Mitsuru Okuda

Characterization of the tufB-secE-nusG-rplKAJL-rpoB Gene Cluster of the Citrus Greening Organism and Detection by Loop-Mediated Isothermal Amplification

Thermal asymmetric interlaced polymerase chain reaction (TAIL-PCR) was performed to amplify the uncharacterized regions adjacent to the nusG-rplKAJL-rpoB gene cluster of citrus greening organism (GO) isolates from different locations in Japan and Indonesia. Conventional PCR was used to amplify the internal nusG-rplKAJL-rpoB gene cluster of these isolates, and the complete sequence of this 6.1-kb fragment was determined. Comparisons with other bacterial sequences showed that the fragment is the tufB-secE-nusG-rplKAJL-rpoB gene cluster. The organization of this gene cluster is similar to that of the homologous cluster found in Escherichia coli. Except for three nucleotide changes, the sequence was identical among Japanese and Indonesian isolates. A loop-mediated isothermal amplification (LAMP) assay based on the conserved sequence of the nusG-rplKAJL-rpoB gene cluster was developed for the detection of the GO. The LAMP product was rapidly detected on nylon membranes by staining with AzurB. LAMP could detect as low as about 300 copies of the nusG-rplKAJL-rpoB fragment of the Japanese and Indonesian isolates of GO. The LAMP-based detection method, which does not depend upon a thermal cycler and electrophoresis apparatus, will be useful for under-equipped laboratories, including those found in extension centers and quarantine offices.

Dynamics of Southern rice black-streaked dwarf virus in rice and implication for virus acquisition.

A novel viral disease of rice caused by Southern rice black-streaked dwarf virus (SRBSDV) has spread throughout East and Southeast Asia since the mid-2000s. Outbreaks of this viral disease occur yearly in southern parts of Japan concurrently with overseas migration of the planthopper vector Sogatella furcifera from southern China during the rainy season (from late June to early July). We examined the dynamics (changes in titer and localization) of SRBSDV on rice using reverse-transcription real-time polymerase chain reaction and determined the relationship between virus titer in plants and virus acquisition by S. furcifera. Under a constant temperature of 27°C, a substantial increase of SRBSDV titer in the leaf sheath together with typical symptoms (stunted growth and twisting of leaf tips) was observed at 20 days after the end of a 7-day exposure of viruliferous S. furcifera. Approximately 40% of S. furcifera acquired SRBSDV through feeding for 5 days on rice plants that were infected following exposure to viruliferous vectors for 10 to 15 days. These results suggest that rice infected by S. furcifera can be a source of SRBSDV before the next generation of S. furcifera emerges.

Host range and complete genome sequence of Cucurbit chlorotic yellows virus, a new member of the genus Crinivirus.

Cucurbit chlorotic yellows virus (CCYV) causes chlorotic yellows on cucumber (Cucumis sativus) and melon (Cucumis melo) and is transmitted by Bemisia tabaci biotype B and Q whiteflies. To characterize the host range of CCYV, 21 cucurbitaceous and 12 other plant species were inoculated using whitefly vectors. All tested Cucumis spp. except Cucumis anguria and Cucumis zeyheri were systemically infected with CCYV, although infection rates varied among species. Citrullus lanatus, Cucurbita pepo, and Luffa cylindrica were susceptible to CCYV; however, the infection rates were low and symptoms were unclear. In addition to the cucurbitaceous plants, Beta vulgaris, Chenopodium amaranticolor, Chenopodium quinoa, Spinacia oleracea, Lactuca sativa, Datura stramonium, and Nicotiana benthamiana were also systemically infected by CCYV. Complete RNA1 and RNA2 were reverse-transcribed, cloned, and sequenced. CCYV RNA1 was found to be 8,607 nucleotides (nt) long and contained four open reading frames (ORFs). The first ORF spanned methyltransferase and RNA helicase domains followed by an RNA-dependent RNA polymerase domain, presumably translated by a +1 ribosomal frameshift. CCYV RNA2 was found to be 8,041 nt long and contained eight ORFs, including the hallmark gene array of the family Closteroviridae. Phylogenetic analysis demonstrated that CCYV was genetically close to Lettuce chlorosis virus, Bean yellow disorder virus, and Cucurbit yellow stunting disorder virus. Amino acid sequence similarities of representative proteins with these viruses indicated that CCYV should be classified as a distinct crinivirus species.

Differentiation of “Candidatus Liberibacter asiaticus” Isolates by Variable-Number Tandem-Repeat Analysis

ABSTRACT Four highly polymorphic simple sequence repeat (SSR) loci were selected and used to differentiate 84 Japanese isolates of “Candidatus Liberibacter asiaticus.” The Neis measure of genetic diversity values for these four SSRs ranged from 0.60 to 0.86. The four SSR loci were also highly polymorphic in four isolates from Taiwan and 12 isolates from Indonesia.

Genetic exchange between two freshwater apple snails, Pomacea canaliculata and Pomacea maculata invading East and Southeast Asia

Two species of apple snails, Pomacea canaliculata and Pomacea maculata (formerly Pomacea insularum), have invaded many countries of East and Southeast Asia from their native range in South America. This study investigated the genetic structure of the two species invading these areas. Phylogenetic analysis based on sequences of the nuclear gene elongation factor 1-alpha (EF1α) detected two well-supported clades (Clade C and Clade M). Both P. canaliculata and P. maculata were represented in each clade. Some snails had both Clade C and Clade M EF1α sequences. These results suggest genetic exchange between snails of the two clades. A mating experiment between P. canaliculata with Clade C EF1α sequences and P. maculata with Clade M EF1α sequences resulted in viable F1 progeny under laboratory conditions. The genetic exchange was also inferred in some populations collected from Argentina, suggesting an existence of hybrid in the native range. Simple identification of EF1α types using a restriction enzyme, ApaLI, detected significant geographical structure of the EF1α variants in the invaded area. The divergent geographical structure could have resulted from either the founder effect or the bridgehead effect, although further genetic analysis is needed to clarify this. Average individual egg weight, which is an indicator of egg size, was higher in P. canaliculata than P. maculata in both field and laboratory reared samples, suggesting that some (probably most) P. canaliculata and P. maculata invading East and Southeast Asia still maintain species-specific populations.

