Tobamoviruses that infect cucurbits are significant pathogens in crops of the Cucurbitaceae family, spreading through seed-borne, mechanical, and soil-transmission methods. The cucumber green mottle mosaic virus (CGMMV) and the kyuri green mottle mosaic virus (KGMMV) have been prevalent in Japan since the 1960s. In contrast, the zucchini green mottle mosaic virus (ZGMMV) has only been reported in South Korea and China. To understand the potential impact of ZGMMV on cucurbit production in Japan, we compared the systemic symptoms and virus quantities in infected plants across four cucurbit crops: cucumber, melon, watermelon, and zucchini. Each of these crops was inoculated with each virus. All the viruses caused systemic infections. However, KGMMV generally induced the most severe leaf symptoms and showed the highest accumulation of the coat protein followed by ZGMMV and CGMMV in terms of symptom severity and protein accumulation. These findings, although derived from a small-scale, short-term experiment, could be instrumental in assessing the risk these tobamoviruses pose to cucurbit production.

In Japan, only one satsuma dwarf virus (SDV) isolate has been fully sequenced to date. This study provides complete genome sequences of six SDV isolates for which only partial sequences had been determined and three for which no sequence data were available. Among all known isolates, nucleotide sequence identity was found to range from 67.9 to 98.6% for RNA1 and from 67.9 to 98.8% for RNA2. The amino acid sequence identity in the coat protein (CP) ranged from 76.9 to 99.2%. Phylogenetic analysis revealed that five of the nine newly sequenced isolates clustered with the citrus mosaic virus (CiMV) strain, two with the SDV strain, and one each with the navel orange infectious mottling virus (NIMV) and hyuganatsu virus (HV) strains.

Viroids inhibit the development of crown gall caused by tumorigenic Allorhizobium/Rhizobium strains in chrysanthemum and tomato plants, demonstrating the microbial interkingdom interactions of viroids in plants. This is the first report of viroids suppressing a bacterial disease.

Since its first occurrence in Israel and Jordan in 2014 and 2015, the tomato brown rugose fruit virus (ToBRFV) has become one of the most concerning pathogens affecting tomatoes and other crops worldwide. Its rapid spread is believed to result from the international trade of contaminated seeds and its seed transmissibility, underscoring the critical importance of seed health testing for ToBRFV to prevent further dissemination of the virus. To this end, reverse transcription-quantitative polymerase chain reaction (RT-qPCR) protocols employing TaqMan probe chemistry have been widely adopted. However, the development of RT-qPCR protocols for ToBRFV seed testing using SYBR Green chemistry remains limited. The SYBR Green method offers the advantage of distinguishing ToBRFV from other tobamoviruses through melt curve analysis. In this study, we developed a SYBR Green-based RT-qPCR detection method using newly designed primer sets, which demonstrated high specificity for ToBRFV and sufficient sensitivity. While this protocol requires further optimization and validation for application in routine seed testing, it establishes a foundational approach for SYBR Green-based RT-qPCR seed testing. Additionally, this study raises an important question regarding the relationship between RT-qPCR results in seed tests and the likelihood of virus contamination or transmission via seeds.

Potato spindle tuber viroid (PSTVd) is a small, single-stranded, and circular RNA molecule and is an important pathogen that significantly affects the production of tomato and potato. PSTVd is transmitted by seeds in tomato, potato and petunia; therefore, seed testing for PSTVd in these crops is necessary to prevent the spread of PSTVd to the uncontaminated regions or countries. Eggplant is also a host plant for PSTVd, and its seeds are widely produced and internationally traded; therefore, these seeds can spread PSTVd contamination. However, seed transmission of PSTVd in eggplants has not yet been reported. In this study, we investigated whether PSTVd is seed-transmitted in commercial eggplant cultivars and the World Eggplant Core (WEC) collection. Seed transmission to germinated eggplant seedlings varied among the progeny of infected plants, ranging between 0–64

