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An increasing number of plant viruses and viroids have been reported from all over the world due largely to metavirogenomics approaches with technological innovation. Herein, the official changes of virus taxonomy, including the establishment of megataxonomy and amendments of the codes of virus classification and nomenclature, recently made by the International Committee on Taxonomy of Viruses were summarized. The continued efforts of the plant virology community of Japan to index all plant viruses and viroids occurring in Japan, which represent 407 viruses, including 303 virus species and 104 unclassified viruses, and 25 viroids, including 20 species and 5 unclassified viroids, as of October 2021, were also introduced. These viruses and viroids are collectively classified into 81 genera within 26 families of 3 kingdoms (Shotokuvirae, Orthornavirae, Pararnavirae) across 2 realms (Monodnaviria and Riboviria). This review also overviewed how Japan’s plant virus/viroid studies have contributed to advance virus/viroid taxonomy.

Rose yellow mosaic virus, which belongs to the Roymovirus genus in the Potyviridae family, was isolated here in Japan from a rose cultivar, Irish Mist. The full-length genomic sequence was similar to a strain in Minnesota, United States, which was the first ever isolated, with 85% nucleotide and 94% amino acid sequence similarities. Unlike the Minnesota strain, which lacks 220 nt including the 6K1 protein region, the Japanese isolate contains this coding region, which is commonly found in those of the Potyviridae.

Shiso (Perilla frutescens var. crispa) is widely grown as an important vegetable or herb crop in Japan. Beginning around the year 2000, occurrences of severe mosaic symptoms on shiso were documented and gradually spread across Kochi Prefecture, one of four major shiso production areas in Japan. Next generation sequencing and cloning indicated the presence of a previously unknown virus related to the members of the genus Emaravirus, for which we proposed the name Perilla mosaic virus (PerMV). The genome of PerMV consists of 10 RNA segments, each encoding a single protein in the negative-sense orientation. Of these proteins, P1, P2, P3a, P3b, P4, and P5 show amino acid sequence similarities with those of known emaraviruses, whereas no similarities were found in P6a, P6b, P6c, and P7. Characteristics of the RNA segments as well as phylogenetic analysis of P1 to P4 indicate that PerMV is a distinct and highly divergent emaravirus. Electron microscopy observations and protein analyses corresponded to presence of an emaravirus. Transmission experiments demonstrated that an eriophyid mite, Shevtchenkella sp. (family Eriophyidae), transmits PerMV with a minimum 30-min acquisition access period. Only plants belonging to the genus Perilla tested positive for PerMV, and the plant−virus−vector interactions were evaluated. The nucleotide sequences reported here are available in the DDBJ/ENA/GenBank databases under accession numbers LC496090 to LC496099.

Two potyviruses, uraria mosaic virus (UMV) and passiflora foetida virus Y (PfVY), have been detected in Japan for the first time, from newly bred passionfruit cultivars. Host range analyses and phylogenetic analyses based on full and partial polyprotein coding regions of UMV and PfVY, respectively, suggested they were distinct from viruses previously isolated from passionfruit. Both species belong to the BCMV subgroup within the genus Potyvirus, which contains other pathogenic passionfruit viruses.

Unfortunately, the inclusion of original names (in non-Latin script) of the following authors caused problems with author name indexing in PubMed. Therefore, these original names were removed from XML data to correct the PubMed record. Mengji Cao, Yuya Chiaki, Hideki Ebihara, Jingjing Fu, George Fú Gāo, Tong Han, Jiang Hong, Ni Hong, Seiji Hongo, Masayuki Horie, Dàohóng Jiāng, Fujio Kadono, Hideki Kondō, Kenji Kubota, Shaorong Li, Longhui Li, Jiànróng Lǐ, Huazhen Liu, Tomohide Natsuaki, Sergey V. Netesov, Anna Papa, Sofia Paraskevopoulou, Liying Qi, Takahide Sasaya, Mang Shi, Xiǎohóng Shí, Zhènglì Shí, Yoshifumi Shimomoto, Jin‑Won Song, Ayato Takada, Shigeharu Takeuchi, Yasuhiro Tomitaka, Keizō Tomonaga, Shinya Tsuda, Changchun Tu, Tomio Usugi, Nikos Vasilakis, Jiro Wada, Lin‑Fa Wang, Guoping Wang, Yanxiang Wang, Yaqin Wang, Tàiyún Wèi, Shaohua Wen, Jiangxiang Wu, Lei Xu, Hironobu Yanagisawa, Caixia Yang, Zuokun Yang, Lifeng Zhai, Yong‑Zhen Zhang, Song Zhang, Jinguo Zhang, Zhe Zhang, Xueping Zhou. In addition, the publication call-out in the supplementary material was updated from issue 11 to issue 12. The original article has been corrected.

