Ryoji Nakaune

Detection of viruses associated with rugose wood in Japanese grapevines and analysis of genomic variability of Rupestris stem pitting-associated virus

In Vitis labruscana ‘Pione’ and ‘Kyoho’, 57 grapevines with rugose wood (RW) symptoms and 31 asymptomatic grapevines were obtained from vineyards in Hiroshima and Okayama prefectures. We surveyed vines for RW-associated viruses and other viruses by reverse transcription-polymerase chain reaction with virus-specific primers and degenerate primers for simultaneous detection of viruses associated with RW or leafroll disease. Rupestris stem pitting-associated virus (RSPaV) was detected from all grapevines with RW symptoms and from eight asymptomatic grapevines. Grapevine leafroll-associated virus-1, -2, -3, Grapevine virus B, Grapevine fleck virus, and Grapevine asteroid mosaic-associated virus were detected from some of the grapevines tested. RSPaV isolates detected were clustered in four groups by phylogenetic analysis using the nucleotide sequences of coat protein gene. These results suggest that RSPaV with diverse genetic variants is highly related to RW in Japanese grapevine cultivars Pione and Kyoho.

Effect of Culture Conditions on Conidia Formation by Elsinoë ampelina, the Causal Organism of Grapevine Anthracnose

Elsinoë ampelina, the causal organism of grapevine anthracnose, can be easily grown in culture, yet its sporulation is poor and unstable in culture. In this study, we sought the optimum conditions for a simple method to stably generate conidia. We first examined the optimum period of incubation for young colonies grown on potato dextrose agar (PDA) in water. The resultant number of conidia showed a logarithmic increase, which slowed at about 8 to 10 h. This suggests that 8 to 10 h of preculture would provide a sufficient number of conidia under the culture conditions used. A high negative correlation between colony density on PDA and the number of resultant conidia existed: colonies grown at >2.5 colonies per cm2 produced few or no conidia, whereas those grown at <1.0 colony per cm2 stably produced as many as 2.9 × 106 conidia. The optimum condition for preculture was to incubate colonies grown for 6 days on PDA at a density of <1.0 colony per cm2. The conidia obtained by our method were pathogenic on the grape cultivar Rizamat.

Novel variants of grapevine leafroll-associated virus 4 and 7 detected from a grapevine showing leafroll symptoms

All grapevine leafroll-associated viruses (GLRaV) identified so far belong to the family Closteroviridae [9]. The genus Closterovirus includes GLRaV-2, and the genus Ampelovirus includes GLRaV-1 and -3 in subgroup I and GLRaV-4, including the genetically divergent variants GLRaV-5, -6, -9, -De, -Pr and -Car, in subgroup II [9]. The provisional genus ‘‘Velarivirus’’ includes GLRaV-7 [9]. In Japan, although GLRaV-1, -2, and -3 are prevalent, GLRaV-4 and -7 have not been found. Recently, a generic nested RT-PCR for detection of the gene for the of heat shock protein 70 homolog (HSP70h) of members of the family Closteroviridae [5], using the method described by Nakaune et al. [11], detected a clear positive amplification from cultivar Chiliaki Chjornyj introduced from Russia in 1967 and planted at the Grape and Persimmon Research Station, NIFTS. Cane samples from the source vine were graftinoculated to Cabernet Franc, an indicator host for grapevine leafroll diseases [3], and these showed clear leafroll symptoms. However, each RT-PCR [10] using the GLRaV-1–6-, and -9-specific primer pairs (Table S1; [2, 4, 8, 10, 12]) was negative. Only RT-PCR [10] using the GLRaV-7-specific primer pair (Table S1; [6]) detected a faint positive amplification under the following PCR conditions: pre-activation at 95 C for 10 min followed by 43 cycles of denaturation (95 C for 20 s), primer annealing (42 C for 60 s), and extension (72 C for 60 s). Direct sequencing of a clear band amplified in a retested GLRaV-7-specific RT-PCR showed that the band had significant sequence identity to GLRaV-7. The amplified fragments from the nested RT-PCR were also cloned and sequenced following the method of Shimizu et al. [14]. Unexpectedly, all seven clones showed sequences related to GLRaV-4 but not GLRaV-7. To analyze the remaining parts of the genomes, 30-RACE was performed. cDNA was synthesized using the 30 RACE adapter of the FirstChoice RLM-RACE Kit (Ambion, Austin, TX, USA) as described previously [10], from RNA extracted using the method of Reid et al. [13]. cDNA was also synthesized using a SMARTer RACE cDNA Amplification Kit (Clontech, Mountain View, CA, USA) from RNA extracted using a Plant/Fungi Total RNA Purification Kit (Norgen, Thorold, ON, Canada) and finally poly-A-tailed with E. coli poly(A) polymerase (New England Biolabs, Ipswich, MA, USA) according to the manufacturer’s instructions. Both RLMand SMARTer-RACE PCRs were carried out using LA Taq (Takara, Shiga, Japan) following the manufacturer’s instructions. The PCR products were purified using QIAEX II (QIAGEN, Hilden, Germany) and then TA-cloned and sequenced as described previously [14] with the modification that LA Taq was used in the colony PCR. During the 30-RACE of the GLRaV-4related virus, two distinct types showing slightly different nucleotide sequences were obtained, each from more than Electronic supplementary material The online version of this article (doi:10.1007/s00705-012-1444-2) contains supplementary material, which is available to authorized users.

Molecular characterization of a novel putative ampelovirus tentatively named grapevine leafroll-associated virus 13

A novel putative ampelovirus was detected in grapevines that showed typical leafroll symptoms and was tentatively named grapevine leafroll-associated virus (GLRaV)-13 following the series of numbering of other GLRaVs. The complete genome of GLRaV-13 comprised 17,608 nt and contained eleven putative open reading frames, showing genetic features similar to those of viruses belonging to subgroup I of genus Ampelovirus. Phylogenetic trees based on the RNA-dependent RNA polymerase, heat shock protein 70 homolog, and coat protein showed that GLRaV-13 had the closest, but still distant, relationship to GLRaV-1 in the subgroup I cluster.

Ampeloviruses associated with incomplete flower syndrome and leaf-edge necrosis in Japanese apricot

Japanese apricot (Prunus mume Sieb. et Zucc.) trees produce popular fruits and are also used in Japan as ornamental flowering trees. Since the 1980s, graft-transmissible symptoms (e.g., incomplete flowers and leaf-edge necrosis) have been observed on ‘Nanko’ trees in Wakayama Prefecture. We here describe the detection of viruses associated with these symptoms in Japanese apricot trees using DECS analysis (i.e., double-stranded RNA [dsRNA] isolation, exhaustive amplification, cloning, and sequencing). We isolated dsRNA from symptomatic flowers, including dsRNA of plum bark necrosis stem pitting-associated virus (PBNSPaV), little cherry virus 2 (LChV-2), and a Luteovirus-like sequence. The reverse transcription polymerase chain reaction results suggested that PBNSPaV and LChV-2 may induce the development of incomplete flowers and leaf-edge necrosis. In contrast, the Luteovirus-like sequence is apparently unrelated to these symptoms. Additional investigations are necessary to clarify the relationship between the symptoms and viral infections.