Viroids, one of the smallest known infectious agents, induce symptoms of varying severity, ranging from latent to severe, based on the combination of viroid isolates and host plant species. Because viroids are transmissible between plant species, asymptomatic viroid-infected plants may serve as latent sources of infection for other species that could exhibit severe symptoms, occasionally leading to agricultural and economic losses. Therefore, predicting the symptoms induced by viroids in host plants without biological experiments could remarkably enhance control measures against viroid damage. Here, we developed an algorithm using unsupervised machine learning to predict the severity of disease symptoms caused by viroids (e.g., potato spindle tuber viroid; PSTVd) in host plants (e.g., tomato). This algorithm, mimicking the RNA silencing mechanism thought to be linked to viroid pathogenicity, requires only the genome sequences of the viroids and host plants. It involves three steps: alignment of synthetic short sequences of the viroids to the host plant genome, calculation of the alignment coverage, and clustering of the viroids based on coverage using UMAP and DBSCAN. Validation through inoculation experiments confirmed the effectiveness of the algorithm in predicting the severity of disease symptoms induced by viroids. As the algorithm only requires the genome sequence data, it may be applied to any viroid and plant combination. These findings underscore a correlation between viroid pathogenicity and the genome sequences of viroid isolates and host plants, potentially aiding in the prevention of viroid outbreaks and the breeding of viroid-resistant crops.

Since the first report of the tobamovirus tomato brown rugose fruit virus (ToBRFV) in 2014, it has become globally distributed. Its rapid spread has been primarily attributed to seed-borne transmission. Here, the seed-borne nature of ToBRFV transmission was investigated in different cultivars of tomato, bell pepper, and eggplant. In situ hybridization to localize the virus in reproductive organs of ToBRFV-infected tomato plants revealed that the virus was not present in shoot apices, flower buds, or in ovules during flower opening, indicating the virus may be restricted to the outer integument and transported in the vascular bundles during seed development. However, during early fruit development, the virus was present in the integuments in the ovule. Seeds of tomato cultivars with or without tobamovirus resistance gene Tm-2 2 transmitted the virus to the progeny seedlings at rates that reflected the ineffectiveness of the gene against ToBRFV. Seeds of bell peppers transmitted ToBRFV at higher rates than tomato seeds, but a bell pepper cultivar that has resistance gene L 3 was not systemically infected, and its seeds did not harbor the virus. Three eggplant cultivars were systemically infected with ToBRFV but without showing any obvious symptoms, and even though ToBRFV was present in their seeds, the seedlings were not infected. ToBRFV was detected in the seed coats of contaminated tomato and bell pepper seeds, but not in eggplant seed coats. These results indicate mechanistic differences in seed-borne transmission among the three Solanaceae crops.

The tobamovirus, tomato brown rugose fruit virus (ToBRFV), is a significant concern in global tomato production due to the ineffectiveness of the widely used Tm-22 resistance gene. Our previous study showed that the tomato variety GCR237, a Tm-1 homozygote, resisted an Israeli isolate of ToBRFV (DSMZ PV-1241) for up to 35 days post inoculation (dpi), suggesting Tm-1-homozygous cultivars could control ToBRFV. In the present study, we inoculated GCR237 plants with ToBRFV and cultivated them for a longer period of time. The plants resisted systemic infection up to 50 dpi, but mosaic symptoms appeared on the upper leaves by 100 dpi. We retrieved the virus from symptomatic leaves and established four single local lesion isolates. These isolates had several amino acid (AA) substitutions in the helicase domain of 126-kDa/183-kDa replication proteins, where the Tm-1 protein likely binds to inhibit viral RNA replication. Back-inoculating these isolates onto GCR237 plants confirmed they had acquired the ability to overcome GCR237’s resistance and induced mosaic symptoms as early as 14 dpi. About 90