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Pospiviroids cause serious economic damage to solanaceous crops, including tomato (Solanum lycopersicum), and cause symptomless infections in many horticultural plants. Symptomless infection and seed transmission are obstacles in visual inspection for plant quarantine. Therefore, in the present study, a comprehensive detection and identification system for molecular diagnosis of eight pospiviroids was developed, based on real-time reverse transcription polymerase chain reaction (RT-PCR). In this system, two real-time RT-PCR methods based on SYBR Green and TaqMan technologies were combined. First, tomato seed samples were screened for all the eight pospiviroids by the SYBR Green method using three primer pairs, including one pair of universal primers. Samples that were positive using the universal primer set were subjected to secondary screening for the identification to species level of six viroids by TaqMan real-time RT-PCR method, with specific primer and probe sets. This two-phase system could specifically detect a viroid in a sample of 400 seeds containing a single contaminated seed. The developed system could be an effective and simple tool to avoid the introduction of pospiviroids through tomato seeds.

A reliable method, based on the double antibody sandwich enzyme-linked immunosorbent assay (DAS-ELISA), was developed for the extraction of viruses from soil and optimized for the detection of Pepper mild mottle virus (PMMoV) in soil taken from green pepper (Capsicum annuum) fields. When added to phosphate buffer, Tween 20 increases extraction efficiency and skim milk increases the specificity for PMMoV. Samples positive by DAS-ELISA were verified by inhibition testing using specific anti-PMMoV antibody, immuno-electron microscopy, reverse transcription-polymerase chain reaction, and inoculation tests on assay plants. Our system for detecting PMMoV in soil was successfully tested on samples from 22 infected and uninfected fields in Japan. When used before seedlings are planted, this method allows for the prediction of possible damage to cultivated plants by soil-borne PMMoV.

Frankliniella cephalica (Crawford) is an invasive species of thrips found in the islands of Yaeyama in the Okinawa Prefecture, Japan. During the late 1990s to early 2000s, a species of thrips was isolated from wild flowers of Bidens pilosa L. and Ipomoea batatas L. growing close to cultivated fields. They were subsequently identified as F. cephalica using fine morphological characteristics with the help of Steve Nakahara (U.S. Department of Agriculture, Beltsville, MD) and Laurence Mound (CSIRO, Australia). Voucher specimens were deposited in the Laboratory of Insect Resources, Faculty of Agriculture, Tokyo University of Agriculture by Shuji Okajima (2). We investigated the ability of F. cephalica to vector Tomato spotted wilt virus (TSWV) by experimentally determining virus transmission efficiency. Newly hatched larvae as much as 12 h old underwent a viral acquisition-access period (AAP) of 24 h, during which they fed on the leaves of Datura stramonium infected with TSWV-O, a Japanese type isolate. Transmission efficiency of adults 4 days after emergence from molt (14 days after the AAP) was determined by a petunia leaf disk assay (3) in which the adults were individually allowed to feed for successive 24-h inoculation access periods (IAP) on two different leaf disks of Petunia × hybrida cv. Polo Blue. Transmission of the virus by the adults was considered positive if at least one of the leaf disks showed viral necrotic spot. We tested 20 randomly selected leaf disks with clear necrotic spots using a simplified rapid immunofilter paper assay. All selected disks were positive for TWSV. The transmission efficiencies were 24.6% for female (n = 57) and 54.4% for male (n = 125) adults. The efficiency was significantly different between sexes (Fisher's exact probability test, P < 0.001). We also examined changes in the virus infection site at different developmental stages in thrips using immunofluorescence microscopy with a polyclonal antibody to N protein of the virus (4). After a 6-h AAP feeding by first instar larvae, the virus was found initially to infect the epithelial cells and then spread throughout the midgut tissue in the second instar larvae 5 days after acquisition of the virus. In viruliferous adults, the virus was present in the salivary glands and on the basement membrane of the midgut tissue. These data indicate that F. cephalica is a new insect vector for TSWV. F. cephalica is a major insect pest of tropical crops in tropical and subtropical coastal belts (1). The presence of a thrips vector in weed hosts surrounding cultivated fields might increase the chance of crops in this habitat becoming infected with viruses. References: (1) M. Lamberts and J. H. Crane. Page 337 in: Advances in New Crops. J. Janick and J. E. Simon, eds. Timber Press, Portland, OR, 1990. (2) M. Masumoto and S. Okajima. Jpn. J. Appl. Entomol. Zool. 48:225, 2004. (3) T. Sakurai et al. Appl. Entomol. Zool. 39:71, 2004. (4) S. Tsuda et al. Ann. Phytopathol. Soc. Jpn. 60:216, 1994.

