Genetic variability of grapevine fabavirus variants and development of a broad-spectrum assay for their detection.

Complete RNA1 and RNA2 sequences of two and nearly complete genome sequences of six new variants of grapevine fabavirus found in Japan were compared to those of previously reported variants. Negative selection pressure was suggested, and no recombination events were detected in either RNA1 or RNA2. The first 18 nucleotides in both RNAs were predicted to form a stem-loop structure. The variants could be genetically divided into four groups based on RNA1 and two based on RNA2. A broad-spectrum reverse transcription polymerase chain reaction assay using a primer pair designed based on an RNA2 consensus sequence was able to detect all of the known variants.

Genome segments encoding capsid protein-like variants of Pyrus pyrifolia cryptic virus.

According to previous studies, three double-stranded (ds) RNA molecules (dsRNA1, 2, and 3) detected in Japanese pear are transmitted to the next generation with high frequency through both ovules and pollen. Nucleotide sequence analysis of dsRNA1-encoding RNA-dependent RNA polymerase (RdRp) has suggested that these dsRNAs are related to a cryptovirus named Pyrus pyrifolia cryptic virus (PpCV). In this study, purified dsRNA prepared from a PpCV-infected Japanese pear cultivar was subjected to next-generation deep sequencing. This sequencing generated two de novo assembled contigs corresponding to dsRNA2 and 3, with BLAST analysis of the predicted amino acid sequences indicating homology to capsid proteins (CPs) of the cryptoviruses persimmon cryptic virus and Sinapis alba cryptic virus 1, respectively. Relationships between the two contigs and dsRNA2 and 3 were confirmed by northern blot hybridization with probes generated using primers designed from the assembled contigs. Rapid amplification of cDNA ends analyses of 5'- and 3'-terminal sequences of dsRNA2 and 3 revealed that these two dsRNAs consist of 1523 and 1481bp, respectively. The 5'-terminal sequences (AGAAUUUC) of dsRNA1, 2 and 3 were found to be conserved. Phylogenetic analysis of deduced amino acid sequences of the two CP-like variants indicated that PpCV belongs to Deltapartitivirus (Partitiviridae). Our results imply that PpCV is tri-segmented.

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.

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.

Identification and characterization of a new vitivirus from grapevine.

New virus-like sequences, TvAQ7 and TvP15, were found in a Japanese grapevine accession of OKY-AQ7 (cv. Aki Queen) and of OKY-P15 (cv. Pione) by nested RT-PCR to simultaneously amplify a segment of the RNA-dependent RNA polymerase (RdRp) gene from members of the genera Vitivirus and Foveavirus. The TvAQ7 genome, except for an exact 5' terminus, is composed of about 7.6 kb containing five potential open reading frames. The genomic organization resembles those of grapevine virus A and other known vitiviruses. The 199-amino-acid sequence deduced from the ORF4 of TvAQ7 has 38.5-54.0% identity with the coat protein (CP) of known grapevine vitiviruses and 86.9% identity with TvP15. Phylogenetic analysis based on amino acid sequences of the CP showed that TvAQ7 and TvP15 were closely related to the vitiviruses. In addition, we confirmed that TvAQ7 and TvP15 were transmitted from infected grapevine to healthy seedlings by the mealybug Pseudococcus comstocki Kuwanae. On the basis of our findings, TvAQ7 and TvP15 should be considered isolates of a new species of the genus Vitivirus, and both isolates are probably genetic variants of the new species. We propose to name this virus grapevine virus E (GVE).

Identification of a new Apscaviroid from Japanese persimmon.

Three viroid-like sequences were detected from Japanese persimmon (Diospyrus kaki Thunb.) by RT-PCR using primers specific for members of the genus Apscaviroid. Based on the sequences, we determined the complete genomic sequences. Two had 92.1-94.3% sequence identity with citrus viroid OS (CVd-OS) and 91.4-96.3% identity with apple fruit crinkle viroid (AFCVd), respectively. Another one, tentatively named persimmon viroid (PVd), had 396 nucleotides and less than 70% sequence identity with known viroids. The secondary structure of PVd is proposed to be rod-like with extensive base pairing and contains the terminal conserved region and the central conserved region characteristic of the genus Apscaviroid. Moreover, we confirmed that the viroids, including PVd, are graft transmissible from persimmon to persimmon and that persimmon is a natural host of these viroids. According to its molecular and biological properties, PVd should be considered a member of a new species in the genus Apscaviroid.

Benomyl resistance of Colletotrichum acutatum is caused by enhanced expression of beta-tubulin 1 gene regulated by putative leucine zipper protein CaBEN1.

Colletotrichum acutatum, the fungus causing anthracnose disease of various fruits and crops, has inherent resistance to benomyl. The mechanism underlying its resistance to benomyl remains unclear. We generated a benomyl-sensitive mutant CAT7-150 by transformation-mediated insertional mutagenesis. Subsequently, a CaBEN1 gene was isolated from CAT7-150 by plasmid rescue and sequenced. CaBEN1 encodes a protein of 818 amino acids containing a leucine zipper motif, and the amino acid sequence has significant similarity to hypothetical proteins found in Gibberella zeae and other fungi. Disruption and complementation of the CaBEN1 gene demonstrated that CaBEN1 was necessary for resistance to benzimidazole fungicides, but was not essential for mycelial growth. Northern analysis suggests that CaBEN1 has an important role in transcriptional activation of CaTUB1 in response to benomyl. Benomyl resistance of CaBEN1-disrupted transformant was recovered by overexpression of CaTUB1. Our results clearly demonstrate that the benomyl resistance is due to enhanced expression of CaTUB1, which is controlled by CaBEN1.

Efficient methods for sample processing and cDNA synthesis by RT-PCR for the detection of grapevine viruses and viroids.

Template preparation is important in reverse-transcription polymerase chain reaction (RT-PCR)-based detection methods. A TissueLyser with tungsten carbide beads was used for simultaneous processing of up to 48 samples under the same conditions in the development of a simple and rapid procedure to prepare a plant extract for RT reaction. A sandpaper method was also developed by which wood tissue of dormant cuttings could be macerated easily to process with minimal time and effort. It was also demonstrated that the combination use of random primers and oligo dT primer in an RT reaction was efficient for simultaneous cDNA synthesis of viral and viroid RNAs in plant extracts. These template preparation methods were used for the amplification of Grapevine leafroll-associated virus-1,-2, and -3; Grapevine virus A and B; Grapevine rupestris stem pitting-associated virus; Grapevine fleck virus; and Grapevine fanleaf virus. All these viruses tested in this study were reliably detected up to a 10(3)-fold or higher dilution of the original extract. Besides, Hop stunt viroid and Grapevine yellow speckle viroid 1 were well amplified in the same manner as the template preparation and following PCR for virus detection. These methods would contribute to cost-effective testing of a large number of samples through the year and help to detect viral pathogens in grapevine.