Grapevine crown gall (GCG) is a significant bacterial disease caused by tumorigenic Allorhizobium vitis (TAV) and is prevalent worldwide. TAV infects grapevines through wounds such as freezing injuries. Although grapevines typically avoid being wounded under snow cover, GCG occurs in many commercial vineyards in snowy regions. This study investigated the TAV population in GCG gall tissues, grapevine skins, and snow on grapevine skins from six infected vineyards located in Hokkaido, Japan, an area known for heavy snowfall. TAV was isolated not only from gall tissues but also from skins and snow on skins throughout the year. Hierarchical Bayesian model (HBM) analysis revealed that the number of TAV cells in gall tissues was affected by cultivar and low temperature, while those in skins were affected by location and low temperature. Additionally, Bayesian changepoint detection (BCD) showed that the number of TAV cells in gall and skin tissues increased during winter, including the snowfall season. Furthermore, the TAV population in grapevine skins under the snow was significantly higher than those above the snow, indicating that TAV under the snow is protected by the snow and can survive well during the snowfall season. This study highlights the ability of TAV to overwinter on/in galls and skins under the snow and act as inoculum for the next season.

Plant viruses induce various disease symptoms that substantially impact agriculture, but the underlying mechanisms of viral disease in plants are poorly understood. Kobu-sho is a disease in gentian that shows gall formation with ectopic development of lignified cells and vascular tissues such as xylem. Here, we show that a gene fragment of gentian Kobu-sho-associated virus, which is designated as Kobu-sho-inducing factor (KOBU), induces gall formation accompanied by ectopic development of lignified cells and xylem-like tissue in Nicotiana benthamiana. Transgenic gentian expressing KOBU exhibited tumorous symptoms, confirming the gall-forming activity of KOBU. Surprisingly, KOBU expression can also induce differentiation of an additional leaf-like tissue on the abaxial side of veins in normal N. benthamiana and gentian leaves. Transcriptome analysis with Arabidopsis thaliana expressing KOBU revealed that KOBU activates signaling pathways that regulate xylem development. KOBU protein forms granules and plate-like structures and co-localizes with mRNA splicing factors within the nucleus. Our findings suggest that KOBU is a novel pleiotropic virulence factor that stimulates vascular and leaf development.IMPORTANCEWhile various mechanisms determine disease symptoms in plants depending on virus-host combinations, the details of how plant viruses induce symptoms remain largely unknown in most plant species. Kobu-sho is a disease in gentian that shows gall formation with ectopic development of lignified cells and vascular tissues such as xylem. Our findings demonstrate that a gene fragment of gentian Kobu-sho-associated virus (GKaV), which is designated as Kobu-sho-inducing factor, induces the gall formation accompanied by the ectopic development of lignified cells and xylem-like tissue in Nicotiana benthamiana. The molecular mechanism by which gentian Kobu-sho-associated virus induces the Kobu-sho symptoms will provide new insight into not only plant-virus interactions but also the regulatory mechanisms underlying vascular and leaf development.

Potato spindle tuber viroid (PSTVd) is an important pathogen that severely affects tomato and potato production. Several Solanaceae species are naturally and experimentally susceptible to PSTVd, implying that Solanaceae weeds can be a reservoir of PSTVd through infected seeds in crop fields. To determine whether PSTVd is seed-transmissible in solanaceous weed species, we examined seed transmission rates and localization of PSTVd in the reproductive organs of Solanum nigrum and Physalis angulata. The results revealed that the seed transmission rate of PSTVd was 0

Viruses that infect Ranunculus asiaticus L., a globally important greenhouse ornamental, have consistently caused serious damage in Japan. We coupled nextgeneration-sequencing (NGS) with visual assessment of symptoms on leaf tissues, and found almost all of the diseases were caused by ranunculus mild mosaic virus (RanMMV) and ranunculus leaf distortion virus (RanLDV). These results were confirmed by reverse transcription-polymerase chain reaction specific to viral genes of ranunculus-infecting viruses. NGS with symptom assessment should be able to provide a rapid and practical diagnostic method focusing on RanMMV and RanLDV in Japan.

