High-Throughput Sequencing Identifies Novel Viruses in Nectarine: Insights to the Etiology of Stem-Pitting Disease.

Recent studies have shown the superiority of high-throughput sequencing (HTS) technology over many standard protocols for pathogen detection. HTS was initiated on fruit tree accessions from disparate sources to improve and advance virus-testing procedures. A virus with genomic features resembling most closely that of the recently described Nectarine stem-pitting-associated virus, putative member of genus Luteovirus, was found in three nectarine trees (Prunus persica cv. nectarina), each exhibiting stem-pitting symptoms on the woody cylinder above the graft union. In these samples, HTS also revealed the presence of a coinfecting virus with genome characteristics typical of members of the genus Marafivirus. The same marafivirus- and luteovirus-like viruses were detected in nonsymptomatic nectarine and peach selections, indicating only a loose relationship between these two viruses with nectarine stem-pitting disease symptoms. Two selections infected with each of these viruses had previously tested free of known virus or virus-like agents using the current biological, serological, and molecular tests employed at the Clean Plant Center Northwest. Overall, this study presents the characterization by HTS of novel marafivirus- and luteovirus-like viruses of nectarine, and provides further insights into the etiology of nectarine stem-pitting disease. The discovery of these new viruses emphasizes the ability of HTS to reveal viruses that are not detected by existing protocols.

Internal effect concentrations of organic substances for early life development of egg-exposed fish.

The present study investigates the likelihood that early life development of marine fish from contaminated areas is affected by maternally transferred persistent organic substances (POPs). The common sole (Solea solea) was used as model species. Fertilized eggs were exposed via the water until hatching, 6 days post fertilization. The newly hatched larvae were allowed to develop further under unexposed conditions until the end of the metamorphosis. Effects on the larvae were determined for the dioxin-like polychlorinated biphenyl PCB 126, the technical PCB-mixture Arochlor 1254, polybrominated diphenylethers (PBDEs), and hexabromocyclododecane (HBCD), for an artificial mixture of PCBs and PBDEs, and for 'field mixtures' extracted from sole from the North Sea and the contaminated Western Scheldt estuary. Effect levels were expressed as tissue concentrations in the newly hatched larvae at the end of the exposure period. Exposure to PCBs, PBDEs, and the artificial and field mixtures caused mortality that started to occur shortly after the larvae became free-feeding (10 days post fertilization) and continued to increase until the onset of metamorphosis, 15 days later. The effects induced by the field mixtures correlated well with the ΣPCB concentrations in the tissue of the exposed larvae. No indications were found for synergistic effects or for substantial contribution of other (unknown) substances in the field mixtures. HBCD did not induce toxic effects. As lipid normalized POP levels in fish eggs are in general comparable to the levels in the tissue of the female fish, fish tissue concentrations are indicative of the internal exposure of the developing larvae as a result maternally transferred POPs will occur in the field. In sole from the Western Scheldt estuary POP levels are about twenty times lower than the larval tissue concentration that produced 50 percent early life stage mortality. Levels in North Sea sole are an order of a magnitude lower. At more heavily contaminated sites negative effect of PCBs, especially of those with dioxin-like toxicity can be expected.

Plastic in north sea fish.

To quantify the occurrence of ingested plastic in fish species caught at different geographical positions in the North Sea, and to test whether the fish condition is affected by ingestion of plastics, 1203 individual fish of seven common North Sea species were investigated: herring, gray gurnard, whiting, horse mackerel, haddock, atlantic mackerel, and cod. Plastic particles were found in 2.6% of the examined fish and in five of the seven species. No plastics were found in gray gurnard and mackerel. In most cases, only one particle was found per fish, ranging in size from 0.04 to 4.8 mm. Only particles larger than 0.2 mm, being the diameter of the sieve used, were considered for the data analyses, resulting in a median particle size of 0.8 mm. The frequency of fish with plastic was significantly higher (5.4%) in the southern North Sea, than in the northern North Sea above 55°N (1.2%). The highest frequency (>33%) was found in cod from the English Channel. In addition, small fibers were initially detected in most of the samples, but their abundance sharply decreased when working under special clean air conditions. Therefore, these fibers were considered to be artifacts related to air born contamination and were excluded from the analyses. No relationship was found between the condition factor (size-weight relationship) of the fish and the presence of ingested plastic particles.

First Report of Transmission of Little cherry virus 2 to Sweet Cherry by Pseudococcus maritimus (Ehrhorn) (Hemiptera: Pseudococcidae).