In 2009, some commercially grown tomato (Solanum lycopersicum) plants in Chiba Prefecture, Japan, exhibited mosaic symptoms. Ten plants from a total of about 72,000 cultivated plants in the greenhouses showed such symptoms. To identify the causal agent, sap from leaves of the diseased plants was inoculated into Chenopodium quinoa and Nicotiana benthamiana plants. Local necrotic lesions appeared on inoculated leaves of C. quinoa, but no systemic infection was observed. Systemic mosaic symptoms were observed on the N. benthamiana plants inoculated. Single local lesion isolation was performed three times using C. quinoa to obtain a reference isolate for further characterization. N. benthamiana was used for propagation of the isolate. Sap from infected leaves of N. benthamiana was mechanically inoculated into three individual S. lycopersicum cv. Momotaro. Symptoms appearing on inoculated tomatoes were indistinguishable from those of diseased tomato plants found initially in the greenhouse. Flexuous, filamentous particles, ~750 nm long, were observed by electron microscopy in the sap of the tomato plants inoculated with the isolate, indicating that the infecting virus may belong to the family Potyviridae. To determine genomic sequence of the virus, RT-PCR was performed. Total RNA was extracted from the tomato leaves experimentally infected with the isolate using an RNeasy Plant Mini kit (QIAGEN, Hilden, Germany). RT-PCR was performed by using a set of universal, degenerate primers for Potyviruses as previously reported (2). Amplicons (~1,500 bp) generated by RT-PCR were extracted from the gels using the QIAquick Gel Extraction kit (QIAGEN) and cloned into pCR-BluntII TOPO (Invitrogen, San Diego, CA). DNA sequences of three individual clones were determined using a combination of plasmid and virus-specific primers, showing that identity among three clones was 99.8%. A consensus nucleotide sequence of the isolate was deposited in GenBank (AB823816). BLASTn analysis of the nucleotide sequence determined showed 99% identity with a partial sequence in the NIb/coat protein (CP) region of Colombian datura virus (CDV) tobacco isolate (JQ801448). Comparison of the amino acid sequence predicted for the CP with previously reported sequences for CDV (AY621656, AJ237923, EU571230, AM113759, AM113754, and AM113761) showed 97 to 100% identity range. Subsequently, CDV infection in both the original and experimentally inoculated plants was confirmed by RT-PCR using CDV-specific primers (CDVv and CDVvc; [1]), and, hence, the causal agent of the tomato disease observed in greenhouse tomatoes was proved to be CDV. The first case of CDV on tomato was reported in Netherlands (3), indicating that CDV was transmitted by aphids from CDV-infected Brugmansia plants cultivated in the same greenhouse. We carefully investigated whether Brugmansia plants naturally grew around the greenhouses, but we could not find them inside or in proximity to the greenhouses. Therefore, sources of CDV inoculum in Japan are still unclear. This is the first report of a mosaic disease caused by CDV on commercially cultivated S. lycopersicum in Japan. References: (1) D. O. Chellemi et al. Plant Dis. 95:755, 2011. (2) J. Chen et al. Arch. Virol. 146:757, 2001. (3) J. Th. J. Verhoeven et al. Eur. J. Plant. Pathol. 102:895, 1996.

Plum pox virus (PPV) is transmitted by infected buds and aphids. It is important to analyze the outbreak trends and viruliferous rate of aphids in areas where the occurrence of PPV is reported, so as to develop strategies for disease control. Between April 2011 and December 2012, yellow insect-trapping adhesive plates were placed for 2 days at a time each week in an area where PPV is occurring in Japan. Outbreak trends were analyzed based on the trapped alate aphid samples, and up to 50 of them were tested per week to identify species and determine the rate of viruliferous specimens. Although the number of aphids varied according to survey year, three peaks were noticeable in each year. Based on the sequence data for the mitochondrial cytochrome c oxidase I region, approximately 40 different species of aphid were trapped in both years. Of the five dominant species of aphids identified during the 2 years, Aphis spiraecola was trapped in large numbers. PPV-positive aphids were higher in fall onward, when the total number of trapped aphids decreased, than in spring and summer, when a larger number of aphids was caught. PPV transmission tests using the most abundant species revealed that A. spiraecola, A. craccivora, A. gossypii, and Rhopalosiphum maidis were transmitters, although A. spiraecola is likely of epidemiological significance.

