Tetsuo Maoka

Mixed infection in tomato to ensure frequent generation of a natural reassortant between two subgroups of Cucumber mosaic virus

Cucumber mosaic virus (CMV) is divided into subgroups I (sub-I) and II (sub-II). Isolate CMV-PF, which had been isolated from tomato by single lesion isolation (SLI), was classified in sub-II by serology, but it had the pathogenicity of sub-I CMVs. Sequence comparisons and phylogenetic analyses showed that CMV-PF was actually a reassortant CMV isolate containing RNAs 1 and 2 from sub-I and RNA 3 from sub-II (I, I, and II). A subgroup-specific reverse transcription–polymerase chain reaction (SS-RT–PCR) was devised to detect reassortants between sub-I and sub-II. Six primer sets specific to genomic RNAs were designed for each subgroup, and those primer sets successfully amplified their target RT–PCR products. Of 38 natural CMV isolates, two isolates (nos. 9-7 and 27-3) contained mixed RNAs of both subgroups. SLI-progenies of 9-7 were subsequently isolated by SLI using Vigna unguiculata and analyzed by SS-RT–PCR; they were found to be a reassortant containing RNAs 1 and 2 from sub-I and RNA 3 from sub-II (I, I, and II). Although natural reassortants between CMV subgroups were thought to be infrequent based on previous observations, we readily found two isolates of a reassortant (I, I, and II) in different tomato fields, implying that tomato may act as a reservoir of mixed infection in the generation of a CMV reassortant perhaps by aphids.

Changes in Serological Reactivity of Papaya leaf distortion mosaic virus Caused by Papain in Carica papaya L. and Its Detection Using Antipain or Papain

Losses in serological reactivity of Papaya leaf distortion mosaic virus (PLDMV) were demonstrated. An antibody, IgG-papaya, raised against PLDMV purified from papaya (Carica papaya L.) did not react with virus particles in Cucumis metuliferus leaf extracts in ELISA or SSEM-PAG (serologically specific electron microscopy using protein A-gold). In addition, IgG-papaya and IgG- Cucumis raised against PLDMV purified from C. metuliferus did not react with virus particles in papaya leaf extracts after western blotting. From results of electrophoresis, the coat protein (CP) of PLDMV purified from papaya had degraded and migrated in two bands. Similar degradation was also observed when virus purified from C. metuliferus was treated with papain. These results indicated that the CP of PLDMV purified from papaya was degraded during the purification process by papain in the host plant. IgG-papaya was reactive to papain-degraded CP, while IgG-Cucumiswas reactive to both intact and degraded CP. Modified serological methods using antipain (a protease inhibitor) or papain were established to detect PLDMV.

Multivirus Detection from Japanese Landraces of Potato by Reverse Transcription–Polymerase Chain Reaction–Microplate Hybridization

In Japan, 12 viruses have been identified as causal agents of virus diseases of potato: Alfalfa mosaic virus, Cucumber mosaic virus, Potato aucuba mosaic virus, Potato leafroll virus (PLRV), Potato mop-top virus (PMTV), Potato virus A (PVA), Potato virus M (PVM), Potato virus S (PVS), Potato virus X (PVX), Potato virus Y (PVY), Tomato ringspot virus (ToRSV), and Tomato spotted wilt virus (TSWV). In a preliminary survey for viruses using ELISA, we found numerous virus species in landrace potatoes but only a few, e.g., PVY, in commercial ware potato crops. We have now modified a reverse transcription–polymerase chain reaction–microplate hybridization technique, developed for PVY, PLRV, and PMTV, to assay for the 12 viruses referred to above simultaneously. In leaf samples from 35 landraces, PVM, PVS, PVY, PLRV, PVA, and PVX were detected in 94%, 91%, 80%, 77%, 57%, and 43% of the landraces, respectively. These results indicate that potato landraces can act as a reservoir for these viruses with some landraces being asymptomatic. It is thus important to inspect and test for these viruses in seed potato production.

