Kenichi Tsuchiya

Infection Dynamics in Viral Spread and Interference Under the Synergism Between Cucumber mosaic virus and Turnip mosaic virus

Mixed infection of Cucumber mosaic virus (CMV) and Turnip mosaic virus (TuMV) induced more severe symptoms on Nicotiana benthamiana than single infection. To dissect the relationships between spatial infection patterns and the 2b protein (2b) of CMV in single or mixed infections, the CMV vectors expressing enhanced green fluorescent or Discosoma sp. red fluorescent proteins (EGFP [EG] or DsRed2 [Ds], respectively were constructed from the same wild-type CMV-Y and used for inoculation onto N. benthamiana. CMV2-A1 vector (C2-A1 [A1]) has a functional 2b while CMV-H1 vector (C2-H1 [H1]) is 2b deficient. As we expected from the 2b function as an RNA silencing suppressor (RSS), in a single infection, A1Ds retained a high level of accumulation at initial infection sites and showed extensive fluorescence in upper, noninoculated leaves, whereas H1Ds disappeared rapidly at initial infection sites and could not spread efficiently in upper, noninoculated leaf tissues. In various mixed infections, we found two phenomena providing novel insights into the relationships among RSS, viral synergism, and interference. First, H1Ds could not spread efficiently from vasculature into nonvascular tissues with or without TuMV, suggesting that RNA silencing was not involved in CMV unloading from vasculature. These results indicated that 2b could promote CMV to unload from vasculature into nonvascular tissues, and that this 2b function might be independent of its RSS activity. Second, we detected spatial interference (local interference) between A1Ds and A1EG in mixed infection with TuMV, between A1Ds (or H1Ds) and TuMV, and between H1Ds and H1EG. This observation suggested that local interference between two viruses was established even in the synergism between CMV and TuMV and, again, RNA silencing did not seem to contribute greatly to this phenomenon.

Analysis of genetic and biological characters of japanese potato strains of ralstonia solanacearum

We assessed the geographic distribution, biovar, phylotype, DNA fingerprints (rep-PCR), and/or endoglucanase sequence of potato bacterial wilt pathogen, Ralstonia solanacearum (Rs), in Japan. Rs has been isolated from potato fields in southwestern, warm, temperate regions. Of the 188 isolates, 74 belonged to biovar N2 (39%), 44 to biovar 3 (24%), and 70 to biovar 4 (37%). Biovars N2 and 4 strains were widely distributed, from northern (Hokkaido) to southern (Okinawa) Japan. Based on the results of multiplex-PCR analysis, every potato strains belonged to either phylotype I or IV. Phylotype I comprised both biovars 3 and 4 strains. On the other hand, phylotype IV included biovar N2 strains. None of the strains belonged to phylotype II or III or biovar 1 or 2. Phylogenetic analysis based on DNA fingerprints and endoglucanase gene sequences clarified the genetic diversity of the Japanese potato strains and the close genetic relationship between the Japanese strains and the Asian strains in phylotypes I and IV.

Endophytic fungi associated with Fallopia japonica (Polygonaceae) in Japan and their interactions with Puccinia polygoni-amphibii var. tovariae, a candidate for classical biological control

Fallopia japonica (Polygonaceae), or Japanese knotweed, is now spreading globally, causing serious problems in Europe and North America in both natural and urban habitats. There is an urgent need for alternative management solutions, and classical biological control, using coevolved natural enemies found in the native range, is currently being investigated. Here, we isolated fungal endophytes from F. japonica in Japan, its natural habitat, to find endophytes that might increase the virulence of a coevolved rust pathogen, Puccinia polygoni-amphibii var. tovariae. A total of 1581 fungal endophytes were recovered from F. japonica and classified into 15 taxa. Five genera (Colletotrichum, Pestalotiopsis, Phoma, Phomopsis, and Alternaria) were dominant as endophytes in F. japonica. A greenhouse study of the dominant endophyte-pathogen interactions revealed three types of reactions: suppressive, synergistic, and neutral. In particular, one Phomopsis isolate--closely related to Diaporthe medusaea, based on ITS sequences--promoted the pathogenic aggressiveness of P. polygoni-amphibii var. tovariae and, therefore, this interaction is potentially useful to increase the effectiveness of the rust fungus as a biological control agent of F. japonica in its invasive range.

Pathogenic characters of Japanese potato strains of Ralstonia solanacearum

Ralstonia solanacearum (Rs) strains in phylotypes I and IV isolated from potato in Japan were investigated for pathogenicity on potato, tomato, eggplant, Solanumintegrifolium, tobacco, groundnut, and pumpkin. The strains were divided into 17 types based on differences in their pathogenicity on the tested plants. Particularly, the pathogenicity of most phylotype I strains on eggplant was distinctly different from that of the phylotype IV strains. When nine potato varieties (included two breeding lines) were inoculated with several Rs strains, phylotype IV strains were highly virulent on the breeding lines that are regarded as resistant to phylotype I strains.