This paper presents a reduction of artificial bee colony algorithm for global optimization. Artificial bee colony algorithm is an optimization technique which refers to the behavior of honeybee swarms, and a multi-point search approach which finds a best solution using multiple bees. For avoiding local minima, a number of bees are initially prepared and their positions are updated by artificial bee colony algorithm. Bees sequentially reduce to reach a predetermined number of them grounded in the evaluation value and artificial bee colony algorithm continues until the termination condition is met. In order to show the effectiveness of the proposed algorithm, we examine the best value by using test functions compared to existing algorithms. Furthermore the influence of best value on the initial number of bees for our algorithm is discussed.

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.

International trade is one of the main ways by which a species can expand its geographical range. Since the beginning of the twenty-first century, the worldwide distribution of many pests has increased through the international circulation of crop seeds and seedlings. In this review, viruses and viroids that have invaded Japan since 2000 are described in order of their prevalence. Measures to prevent invasion by such viruses and viroids and protect agricultural production in Japan from alien pests are discussed.

Pepper mild mottle virus (PMMoV), which causes mosaic disease of green pepper (Capsicum annuum L.), is transmitted via the soil or sap during cultural practices such as pruning and harvesting. Here we examined the use of the attenuated PMMoV isolate L3-163, which can systemically infect pepper crops carrying the L3 resistance gene, for cross protection. During the greenhouse cultivation of green peppers in commercial production in plastic houses, the L3-163 isolate provided a high-level protection in previously inoculated pepper plants against the wild-type virus infection, resulting in acceptable economic fruit yields compared with non-inoculated control plants. Moreover, the L3-163 isolate was compatible to all of the main pepper cultivars grown in Japan. The level of cross protection (% infection with the attenuated isolate) needed to prevent or minimize economic loss from wild-type PMMoV was from 50% to 75%. These results indicate that the attenuated isolate effectively protected commercially cultivated pepper plants against PMMoV infection, thereby providing an alternative to methyl bromide soil fumigation in Japan.

The chytrid fungus Olpidium bornovanus is an obligate plant parasite that acts as a vector to transmit Melon necrotic spot virus in cultivated soil. Here, we conducted a molecular phylogenetic analysis of 16 isolates of O. bornovanus taken from soil in which Cucurbitaceae plants had been grown in various locations throughout Japan. The ribosomal DNA and internal transcribed spacer region sequences of the 16 O. bornovanus isolates were divided into four molecular phylogenetic groups, designated O.bor-A to O.bor-D. Biological features of O. bornovanus isolates such as host specificity varied consistently with their molecular phylogenetic groups.

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 (L 1a , L 2 , L 3 , and L 4 ) and tomato (Tm-1, Tm-2, and Tm-2 a ) conferred resistance against ReMV.

We investigated what gene(s) in the plant roots have the positive role against repressing root-knot nematode (RKN) infection. We investigated the interaction between RKN infection and gene expression in the plant roots induced by methyl jasmonate (MeJA). We focused on the induced resistance response and the duration after foliar treatment with MeJA of 0.1, 0.5, 1.0, and 5.0mM at 1, 24, 48, and 72h prior to the inoculation of RKN. As a result, the foliar treatment with MeJA at 0.5mM or higher concentrations significantly reduced the infection of RKN in plants and the effect lasted for about 1 week. The repressing effect on RKN population declined to the lowest level in two weeks after MeJA treatment. The expression of proteinase inhibitors (PIs) and multicystatin (MC) were induced while the repressing effect on RKN was valid and a negative correlation was found between the expression of PIs or MC and RKN infection. In addition, when tomato plants no longer expressing MC and PIs were treated again with MeJA, the repressing effect revived. These phenomena appeared to be regardless of the existence of Mi-genes or isolate of RKN. Our results indicate that the expression level of MC and PIs may be effective as marker genes for estimating the induced resistance response against RKN infection.