Little cherry virus 2 (LChV2; genus Ampelovirus, family Closteroviridae) is associated with Little Cherry Disease (LCD), one of the most economically destructive diseases of sweet cherry (Prunus avium (L.)) in North America (1). Since 2010, incidence of LCD associated with LChV2 confirmed by reverse transcription (RT)-PCR assays has increased in orchards of Washington State. LChV2 was known to be transmitted by the apple mealybug (Phenacoccus aceris (Signoret)) (3). However, the introduction of Allotropus utilis, a parasitoid platygastrid wasp (2) for biological control, contributed to keeping insect populations below the economic threshhold. In recent years, the population of grape mealybug (Pseudococcus maritimus (Ehrhorn)) increased in cherry orchards of Washington State (Beers, personal observation). Since grape mealybug is reported to transmit Grapevine leafroll associated virus 3 (Ampelovirus) in grapevine (4), this study investigated whether this insect would also transmit LChV2. A colony of grape mealybugs on Myrobalan plum (Prunus cerasifera Ehrh.) trees was identified visually and morphologically from slide mounts. In a growth chamber, first and second instar crawlers were fed on fresh cut shoots of sweet cherry infected with a North American strain (LC5) of LChV2. After an acquisition period of 7 days, 50 crawlers were transferred to each young potted sweet cherry trees, cv. Bing, confirmed free from LChV2 by RT-PCR. This process was repeated in two trials to yield a total of 21 potted trees exposed to grape mealybug. One additional tree was left uninfested as a negative control. After 1 week, the trees were treated with pesticide to eliminate the mealybugs. Two to four months after the inoculation period, leaves were collected from each of the recipient trees and tested by RT-PCR for the presence of LChV2. To reduce the possibility of virus contamination from residual mealybug debris on leaf surfaces, the trees were allowed to defoliate naturally. After a 3-month dormant period, the new foliage that emerged was then tested. Two sets of primers: LC26L (GCAGTACGTTCGATAAGAG) and LC26R (AACCACTTGATAGTGTCCT) (1); and LC2.13007F (GTTCGAAAGTGTTTCTTGA) and LC2.14545R (CATTATYTTACTAATGGTATGAC) (this study) were used to amplify a partial segment of the replicase gene (409 bp) and the complete (1,080 bp) coat protein gene of LChV2, respectively. Of 21 trees tested, 18 yielded positive results for LChV2. The reaction products from six randomly selected trees were cloned and the virus identity was verified by sequencing. The sequences of RT-PCR amplicons from both primer pairs showed ≥99% identity to LChV2, strain LC5 (GenBank Accession No. AF416335). The result confirmed that P. maritimus transmits LChV2, a significant finding for this cherry production region. Grape mealybug is of increasing concern in the tree fruit industry because it is difficult to control in established orchards. The presence of infested orchards that serve as reservoirs of both LCD and this insect vector present a challenge for management. To the best of our knowledge this is the first report to show transmission of LChV2 by grape mealybug. References: (1) K. C. Eastwell and M. G. Bernardy. Phytopathology 91:268, 2001. (2) C. F. W. Muesbeck. Can Entomol. 71:158, 1939. (3) J. R. D. Raine et al. Can. J. Plant Pathol. 8:6, 1986. (4) R. Sforza et al. Eur. J. Plant Pathol. 109:975, 2003.

First Report of Grapevine fleck virus from Washington Vineyards.

Grapevine fleck virus (GFkV) is a positive-sense, single-stranded RNA virus with a genome size of 7,564 nucleotides (3). The virus is present in many grape-growing regions (1,2,4). GFkV is phloem-limited and graft transmissible, but a biological vector is not yet known (4). It causes latent infections in Vitis vinifera cultivars, but induces specific foliar symptoms in the indicator host, V. rupestris. While testing samples from wine grape cultivars, samples from cv. Chardonnay tested positive for GFkV in single-tube one-step reverse transcription (RT)-PCR assay using forward primer GFkV585F (5'-CTCAGCCTCCACCTTGCCCCGT-3') and reverse primer GFkV1117R (5'-CAATTTGGCTGGGCGAGAAGTACA-3'). The forward primer is identical to nt 585 to 606 and the reverse primer is complementary to nt 1094 to 1117 in the GFkV genome (Accession No. AJ309022) and encompass a portion of the RNA-dependent RNA polymerase. The primer pair amplified a 533-nt fragment from 15 of 37 individual grapevines in the Chardonnay block. The amplicons obtained from five grapevines were cloned individually into the pCR2.1 plasmid (Invitrogen Corp., Carlsbad, CA). Three independent clones per amplicon were sequenced in both orientations. Sequences were edited and assembled using ContigExpress project in the Vector NTI Advance 11 sequence analysis software packages (Invitrogen). Pairwise comparisons of these sequences (Accession Nos. GU372367 to GU372371) with a corresponding sequence of a GFkV isolate deposited in the GenBank (Accession No. AJ309022) showed 89 to 97% identity at the nucleotide and 95 to 98% identity at the amino acid level. To further support these results, we amplified a 714-nt fragment specific to the complete coat protein (CP) gene of GFkV from three of the five isolates sequenced above using primers GFkV-6351F (5'-CTCTCCGCCTCGTCTGATGA-3') and GFkV-7064R (5'-TCGGTTCATGACGAGGGAGT-3'). The amplicons were cloned and sequenced as described above. A comparison of these sequences (Accession Nos. GU372372 to GU372374) with the CP sequence of GFkV available in the GenBank (Accession No. AJ309022) showed 94 to 95% and 98 to 100% identity, respectively, at the nucleotide and amino acid level. ELISA with GFkV-specific antibodies (BIOREBA AG, Reinach, Switzerland) further confirmed the presence of the virus in samples that were positive in RT-PCR. ELISA results validated the data described above and confirmed the presence of GFkV in Chardonnay samples that tested positive by RT-PCR assay. Previously, GFkV was documented in grapevines in California and Missouri (2), Australia (4) and Europe (1). To our knowledge, this is the first confirmed report of the occurrence of GFkV in Washington vineyards. The results expand our current knowledge on the distribution of GFkV and help to prevent its dissemination through the supply of grapevine cuttings by 'clean' plant programs. References: (1) G. P. Martelli et al. Arch Virol. 147:1847, 2002. (2) B. N. Milkus and R. N. Goodman. Am. J. Enol. Vitic. 50:133, 1999. (3) S. Sabanadzovic et al. J. Gen. Virol 82:2009, 2001. (4) B. J. Shi et al. Ann. Appl. Biol. 142:349, 2003.

