Yosuke Matsushita

Distribution of tomato chlorotic dwarf viroid in floral organs of tomato

In situ hybridization was used to analyze the distribution pattern of Tomato chlorotic dwarf viroid (TCDVd) in floral organs of tomato plants. Following TCDVd invasion of floral organs, it became localized only in sepals at an early developmental stage, then reached other floral organs at the flower opening stage, with the exception of part of the placenta and ovules. When distribution of TCDVd was compared with that of Potato spindle tuber viroid (PSTVd), TCDVd was not detected in the outer integument around the embryo sac even though PSTVd was able to invade there, suggesting that such specific distribution might reflect the frequent occurrence of viroid disease on crops caused by PSTVd-seed transmission.

Distribution of Potato spindle tuber viroid in Reproductive Organs of Petunia During Its Developmental Stages

Embryo infection is important for efficient seed transmission of viroids. To identify the major pattern of seed transmission of viroids, we used in situ hybridization to histochemically analyze the distribution of Potato spindle tuber viroid (PSTVd) in each developmental stage of petunia (flowering to mature seed stages). In floral organs, PSTVd was present in the reproductive tissues of infected female × infected male and infected female × healthy male but not of healthy female × infected male before embryogenesis. After pollination, PSTVd was detected in the developed embryo and endosperm in all three crosses. These findings indicate that PSTVd is indirectly delivered to the embryo through ovule or pollen during the development of reproductive tissues before embryogenesis but not directly through maternal tissues as cell-to-cell movement during embryogenesis.

Host ranges of Potato spindle tuber viroid, Tomato chlorotic dwarf viroid, Tomato apical stunt viroid, and Columnea latent viroid in horticultural plants

Host ranges of Potato spindle tuber viroid (PSTVd), Tomato chlorotic dwarf viroid (TCDVd), Tomato apical stunt viroid (TASVd), and Colmunea latent viroid (CLVd) were investigated in 30 species from 12 genera of horticultural plants that are frequently traded internationally. They were mechanically inoculated, and viroid infection was checked using both ways of reverse-transcription polymerase chain reaction and back-inoculation to tomato. Among 30 species examined, eight to 10 were susceptible to PSTVd, TCDVd, TASVd, and CLVd. Most of these species belong to the Compositae and Solanaceae families, and symptoms were only observed in Calendula officinalis, Petunia × hybrida, and Solanum melongena. These data indicate a risk of unexpected viroid expansion by international trading of vegetatively propagated asymptomatic plant materials.

Root and stem rot of chrysanthemum caused by five Pythium species in Japan

Root and stem rot with wilt of above ground parts of cultivated chrysanthemums was first found in Ibaraki, Toyama and Kagawa prefectures, Japan in 2002 and 2003. Pythium species were isolated from the diseased tissues and identified as P. dissotocum, P. oedochilum, P. sylvaticum, P. ultimum var. ultimum and asexual strains of P. helicoides based on their morphologies and sequences of rDNA-ITS region. All the Pythium species were strongly pathogenic to chrysanthemums in pot conditions and were reisolated from the inoculated plants. Because Pythium root and stem rot of chrysanthemum has never been reported in Japan, we propose that this is a new disease that can be caused by the five Pythium species.

Cutting rot of chrysanthemum (Chrysanthemum morifolium) caused by Plectosporium tabacinum

Cutting rot of chrysanthemum was found on cuttings of cv. Jimba No.2 in 2008. The cuttings were imported, then transplanted in Aichi Prefecture. Root development was not initiated in about 30% of the cuttings. The cut stem ends developed black discolouration and decay. When healthy cuttings were the fungus isolated from diseased cuttings, these cuttings developed the same disease symptoms. The characteristics and morphology of the fungal culture were identical to those of Plectosporium tabacinum. We propose that the new disease be named cutting rot of chrysanthemum.

Characterization of Rehmannia mosaic virus isolated from chili pepper ( Capsicum annuum ) in Japan

In 2010, severe necrotic mosaic disease and fruit distortion were observed on greenhouse-grown chili pepper (Capsicum annuum cv. Fushimi-amanaga) plants in Kyoto Prefecture, Japan. Electron microscopic imaging and genomic RNA sequencing indicated that the virus responsible was a new isolate of Rehmannia mosaic virus (ReMV), which had not been previously reported in Japan. Although ReMV systemically infected many Solanaceae species, including chili pepper and tomato (Solanum lycopersicum), tobamovirus-resistance genes from species of Capsicum (L1a, L2, L3, and L4) and tomato (Tm-1, Tm-2, and Tm-2a) conferred resistance against ReMV.