The location of Pepper mild mottle virus (PMMoV) within seeds as they developed on inoculated seedlings of pepper (Capsicum annuum) was followed over time by detecting the viral coat protein using immunofluorescence microscopy. Seedlings were inoculated with PMMoV when the flower buds on the first and second branching nodes were in bloom. Fluorescence indicating the presence of PMMoV was first observed around immature seeds and placentas in the ovaries on the fourth branching node at 20 days post-anthesis (20 DPA), which corresponded to 39 days post-inoculation (39 DPI). The area with fluorescence gradually expanded from the placenta into the integument and the parenchyma, and finally reached the tip of the immature seeds by 34 DPA (53 DPI). The embryo or endosperm beyond the endothelium never fluoresced during the experiment [i.e., ending at 81 DPA (102 DPI)]. For visualizing viral routes of invasion from seeds into new seedlings, PMMoV-infected C. annuum seeds that were heterozygous for the L 3 tobamovirus-resistance gene were sown in soil at 30°C. After ~2 weeks, the cotyledon developed virally induced necrosis. These findings shed light on the infection cycle of PMMoV through vertical transmission in C. annuum.

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.

Cucurbit chlorotic yellows virus (CCYV), a whitefly-transmitted virus, is a new member of the Crinivirus genus, that causes major economic losses in cucumber, melon and watermelon crops. To develop immunodiagnostic methods for CCYV, we produced an antiserum by immunizing rabbits with bacterially expressed recombinant coat protein of CCYV and detected CCYV from CCYV-infected plants with western blotting and immunoelectron microscopy. CCYV in extracts from infected plants was also readily detected by double antibody sandwich enzyme-linked immunosorbent assay with high sensitivity and specificity. A tissue blot immunoassay indicated that CCYV localizes in the phloem tissues of the petiole and is distributed in tiny spots within the leaf lamina.

We investigated the effectiveness of various chemical or biological agents in disinfecting Tomato chlorotic dwarf viroid (TCDVd), which causes extensive damage to tomato crops. Among seven tested chemicals, sodium hypochlorite (NaOCl) was the most effective in disinfecting TCDVd-contaminated scalpels with a 15-s dipping. TCDVd-contaminated scalpels were effectively disinfected by sodium hypochlorite solution at a concentration of 0.5% or more. Sodium hypochlorite at 0.25% and 0.125% could also reduce rates of infection via contaminated scalpels, suggesting that solutions of more than about 0.2% could also be used to suppress infection via TCDVd-contaminated tools. Low-pH sodium hypochlorite solution, which has powerful oxidizing activity, was found to degrade TCDVd dramatically even at low concentrations (below 0.1%). However, the effectiveness of such low-pH solutions in disinfecting TCDVd decreased to a level similar to that of high-pH solutions in the presence of plant tissue residues on tool surfaces, probably because of a reduction in cleaning activity. Although trisodium phosphate (Na 3 PO 4 ) at a concentration of 5% was effective against TCDVd, a 2.5% solution of this chemical was not sufficiently effective against the viroid. Among the biological agents tested, crude sap of pepper plants significantly suppressed TCDVd infection.

Potato spindle tuber viroid (PSTVd) and Tomato chlorotic dwarf viroid (TCDVd) are two closely related Pospiviroids which cause economically important diseases on tomato (Solanum lycopersicum). Until now, however, there have been no molecular diagnostic methods available for discriminating between them except sequencing. We have developed a multiplex reverse transcription-polymerase chain reaction (RT-PCR) system that simultaneously detects and discriminates between both viroids in one reaction. Using this system, amplified cDNAs resulted in a 271 bp PCR product when PSTVd is detected as the template and 191 bp when TCDVd is detected. This multiplex RT-PCR system was used to accurately detect both viroids in field cultivated tomato and petunia (Petunia × hybrida) plants. This is the first finding of PSTVd in field grown tomatoes in Japan.

We improved a one-step multiplex reverse transcription-polymerase chain reaction (multi-PCR) method previously published for the simultaneous identification of five tospovirus species that mainly occur on Solanaceae and Compositae plants. The multi-PCR system is composed of a universal degenerate primer and five virus-species-specific primers that amplify bands unique to Iris yellow spot virus (837 bp), Tomato spotted wilt virus (709 bp), Impatiens necrotic spot virus (589 bp), Chrysanthemum stem necrosis virus (485 bp), and Capsicum chlorosis virus (366 bp). This technique is potentially useful for epidemiological studies of several tospoviruses that have recently been detected in previously uninfected fields of ornamental crops.