First Report of Grapevine Virus Sequences Highly Similar to Grapevine Syrah virus-1 from Washington Vineyards.

Grapevine Syrah virus-1 (GSyV-1), a tentative member of the genus Marafivirus in the family Tymoviridae, has recently been found in a declining Syrah grapevine in California vineyards (1). To determine if GSyV-1 is present in grapevines grown in Washington State vineyards, extracts prepared from individual grapevines of six cultivars (Merlot, Chardonnay, Pinot Noir, Lemberger, Cabernet Sauvignon, and Syrah/Shiraz) were tested by single-tube reverse transcription (RT)-PCR using the primer pair GSyV-1 Det-F (5'-CAAGCCATCCGTGCATCTGG-3') and GSyV-1 Det-R (5'-GCCGATTTGGAACCCGATGG-3'). The primer GSyV-1 Det-F is identical to nucleotides (nt) 1125 to 1144 and GSyV-1 Det-R complementary to nt 1401 to 1420 of the GSyV-1 genome (GenBank Accession No. NC_012484) in the putative movement protein encoding gene (1). DNA fragment of approximately 296 base pairs (bp) was amplified only from 7 of 60 and 2 of 20 individual grapevines of cv. Syrah/Shiraz and Chardonnay, respectively, obtained from geographically separate vineyards. The 296-bp fragments from three Syrah/Shiraz and two Chardonnay grapevines were cloned individually into the pCR2.1 plasmid (Invitrogen Corp., Carlsbad, CA). Three independent clones derived from each DNA fragment were sequenced from both orientations and the sequences edited and assembled using ContigExpress project in the Vector NTI Advance 11 sequence analysis software packages (Invitrogen). Pairwise comparison of four of these sequences (Accession Nos. GU372349-52) showed 99 to 100% amino acid (aa) sequence identity among themselves and with corresponding sequences of GSyV-1. Because of the lack of antibodies, an additional 611-bp fragment specific to the capsid protein (CP) gene of GSyV-1 was amplified from six isolates (five from cv. Syrah/Shiraz, and one from cv. Chardonnay) (Accession Nos. GU372353-66) using primers GSyV-1-F (5'-TGTCGACGCTCCAATGTCTGA-3') and GSyV-1-R (5'-CATTGCTGCGCTTTGGAGGCTTTA-3'). GSyV-1-F is identical to nt 5775 to 5795 and GSyV-1-R is complementary to nt 6385 to 6408 of the GSyV-1 genome. The amplicons were cloned and sequenced as described above. Comparison of these sequences among themselves and with corresponding sequences of GSyV-1 showed 96 to 99% aa sequence identity, further complementing the results obtained above. To our knowledge, this is the first report of the occurrence of viral sequences closely related to GSyV-1 in Washington vineyards. Together with other reports (1,2), this study suggests that viruses similar to GSyV-1 could be widely distributed in wine grape cultivars across grape-growing regions. References: (1) M. Rwahnih et al. Virology 387:395, 2009. (2) S. Sabanadzovic. Virology 394:1, 2009.

First Report of Grapevine leafroll-associated virus-3 in Six Wine Grape Cultivars in Idaho.

