine 1.0g, cefazoline 10mg and agar 14g per l of water (pH 7.2). Colonies of X. axonopodispv.vignicola on CCM medium were whitish, round, raised and 0.2–1.8mm in diameter 96h after incubation. CCM medium generally inhibited growth of Pantoea agglomerans, Bacillus subtilis and saprophytes isolated from cowpea leaves. Colonies of Pseudomonas fluorescens and a saprophytic bacterium, which were not completely suppressed by CCM, could be differentiated from X. axonopodispv.vignicola by their smaller size and different color. The CCM medium proved useful for isolation of X. axonopodispv.vignicola from cowpea plant and soil samples. This is the first report of a semi-selective medium developed for detection of X. axonopodispv.vignicola.     

Tobacco cultivars vary in induction of systemic resistance against <Emphasis Type="Italic">Cucumber mosaic virus</Emphasis> and growth promotion by <Emphasis Type="Italic">Pseudomonas chlororaphis</Emphasis> O6 and its <Emphasis Type="Italic">gacS</Emphasis> mutant

The colonization of plant roots with certain rhizosphere bacteria promotes plant growth and induces long lasting systemic protection against a broad spectrum of plant pathogens. The role of the global regulator, GacS, in the rhizosphere colonist Pseudomonas chlororaphis O6 in stimulating growth promotion and induced resistance against Cucumber mosaic virus was examined in tobacco. Responses were compared in tobacco cvs Samsun and GX3. Root colonization of Samsun with wild-type O6 and the gacS complemented mutant-elicited reduced viral symptoms and viral titre. On GX3, there was little affect on symptoms when roots were colonized by the wild-type, gacS mutant or complemented mutant but colonization by both the wild-type and the gacS mutant lowered viral titre. Wild-type O6 and the gacS mutant caused plant growth to be maintained in both tobacco cultivars after viral infection, although the affect was stronger with GX3 than Samsun. In contrast, although a chemical inducer, benzothiadiazole, reduced symptoms and viral titre in both cultivars, plant growth was suppressed. Our results indicate rhizobacteria-elicited induced viral resistance without a negative impact on growth but there was a differential response between cultivars. Detailed knowledge regarding the mechanisms inherent to these differences between cultivars requires further investigation.     

A novel pathosystem to study the interactions between <Emphasis Type="Italic">Lotus japonicus</Emphasis> and <Emphasis Type="Italic">Fusarium solani</Emphasis>

A wilt disease of the model legume Lotus japonicus was observed in a greenhouse in Tokyo, Japan in May 2004. Roots of diseased plants were rotted and dark brown with lesions spreading to lower stems and leaves, resulting in rapid plant death. The causal agent was identified as Fusarium solani based on the morphology. Sequence analysis of rDNA supported the identification. Inoculation of roots of healthy plants with conidia reproduced characteristic disease symptoms, and F. solani was reisolated from lesions, satisfying Koch’s postulates. The isolate also caused chlorotic to necrotic lesions on leaves of healthy plants after wound-inoculation. Infection by F. solani of leaves of L. japonicus was confirmed histologically. Mycelia were observed in the intercellular spaces of parenchymatous tissues in the lesion area and the surrounding tissues. This is the first report of fungal disease on L. japonicus satisfying Koch’s postulates. We named it “Fusarium root rot of L. japonicus” as a new disease. The compatibility of L. japonicus and F. solani is expected to form a novel pathosystem for studying interactions between legumes and fungal pathogens. The nucleotide sequence data reported are available in the DDBJ/EMBL/GenBank databases under accession numbers AB258993 and AB258994.     

Host status and reaction of <Emphasis Type="Italic">Medicago truncatula</Emphasis> accessions to infection by three major pathogens of pea (<Emphasis Type="Italic">Pisum sativum</Emphasis>) and alfalfa (<Emphasis Type="Italic">Medicago sativa</Emphasis>)

Ditylenchus dipsaci, the stem nematode of alfalfa (Medicago sativa), Mycosphaerella pinodes, cause of Ascochyta blight in pea (Pisum sativum) and Aphanomyces euteiches, cause of pea root rot, result in major yield losses in French alfalfa and pea crops. These diseases are difficult to control and the partial resistances currently available are not effective enough. Medicago truncatula, the barrel medic, is the legume model for genetic studies, which should lead to the identification and characterization of new resistance genes for pathogens. We evaluated a collection of 34 accessions of M. truncatula and nine accessions from three other species (two from M. italica, six from M. littoralis and one from M. polymorpha) for resistance to these three major diseases. We developed screening tests, including standard host references, for each pathogen. Most of the accessions tested were resistant to D. dipsaci, with only three accessions classified as susceptible. A very high level of resistance to M. pinodes was observed among the accessions, none of which was susceptible to this pathogen. Conversely, a high level of variation, from resistant to susceptible accessions, was identified in response to infection by A. euteiches.