Quantitative PCR revealed that Tomato chlorotic dwarf viroid (TCDVd) was present in substantial amounts in viroid-infected tomato flowers. Healthy tomato plants were arranged in two different glasshouses, and plants were mechanically inoculated with TCDVd. Bumblebees (Bombus ignitus) were then introduced into the glasshouses to reveal whether the viroid was transmitted from infected source plants to neighbouring healthy plants. TCDVd infection was found in neighbouring tomato plants more than 1 month after the introduction of the bees, some of which expressed symptoms, in both glasshouses. Thus, bumblebees transmitted TCDVd from tomato to tomato by pollination activities.

The L11Y strain of Tomato mosaic virus (ToMV) causes severe chlorosis on infected tobacco leaves. Sequencing analysis for the genome showed that L11Y contained multiple nucleotide changes and that some led to amino acid substitutions, when compared with that of the common L strain of ToMV. The chimeric virus, which has the CP of L11Y in the context of the L strain RNA genome, caused severe chlorosis on infected tobacco plants, suggesting that the CP of L11Y containing three amino acid changes (E33S, A86T and E97K) was the determinant of the chlorosis. Two of these amino acid changes (A86T and E97K) were associated with the induction of chlorosis when present together in the CP. Severe destruction and deformation of chloroplasts and the formation of discrete dark-staining materials adjacent to chloroplasts were observed with electron microscopy in L11Y-infected plants. Fewer virus particles accumulated in the cytoplasm of L11Y-infected plant cells. The level of accumulation of CP subgenomic RNA and CP in the infected protoplasts was similar between L and L11Y. Fewer virus particles accumulated in L11Y-infected protoplasts, and many of them were shorter-than-full-length.

We characterised the host range and physical properties of Tomato chlorotic dwarf viroid. Among the 46 plant species inoculated with the viroid, two in the family Compositae and 23 in the family Solanaceae were found to be systemic hosts. The viroids in the crude sap from diseased tomato plants were thermally inactivated by heating to 100°C for at least 40 min. These viroids also lost their infectivity when diluted in phosphate buffer to at least 10−6, or after 3 days of incubation at room temperature. However, the infectivity of the viroids in dried crude sap from the plants persisted throughout the 50-day test period.

We report a new strain of Melon necrotic spot virus (MNSV) that is unable to systemically infect Cucumis melo. A spherical virus (W-isolate), about 30 nm in diameter like a carmovirus, was isolated from watermelons with necrotic symptoms. The W-isolate had little serological similarity to MNSV, and it did not cause any symptoms in six melon cultivars susceptible to MNSV; however, the host range of the W-isolate was limited exclusively to cucurbitaceous plants, and transmission by O. bornovanus was confirmed. Its genomic structure was identical to that of MNSV, and its p89 protein and coat protein (CP) showed 81.6 to 83.2% and 74.1 to 75.1% identity to those of MNSV, respectively. Analysis of protoplast showed that the W-isolate replicated in melons at the single-cell level. Furthermore, chimeric clones carrying the CP of MNSV induced necrotic spots in melons. These results suggested that the absence of symptoms in melons was due to a lack of ability of the W-isolate to move from cell to cell. In view of these findings, we propose that the new isolate should be classified as a novel MNSV watermelon strain.

Mosaic is a common disease symptom caused by virus infection in plants. Mosaic leaves of Tomato mosaic virus (ToMV)-infected tobacco plants consist of yellow-green and dark green tissues that contain large and small numbers of virions, respectively. Although the involvement of RNA silencing in mosaic development has been suggested, its role in the process that results in an uneven distribution of the virus is unknown. Here, we investigated whether and where ToMV-directed RNA silencing was established in tobacco mosaic leaves. When transgenic tobaccos defective in RNA silencing were infected with ToMV, little or no dark green tissue appeared, implying the involvement of RNA silencing in mosaic development. ToMV-related small interfering RNAs were rarely detected in the dark green areas of the first mosaic leaves, and their interior portions were susceptible to infection. Thus, ToMV-directed RNA silencing was not effective there. By visualizing the cells where ToMV-directed RNA silencing was active, it was found that the effective silencing occurs only in the marginal regions of the dark green tissue (∼0.5 mm in width) and along the major veins. Further, the cells in the margins were resistant against recombinant potato virus X carrying a ToMV-derived sequence. These findings demonstrate that RNA silencing against ToMV is established in the cells located at the margins of the dark green areas, restricting the expansion of yellow-green areas, and consequently defines the mosaic pattern. The mechanism of mosaic symptom development is discussed in relation to the systemic spread of the virus and RNA silencing.