Field survey of ranunculus mild mosaic virus, tomato spotted wilt virus and cucumber mosaic virus infections in Ranunculus asiaticus L. in Japan by newly developed multiplex RT-PCR

Viral symptoms are frequently observed in production fields of ranunculus (Ranunculus asiaticus L.) in Japan. Based on incidence of diseases caused by a large number of ranunculus-infective viruses, ranunculus mild mosaic virus (RanMMV), tomato spotted wilt virus (TSWV) and cucumber mosaic virus (CMV) infections were the focus of an epidemiological field survey in Japan. To efficiently investigate the incidence and distribution of the three viruses, we first developed a new multiplex reverse transcription-polymerase chain reaction method that enables simultaneous detection of RanMMV, TSWV, and CMV in ranunculus. A field survey of virus infections in ranunculus production fields in Tohoku and Kyushu regions revealed that the infection rate of RanMMV was much higher than that of the other viruses in all fields sampled. Interestingly, the infection rate of RanMMV showed an increasing trend in proportion to the number of vegetative propagation cycles of ranunculus, implying virus transmission by aphids. Taken together, this method was proven to be effective for simultaneous detection of RanMMV, TSWV and CMV in ranunculus plants, and RanMMV was recognized as one of the most prevalent plant viruses in ranunculus fields in Japan.

Downy mildew of coleus caused by Peronospora belbahrii in Japan

In April 2007, coleus (Solenostemon scutellarioides) plants cultivated in a greenhouse in Chiba Prefecture, Japan were observed to have poor growth, with yellowing of the adaxial leaf surface and a gray to black downy growth on the abaxial leaf surface. The causal pathogen was identified as Peronospora belbahrii based on its morphology and rDNA-ITS sequences. Uninfected coleus was inoculated with the pathogen, the original symptoms were reproduced, and the isolate was reisolated. This is the first report of downy mildew of coleus in Japan.

Downy mildew of busy lizzie caused by Plasmopara obducens in Japan

In June 2010 and March 2012, busy lizzie (Impatiens sultanii) plants in a glasshouse in Yamagata Prefecture and in nurseries in a plastic house in Shimane Prefecture, Japan were observed with yellowing of the adaxial surface of leaves or cotyledons and a white downy growth on the abaxial surface of leaves or cotyledons. We identified the causal pathogen as Plasmopara obducens based on the morphologies and rDNA-large subunit sequences. This new disease was named “downy mildew of busy lizzie” in Japan.

Characteristics of viroids infecting horticultural plants

Viroids are the smallest and simplest plant pathogens, consisting of a single-stranded, circular, naked RNA genome,246–401 nucleotides long, lacking any protein-coding sequences. Potato spindle tuber viroid (PSTVd) and Tomato chlorotic dwarf viroid (TCDVd), belonging to the genus Pospiviroid, are two closely related pospiviroids. Tomato plants infected with the viroids are severely stunted, and their fruits are reduced in size, leading to economic losses. International trade may disperse the viroids globally via infected plants and seed. In July 2006, we found TCDVd-infected tomato plants in a farmer’s greenhouse in Hiroshima Prefecture, Japan. We characterized the host range and physical properties of the viroid. Finally, the disease was successfully eradicated from the farmer’s greenhouse by this urgent eradication project. We developed a multiplex RT-PCR for the simultaneous detection of PSTVd and TCDVd and detected PSTVd in field-cultivated tomato plants in Fukushima Prefecture, Japan using this method . This is the first finding of PSTVd in field-grown tomatoes in Japan. Although PSTVd is transmitted at high frequency by contaminated seeds, the molecular biology and histochemistry of this mechanism have not yet been fully analyzed. We therefore used in situ hybridization to analyze any differences in the distribution patterns of PSTVd and TCDVd in floral organs at each developmental stage of tomatoes. When distribution of TCDVd was compared with that of PSTVd, TCDVd was not detected in the outer integument around the embryo sac even though PSTVd was able to invade the outer integument, suggesting that such specific distribution reflects the frequent occurrence of viroid diseases in crops caused by PSTVd transmitted through seeds . Furthermore, we observed the patterns of PSTVd in floral organs after fertilization and mature seeds in each developmental stage of infected petunia, resulting that PSTVd is indirectly delivered to the embryo through the ovule or pollen during the development of reproductive tissues before embryogenesis. These results thus contribute to developing the best practices for producing healthy seeds by the seed industry. Our studies also characterized pospiviroids that infect horticultural plants, contributing to the enhancement of seed inspection for plant quarantine systems and seed companies.