Molecular and biological characterization of Chrysanthemum stem necrosis virus 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.

Estimation of the nitrogen fixation by sugarcane cultivar NiF-8 using 15N dilution and natural 15N abundance techniques

Abstract The high population of endophytic diazotrophs in the stalk of the Japanese sugarcane cultivar NiF-8 described in our previous report suggests the possible significant contribution of biological nitrogen fixation (BNF) to the crop nitrogen nutrition. To estimate the amount of BNF in this cultivar, the proportion of biologically fixed nitrogen (%Ndfa) to the plant total nitrogen uptake was determined by 15N dilution and natural 15N abundance (δ15N) techniques. Using the δ15N method, the estimated %Ndfa by the sugarcane cv. NiF-8 under field lysimeter conditions ranged from 32 to 38% at 6 months after planting (MAP) and from 27 to 34% at 7 MAP. With the 15N dilution method, the estimated %Ndfa by the sugarcane plants was 26.0% for the roots, 14.1% for the stem, and 20.5% for the leaves. These results indicate that the sugarcane cv. NiF-8 obtained part of its nitrogen requirements from BNF.

Genetic diversity of Fusarium oxysporum f. sp. spinaciae in Japan based on phylogenetic analyses of rDNA-IGS and MAT1 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.

Induction of antiviral responses by acibenzolar-s-methyl against cucurbit chlorotic yellows virus in Melon

Cucurbit chlorotic yellows virus (CCYV) (family Closteroviridae, genus Crinivirus) is an emerging virus which causes severe diseases on melon (Cucumis melo) plants. CCYV-infected melon plants display yellowing, mottling, chlorosis, or chlorotic spots on leaves. To develop a new control strategy, the potential for 1,2,3-benzothiadiazole-7-thiocarboxylic acid-S-methyl-ester (ASM) to suppress CCYV infection was evaluated. ASM treatment on melon plants greatly increased the expression levels of pathogenesis-related 1a gene, a marker gene for systemic acquired resistance. ASM treatment on melon plants before inoculation of CCYV suppressed systemic symptoms and decreased CCYV accumulation. ASM treatment on melon even after inoculation of CCYV reduced disease severity and accumulation levels of CCYV. The results show the potential for ASM treatment on attenuation of the CCYV disease symptoms.

Taxonomic re-examination of several Japanese Stemphylium strains based on morphological and molecular phylogenetic analyses

Most of the many Stemphylium species on record as plant pathogens in Japan have been identified by morphology. Using molecular phylogenetic analysis of four loci (rDNA-ITS, EF-1α, GPD, and vmaA-vpsA) combined, we re-examined the taxonomy of 31 Stemphylium strains that had been identified morphologically before or after their deposit in the NIAS Genebank, Japan. In the present study, these Japanese strains were separated into three groups (Groups A, C, and E). Sixteen Japanese strains formed a new subgroup (designated Subgroup C2) that contained no type or ex-type strains of existing species. Four of the strains had been identified correctly, whereas the other 27 strains appear to have been misidentified based on our phylogenetic analysis. We examined the conidial morphology of four representative misidentified strains selected from each clade. While the morphological characteristics of a strain isolated from Welsh onion resembles those of the S. vesicarium type, this strain belongs to Subgroup C2 rather than Subgroup C1 and, therefore, could not be identified at the species level. Conversely, three pathogenic strains isolated from aster, asparagus, and Chinese chive were re-identified based on our combined morphological and phylogenetic data. We propose the scientific names of the aster leaf spot pathogen as S. lycopersici, and the asparagus Stemphylium leaf spot and Chinese chive brown leaf blight pathogens as S. herbarum.

Pathogenicity of Stemphylium lycopersici isolated from rotted tobacco seeds on seedlings and leaves

A preemergence damping-off of tobacco (Nicotiana tabacum L.) was found in Fukuoka, Japan, in 2009. A fungus repeatedly isolated from the black-rotted seeds reproduced the symptoms in tobacco seeds, and the preemergence damping-off developed after flower and soil inoculation with the fungus. Brown spots and blight developed on leaves of tobacco seedlings after inoculation with the isolate. The fungus was reisolated from the rotted seeds and blighted leaves. The causal fungus was identified as Stemphylium lycopersici based on morphology and phylogenetic analysis. This is the first report to confirm the pathogenicity of S. lycopersici in tobacco in the world.