Melon necrotic spot virus (MNSV) is transmitted by the fungus Olpidiumbornovanus. In this study, we used immunofluorescence microscopy to detect MNSV particles over the entire surface of the O. bornovanus zoospore; MNSV particles were not detected on the related fungus O. virulentus, which cannot transmit MNSV. The amino acid substitution Ile → Phe at position 300 in the MNSV coat protein resulted in loss of both specific binding and fungal transmission, while virion assembly and biological aspects were unaffected. Taken together, these results suggest that the MNSV coat protein acts as a ligand to the O. bornovanus zoospore as part of a fungal-vector transmission system.

Kido, K., Tanaka, C., Mochizuki, T., Kubota, K., Ohki, T., Ohnishi, J., Knight, L. M., and Tsuda, S. 2008. High temperatures activate local viral multiplication and cell-to-cell movement of Melon necrotic spot virus but restrict expression of systemic symptoms. Phytopathology 98:181-186. The infection of melon plants by Melon necrotic spot virus (MNSV) and the development of necrotic disease symptoms are a seasonal occurrence in Japan, which take place between winter and early summer, but not during mid-summer. In this paper we investigate the effect of three different temperatures (15, 20, and 25°C) on the local and systemic expression of MNSV in melon plants. Previously, the incidence of plants expressing systemic symptoms caused by MNSV and other viruses was found to be greater at temperatures less than 20°C. In this study, our temperature-shift experiments support previous studies that found the expression of systemic symptoms increases as temperature falls from 25 to 20°C and decreases as temperature rises from 20 to 25°C. However, MNSV replication in melon cells and local viral movement within leaves following the inoculation of melon protoplasts or cotyledons were more frequent at 25°C than at 15 or 20°C.

Considerable losses in pepper production by Tomato spotted wilt virus (TSWV) have been reported worldwide. In breeding programs, an assay for resistance that accurately estimates field occurrence of TSWV during pepper production is critical because the virus is vector transmitted. Here, we establish an assay system of TSWV-resistant Capsicum spp. using insect-mediated inoculation within an acrylic chamber in which environmental conditions such as temperature, light intensity, and nutrient supply are controlled. This chamber enables transmission of TSWV from viruliferous plants to plants used in the resistance assay with Frankliniella occidentalis safely, quickly, and precisely.

The single recessive gene, nsv, which confers resistance against Melon necrotic spot virus (MNSV), has recently been used to develop virus-resistant melon cultivars in Japan. However, the Chiba isolate of MNSV, a common isolate in Japan, infected resistant cultivars when inoculated melon plants were grown at 15°C. Viral RNAs accumulated in protoplasts from resistant cultivars at both 15 and 20°C. Mechanical inoculation of the cotyledons caused MNSV to spread throughout the leaves at 15°C, but not at 20°C. These results support our novel hypothesis that a temperature-sensitive inactivation of disease resistance genes occurs at the nsv locus in melon cultivars with the resistance gene grown at temperatures below 20°C.

To determine the pathogenicity domain and to apply cross protection, Pepper mild mottle virus (PMMoV) point-mutations in the replicase (REP) gene between the methyl-transferase and helicase domains, and deletions truncating pseudoknots in the 3′ non-coding region (NCR), were constructed. Some mutants substituting a single amino acid in REP residue 348 exhibited mild symptoms in Nicotiana benthamiana or pepper plants. Accumulation of these mutants was higher than that of other REP mutants or wild-type PMMoV. Deletion mutants in the 3′ NCR pseudoknot showed the lowest virus replication and accumulation among the mutants tested. Six attenuated mutants, which combined 3′ NCR deletions and single or double REP substitution mutations were constructed to investigate cross protection effects on pepper plants. All six of the attenuated mutants showed milder symptom development than wild-type virus. These results suggest that REP and the pseudoknot in the 3′ NCR are major pathogenicity determinants of the virus, and engineered PMMoV attenuated mutants can be useful for protection against the virus in pepper plants.

Alfalfa mosaic virus (AMV), Cucumber mosaic virus (CMV), Potato virus Y (PVY), Tomato bushy stunt virus nipplefruit strain (TBSV-Nf), and an unknown spherical virus were isolated from nipplefruit (Solanum mammosum) cultivated in Chiba Prefecture, Japan. The spherical virus was identified as Grapevine Algerian latent virus nipplefruit strain (GALV-Nf) from the genus Tombusvirus, based on its physical properties, serological relationships, and analysis of genomic RNA. The genomic RNA of GALV-Nf is 4731 nucleotides long and encodes five open reading frames as well as those of other tombusviruses. Nipplefruit infected with GALV-Nf had severe stunting, leaf deformation, and clear mosaic symptoms. This is the first report of an isolation of GALV in Japan.