Biochemical, physiological, and molecular characterization of Dickeya dianthicola (formerly named Erwinia chrysanthemi) causing potato blackleg disease in Japan

The taxonomic assignment of Japanese potato blackleg isolates of Dickeya spp. has not been confirmed after the changes in their former name, Erwinia chrysanthemi. Therefore, we investigated and identified 23 representative isolates of Dickeya spp. from symptomatic stems of potatoes in Japan, with biochemical tests and phylogenetic sequence analysis using recA, dnaX, rpoD, gyrB, and 16S rDNA sequences. Results of our biochemical tests showed that all isolates can be assigned to phenon 5 and biovar 1, which are associated with D. dianthicola. Based on the recA, dnaX, rpoD, gyrB, and 16S rDNA sequences, all isolates are in the same clade with D. dianthicola and were clearly distinguished from D. chrysanthemi, D. dadantii, D. dadantii subsp. dieffenbachiae, D. solani, D. zeae, and D. paradisiaca. Therefore, we conclude that Dickeya spp. isolated from potatoes with blackleg symptoms in Japan are D. dianthicola.

Molecular characterization of Globodera pallida found in Japan using ribosomal DNA and mitochondrial cytochrome b gene sequences

In Japan, Globodera pallida was first reported in Abashiri city, Hokkaido in 2015. Sequences of ribosomal DNA (rDNA) and mitochondrial cytochrome b gene (cytb) from the Japanese population of G. pallida were compared with those of overseas populations. The representative sequences for rDNA and for cytb from the Japanese population had 100% identity with the sequences from G. pallida populations of North America and Europe in GenBank. Phylogenetic analysis also showed that they clustered in the clade that included populations from Europe and North America. From these results, we concluded that the Japanese population of G. pallida is close to populations that have been reported in Europe and North America. Recent studies suggested that a few populations of G. pallida were introduced into Europe from South America and have spread to various places around the world, and according to our results, into Japan also.

Species Composition of Alate Aphids (Hemiptera: Aphididae) Harboring Potato Virus Y and the Harbored Virus Strains in Hokkaido, Northern Japan

Abstract Many studies have evaluated transmission abilities of laboratory-reared aphids for potato virus Y (PVY), but few have focused on PVY-harboring species of field-collected aphids and the strains of PVY harbored by aphids. In the present study, we collected alate aphids in yellow pan traps in potato fields with Japanese commercial cultivars in Hokkaido, northern Japan in single 24-h periods during the tuber bulking stage and examined whether individual whole aphids harbored PVY by nested RT-PCR. PVY-positive individuals were identified to species using the gene sequence for cytochrome c oxidase subunit I and, when needed, morphological data and distribution records. In addition, individual strains of PVY harbored were determined using partial sequences of coat protein. Among 1,857 aphids trapped, 195 aphids had PVY and comprised 19 species; 17 species were identified to species-group taxa. Most of the aphid species detected as PVY positive colonize weeds that are common around potato fields in Hokkaido. Five species-group taxa had not been reported previously as a vector aphid of PVY and might be new PVY-vector species. PVYNTN was most frequently detected from PVY-positive aphids as found recently in PVY-infected potatoes in commercial fields in Hokkaido. Two or three PVY strains were rarely detected from a single aphid, and no obvious difference was found in the proportion of the harbored PVY strains among positive aphid species. The first documentation of the species composition of PVY-harboring aphids and the strains of PVY harbored in East Asia should aid understanding of the epidemiology of PVY in Japan.

Increased susceptibility of potato to Rhizoctonia diseases in Potato leafroll virus-infected plants

In Hokkaido potato fields, tubers produced from the plants with leaf curl symptoms caused by potato leaf roll virus (PLRV) were noted to be more densely covered with Rhizoctonia sclerotia. This observation led us to hypothesize that potato infected with PLRV would have an increased susceptibility to Rhizoctonia solani. To test this hypothesis, in a pot experiment, we inoculated PLRV-infected mother tubers with Rhizoctonia. As a result, PLRV-infected plants produced significantly fewer and smaller tubers than virus-free plants did, suggesting that PLRV-infected plants are more susceptible than virus-free plants to R. solani. Virus-free seed tubers should thus be used to reduce Rhizoctonia diseases.