Overwintering viruliferous Frankliniella occidentalis (Thysanoptera: Thripidae) as an infection source of Tomato spotted wilt virus in green pepper fields

Populations of overwintering viruliferous Frankliniella occidentalis were evaluated in Tomato spotted wilt virus (TSWV)-affected green pepper fields in Bungo-Ohno City, Oita Prefecture, Japan. A survey of TSWV-infected weeds showed that the incidence of infection was low in weeds. Stellaria aquatica was infected frequently; however, the infections were considered secondary cases since S. aquatica appeared in the fields around late February to early March. In contrast, TSWV was frequently detected from green pepper fruits until they rotted. F. occidentalis primarily inhabited and reproduced on the green pepper fruits and moved to Lamium amplexicaule when the fruits rotted and subsequently spread to other weed species as young shoots or flowers appeared. The flying activity level of F. occidentalis rose in late February, and viruliferous F. occidentalis transmitted TSWV to green pepper plants. We concluded that TSWV-infected green pepper fruits discarded in greenhouses and fields are the major source of infection.

First Report of Chrysanthemum stem necrosis virus on Chrysanthemums in Japan

The chrysanthemum (Dendranthema grandiflorum), whose planted area comprises more than 6,000 ha in Japan, is one of the most important ornamental cut flower crops. In August 2006, necrotic streaks on stems, chlorotic and necrotic spots and rings on leaves, and leaf distortions were observed on chrysanthemum cvs. Jimba and Seinotama, with a disease incidence of more than 70% (approximately 30,000 plants), which represents approximately 1,000 m2 of greenhouses of one grower in Hiroshima Prefecture, western Japan. Symptoms were similar to those caused by Tomato spotted wilt virus (TSWV) (genus Tospovirus, family Bunyaviridae). Frankliniella occidentalis was the major thrips species observed on symptomatic plants, followed by F. intonsa. Tospovirus-like spherical particles that were 80 to 100 nm in diameter were found in the infected leaves. After mechanical inoculation, a single lesion isolate reproduced the original symptoms observed in nature on healthy chrysanthemum plants (cv. Jimba). As determined by mechanical inoculation, host range and symptomatology of the isolate were similar to those described previously for Chrysanthemum stem necrosis virus (CSNV), including necrotic spots on Petunia hybrida (1). The isolate caused stunting, severe necrotic lesions on stems, necrotic spots, rings, and vein necrosis on systemically infected leaves of Lycopersicon esculentum (cv. House-momotaro). This virus reacted strongly with CSNV antiserum (DSMZ, Braunschweig, Germany) by indirect dot immuno-binding assay, and cross-reacted weakly with a monoclonal antibody to N protein of TSWV (3) using double-antibody sandwich-ELISA. Reverse transcription (RT)-PCR was conducted to verify virus infection. No amplification was observed from extracts of symptomatic plants (n = 10) by multiplex RT-PCR using TSWV and Impatiens necrotic spot virus specific primer sets (4), indicating that the diseased chrysanthemums were not doubly infected with these viruses. However, a DNA fragment of approximately 450 bp was amplified in samples by RT-PCR using tospovirus universal primers, BR60/65 (2). The nucleotide sequence of the amplified fragment had 98.1% identity with the corresponding region of the CSNV nucleocapsid protein gene (GenBank Accession No. AF067068). The above results indicate that the virus associated with a stem necrosis disease of chrysanthemums in Hiroshima is an isolate of CSNV. To our knowledge, this is the first report of CSNV in Japan. References: (1) I. C. Bezerra et al. Phytopathology 89:823, 1999. (2) M. Eiras et al. Fitopatol. Bras. 26:170, 2001. (3) S. Tsuda et al. Ann. Phytopathol. Soc. Jpn. 60:216, 1994. (4) H. Uga and S. Tsuda. Phytopathology 95:166, 2005.

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.

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.

Resistance in melon to Cucurbit chlorotic yellows virus, a whitefly-transmitted crinivirus

Fifty-one melon (Cucumis melo) accessions that originated from India, Pakistan and Bangladesh were evaluated for resistance to Cucurbit chlorotic yellows virus (CCYV), a newly emerged species in the genus Crinivirus. CCYV was inoculated using sweet potato whitefly (Bemisia tabaci) biotype Q. Accessions, JP 138332, JP 216154, JP 216155, JP 216751 and JP 91204, showed no or faint symptoms, although CCYV was detected from the non-inoculated upper leaves by reverse transcription-polymerase chain reaction (RT-PCR). The five accessions were subjected to quantitative RT-PCR to analyze relative accumulation of CCYV RNA. All accessions except JP 138332 had levels of CCYV RNA accumulation comparable to the commercial variety, ‘Earl’s Seine’, which was used as a control. JP 138332 showed a much lower CCYV RNA accumulation. Numbers of B. tabaci biotype Q on JP 138332 did not differ from ‘Earl’s Seine’, in antixenosis tests, and the result suggested the resistance to CCYV was not due to antixenosis. Consequently, five accessions are of interest for development of resistant varieties. In particular, JP 138332 possesses a promising resistant trait for CCYV, which might be associated with inhibition of virus multiplication.