Two homologous Nicotiana tabacum genes NtTOM1 and NtTOM3 have been identified. These genes encode polypeptides with amino acid sequence similarity to Arabidopsis thaliana TOM1 and TOM3, which function in parallel to support tobamovirus multiplication. Simultaneous RNA interference against NtTOM1 and NtTOM3 in N. tabacum resulted in nearly complete inhibition of the multiplication of Tomato mosaic virus and other tobamoviruses, but did not affect plant growth or the ability of Cucumber mosaic virus to multiply. As TOM1 and TOM3 homologues are present in a variety of plant species, their inhibition via RNA interference should constitute a useful method for generating tobamovirus-resistant plants.

An enhanced attenuated strain of Pepper mild mottle virus (PMMoV) was constructed by incorporating mutations that affect viral attenuation from three reported attenuated strains of PMMoV, which causes serious economic losses in the production of green pepper in Japan. The new strain caused no symptoms on pepper plants and protected them from infection by a wild-type strain. The mutations responsible for viral attenuation were located in the intervening region (IR) of the 126-kDa/183-kDa proteins. The mutations had synergistic effects in terms of the attenuation of symptoms and decreased the accumulation of the viral coat protein in infected pepper plants. In this paper, we propose an efficient method for the improvement of attenuated viruses by reverse genetics in plant viruses.

To clarify the mechanism of seed transmission of Pepper mild mottle virus (PMMoV), the virus was immunolocalized in Capsicum annuum seeds using fluorescence microscopy. Two distinct patterns were observed: In the first, PMMoV was present in the epidermis and parenchyma but not in the endosperm or embryo; in the second, the virus was restricted to the surface of the epidermis and parenchyma. These findings shed light on the fundamental mechanisms of seed transmission of tobamoviruses and may aid in the design of new methods to prevent the spread of seedborne virus diseases.

A one-step reverse transcription-polymerase chain reaction (RT-PCR) method has been developed for the simultaneous detection and identification of multiple tospoviruses that infect plants. The RT-PCR system is composed of six primers in a single tube: a universal degenerate primer and five virus species-specific primers. Amplifications resulted in an 848-bp PCR product for Watermelon silver mottle virus, 709-bp for Tomato spotted wilt virus, 589-bp for Impatiens necrotic spot virus, 511-bp for Melon yellow spot virus, and a 459-bp amplicon for Iris yellow spot virus. This system enables the simultaneous detection of at least three types of tospovirus infections, in addition to their species identities, from five possible tospoviruses studied, on the basis of their S RNA combinations. This multiplex RT-PCR system was applied to the detection of tospovirus in ornamental crops cultivated in fields and shows potential for epidemiological studies.

Posttranscriptional gene silencing (PTGS), a homology-dependent RNA degradation system, has a role in defending against virus infection in plants, but plant viruses encode a suppressor to combat PTGS. Using transgenic tobacco in which the expression of green fluorescent protein (GFP) is posttranscriptionally silenced, we investigated a tomato mosaic virus (ToMV)-encoded PTGS suppressor. Infection with wild-type ToMV (L strain) interrupted GFP silencing in tobacco, coincident with visible symptoms, whereas some attenuated strains of ToMV (L 11 and L 11 A strains) failed to suppress GFP silencing. Analyses of recombinant viruses containing the L and L 11 A strains revealed that a single base change in the replicase gene, which causes an amino acid substitution, is responsible for the symptomless and suppressor-defective phenotypes of the attenuated strains. An agroinfiltration assay indicated that the 130K replication protein acts as a PTGS suppressor. Small interfering RNAs (siRNAs) of 21 to 25 nucleotides accumulated during ToMV infection, suggesting that the major target of the ToMV-encoded suppressor is downstream from the production of siRNAs in the PTGS pathway. Analysis with GFP-tagged recombinant viruses revealed that the suppressor inhibits the establishment of the ToMV-targeted PTGS system in the inoculated leaves but does not detectably suppress the activity of the preexisting, sequence-specific PTGS machinery there. Taken together, these results indicate that it is likely that the ToMV-encoded suppressor, the 130K replication protein, blocks the utilization of silencing-associated small RNAs, so that a homology-dependent RNA degradation machinery is not newly formed.