Chrysanthemum stunt viroid (CSVd) is an important pathogen that decreases dahlia yields due to stunting symptoms. Here, we evaluated the effect of nucleotide sequence of CSVd, cultivation conditions, inoculum type and infection timing on CSVd-infected dahlia growth. Inoculation with CSVd after planting resulted in reduced plant height in the second cut flower, but not the first, indicating that the infection may affect the growth of dahlias after approximately 3-5 months. Additionally, there was no significant difference in the degree of symptom severity among the plants infected with the two types of inoculum, synthesized CSVd RNA and CSVd-infected plant sap. All three CSVd variants affected the growth of dahlias. The reduction in plant height was almost similar among the three variants. However, the reduction in plant fresh weight and compound leaf size in variant JP1 was significantly higher than that in the other variants in the spring experiment. The rates of blown centre in JP1 were also higher than those in the other variants in the spring and winter experiments under the 14.5-h photoperiod. These results suggest that JP1 is a more severe variant than JP2 and JP3. Dark period interruption improved the reduction in plant height and weight and flower weight, as well as the rate of blown centre in CSVd-infected dahlias in the winter experiment compared with the 14.5-h photoperiod. Our results indicate that the three CSVd variants incite stunting symptoms in dahlias and nucleotide sequence of CSVd, photoperiod and infection timing affect the degree of symptom severity.

Crown gall is a globally distributed and economically important disease of grapevine and other important crop plants. The causal agent of grapevine crown gall is tumorigenic Allorhizobium vitis (Ti) strains that harbor a tumor-inducing plasmid (pTi). The epidemic of grapevine crown gall has not been widely elucidated. In this study, we investigated the genetic diversity of 89 strains of Ti and nonpathogenic A. vitis to clarify their molecular epidemiology. Multi-locus sequence analysis (MLSA) of the partial nucleotide sequences of pyrG, recA, and rpoD was performed for molecular typing of A. vitis strains isolated from grapevines with crown gall symptoms grown in 30 different vineyards, five different countries, mainly in Japan, and seven genomic groups A to F were obtained. The results of MLSA and logistic regression indicated that the population of genetic group A was significantly related to a range of prefectures and that the epidemic of group A strains originated mainly in Hokkaido in Japan through soil infection. Moreover, group E strains could have been transported by infected nursery stocks. In conclusion, this study indicates that both soil infection and transporting of infected nursery stocks are working as infection source in Hokkaido.

To investigate the current status of viroid infection in potato fields in Russia, potato spindle tuber viroid (PSTVd) and chrysanthemum stunt viroid (CSVd) were detected in potato (Solanum tuberosum) and S. nigrum plants growing in the Volga (V), North-Western (NW), and Far Eastern (FE) federal districts in Russia. While both PSTVd and CSVd were detected in potato samples from the V and FE regions, only CSVd was detected in potato samples from NW. CSVd was detected in S. nigrum from FE, and this was the first detection of CSVd in S. nigrum. Analysis of the complete genomes of 30 PSTVd and 10 CSVd variants revealed that 5 and 7 PSTVd variants from V and FE, respectively, and 2, 4, and 3 CSVd variants from V, NW, and FE, respectively, were new. Analysis of the infectivity of and symptoms caused by 15 Russian PSTVd isolates in tomato revealed that 14 PSTVd isolates caused intermediate or severe symptoms, and only FP14 caused mild symptoms. Our survey revealed that most of the Russian PSTVd isolates from V and FE caused intermediate symptoms in tomato, and the genome sequences were identical to that of PSTVd isolated from S. demissum. Phylogenetic analysis of nucleotide sequences of the Russian PSTVd variants showed their close relationship to previously studied PSTVd isolates, suggesting that most strains in Russia have not substantially changed genetically, but have widely spread in potato fields.

Potato viral disease has been a major problem in potato production worldwide including Russia. Here, we detected Potato Virus M (PVM), P (PVP), S (PVS), Y (PVY), and X (PVX) and Potato Leaf Roll Virus (PLRV) by RT-PCR on potato leaves and tubers from the Northwestern (NW), Volga (VF), and Far Eastern (FE) federal districts of Russia. Each sample was co-infected with up to five viruses. RT-PCR disclosed all six viruses in NW, three in VF, and five in FE. Phylogenetic analyses of PVM and PVS strains resolved all PVM isolates in Group O (ordinary) and all PVS isolates in Group O. Seven PVY strains were detected, and they included only recombinants. PVY recombinants were thus the dominant potato virus strains in Russia, although they widely varied among the regions. Our research provides insights into the geographical distribution and genetic variability of potato viruses in Russia.

Chrysanthemum stunt viroid (CSVd) is a disease agent caused by a viroid from the genus Pospiviroid. The CSVd pathogen infects chrysanthemum plants and causes severe symptoms of stunting and significant yield loss in cultivation. Although many studies have focussed on this issue, the mechanism of CSVd-infection remains to be clarified. In particular, the effects of CSVd on the underground parts of infected plants are not yet fully understood in Chrysanthemum. Here, we investigated whether the effects of CSVd on the root formation of infected plants varies among cultivars and wild Chrysanthemum species, C. morifolium and C. seticuspe, using transcribed RNA and in vitro assays. Furthermore, to compare infected plants with uninoculated plants, we assessed the symptoms and measured the CSVd RNA accumulation levels in several tissues using RT-qPCR. Our findings indicated that CSVd-infected plants exhibited severe root formation symptoms in both C. morifolium and C. seticuspe. RT-qPCR revealed CSVd accumulation in the root tips and similar patterns of CSVd accumulation in each organ of C. morifolium and C. seticuspe. We propose that in vitro assays are useful for determining root formation in CSVd-infected chrysanthemum plants. Such investigations are necessary to better understand CSVd infection in Chrysanthemum species and plant defences against such diseases.

Chrysanthemum stunt viroid (CSVd) is known to infect a wide range of Dahlia spp; however, to the best of our knowledge, no detailed study has been conducted on the distribution of CSVd variants, the infection efficacy of sap inoculations, and the symptoms caused by infection of CSVd in dahlias. We have detected CSVd in 94 of 214 dahlia samples, from across eight different Japanese Prefectures, using RT-PCR. Six sequence variants of CSVd were detected from 32 CSVd-infected dahlia samples including 3 variants which have not been previously reported and three previously isolated variants from dahlias or chrysanthemums. Five CSVd variants infected and replicated in both dahlia and chrysanthemum plants by sap and RNA inoculation three months post inoculation. The sequence mutations among those variants did not significantly affect the infection efficacy. Infection of CSVd significantly reduced fresh plant weight, plant height, size of compound leaves, flower diameter, and fresh flower weight of dahlias in two cultivars, though it did not affect the number of nodes. In addition, it altered the color of ray florets and increased the rate at which flowers exposed their disks and that of disk florets. The altered color of ray florets in the 'Magokoro' cultivar were darker pink and those of the 'Portlight pair beauty' cultivar were darker orange than the uninfected samples. Our results show that CSVd infects a wide range of dahlias in the field and is an important pathogen to manage for their cultivation as it negatively affects their productivity.

A pospiviroid, detected from asymptomatic potato plants (Solanum tuberosum) from Russia, was identified as chrysanthemum stunt viroid (CSVd). The genomic sequence of this potato isolate showed 100% identity with that of the chrysanthemum isolates from Japan. The CSVd RNA transcribed from the cDNA, having the sequence of the isolate from Russia, was able to infect potato, tomato, and chrysanthemum plants. The infected chrysanthemum plants exhibited stunting symptom. This is the first report of the occurrence of CSVd in Russia, its natural infection of potato plants and the potential spread of CSVd through propagation of infected potato.

Detailed information on viroid invasion of the shoot apical meristem is important for the efficient production of viroid-free plants by meristem tip culture. Additionally, knowledge on viroid distribution in plant reproductive tissues, especially the embryo, is crucial to assess seed transmission of viroids. In this study, we analyzed histochemically the distribution of chrysanthemum chlorotic mottle viroid (CChMVd) in the shoot meristems and flower buds of CChMVd-infected chrysanthemum plants (cv. ‘Okayama-heiwa’). In situ hybridization revealed that CChMVd invaded the shoot apical meristems and leaf primordia in the shoot meristem at a high frequency (ca. 70%). In flower buds, CChMVd was found in the bract, receptacle vascular, petals, ovules, and immature stamens and pistils. Furthermore, we showed that seed transmission of CChMVd occurred at a high frequency when CChMVd-infected plants were used as female parents (♀). Overall, our findings suggest that CChMVd infection of ovules in flower buds is involved in seed transmission of the viroid.