In recent years, wine grape (Vitis vinifera) acreage in Idaho has expanded because of favorable climatic conditions for premium wine production. Nearly 95% of the 491.7 ha (1,215 acres) of wine grapes are in the Snake River Valley with Canyon County accounting for 81% of the vines. Previous studies have shown that grapevine leafroll disease (GLD) is the most widespread and economically significant virus disease in wine grapes in Washington and Oregon (1,2). However, little is known about the incidence and economic impact of GLD on wine grapes in Idaho. During the 2008 growing season, leaf samples were collected from approximately 25 individual grapevines of red-berried cultivars (Cabernet Sauvignon, Merlot, Syrah, and Petit Syrah) showing GLD symptoms and white-berried (Chardonnay) cultivars with suspected GLD symptoms growing in 10 geographically separate vineyards in Canyon County. An additional five samples were collected from a Lemberger block in Elmore County. Petiole extracts from these samples were tested by single-tube reverse transcription (RT)-PCR with primers LC 1 (5'-CGC TAG GGC TGT GGA AGT ATT-3') and LC 2 (5'-GTT GTC CCG GGT ACC AGA TAT-3') specific for the heat shock protein 70 homologue (HSP-70 gene) of Grapevine leafroll-associated virus-3 (GLRaV-3) (3). All samples, except the Petit Syrah, produced a single band of the expected size of 546 bp. ELISA with GLRaV-3-specific antibodies (BIOREBA AG, Reinach, Switzerland) confirmed the presence of the virus in samples that were positive in RT-PCR. GLRaV-3-specific amplicons were cloned in pCR2.1 plasmid (Invitrogen Corp., Carlsbad, CA) and 2 to 3 independent clones per isolate were sequenced in both orientations. A pairwise comparison of 22 sequences, six from Chardonnay (GenBank Accessions GQ344810, GQ344811, GQ344823, GQ344824, GQ344825, and GQ344826), five from Cabernet Sauvignon (GQ344807, GQ344808, GQ344809, GQ344827, and GQ344828), four each from Merlot (GQ344815, GQ344816, GQ344817, and GQ344818) and Syrah (GQ344819, GQ344820, GQ344821, and GQ344822), and three from Lemberger (GQ344812, GQ344813, and GQ344814) showed 87 to 100% identity at the nucleotide level and 92 to 100% identity at the amino acid level. A pairwise comparison of HSP-70 sequences of GLRaV-3 isolates from Idaho with corresponding sequences of GLRaV-3 isolates from GenBank showed nucleotide sequence identities between 88% (AJ748519) and 100% (DQ780885). Phylogenetic analysis of HSP-70 sequences from Idaho and GenBank showed clustering of Idaho sequences into five groups, with 12 sequences clustering with a Washington isolate (DQ780885), six sequences in a second group clustering with an isolate from Tunisia (AJ748522), two sequences in a third group clustering with an isolate from Austria (AJ748513), and one sequence each in groups four and five clustering with isolates from Italy (AJ748520) and Washington (DQ780889), respectively. The clustering was not cultivar- or vineyard-specific, suggesting separate introductions of different GLRaV-3 isolates in planting materials. To our knowledge, this is the first report of GLRaV-3 in grapevines grown in Idaho. These and previous results (1,2), indicate the wide distribution of GLRaV-3 in several grapevine cultivars in the Pacific Northwest Region. References: (1) R. R. Martin et al. Plant Dis. 89:763, 2005. (2) R. A. Naidu et al. (Abstr.) Phytopathology 96(suppl.):S83, 2006. (3) M. J. Soule et al. Plant Dis. 90:1461, 2006.

First Report of the Occurrence of Grapevine fanleaf virus in Washington State Vineyards.

Grapevine fanleaf virus (GFLV; genus Nepovirus, family Comoviridae), responsible for fanleaf degeneration disease, is one of the most important viruses of grapevines worldwide (1). During our reconnaissance studies during 2007, dormant wood cuttings from individual grapevines of wine grape cv. Chardonnay were collected randomly from two geographically separate vineyards in eastern Washington State. Extracts made from cambial scrapings of these cuttings were tested separately for different viruses by single-tube reverse transcription (RT)-PCR using virus-specific primers. Two of the thirty-one grapevines in one vineyard tested positive for GLFV as mixed infection with Grapevine leafroll-associated virus (GLRaV)-3. In another vineyard, six of the twenty-six grapevines tested positive for GFLV as mixed infection with GLRaV-1, GLRaV-3, and Grapevine virus A (GVA) A forward primer (5'-ACCGGATTGACGTGGGTGAT, corresponding to nucleotides [nt] 2231-2250) and reverse primer (5'-CCAAAGTTGGTTTCCCAAGA, complementary to nt 2533-2552) specific to RNA-2 of GFLV-F13 isolate (GenBank Accession No. X16907) were used in RT-PCR assays for the detection of GFLV (4). Primers used for RT-PCR detection of GLRaV-1, GLRaV-2, and GVA were described in Martin et al (2) and Minafra et al (3). The RT-PCR results indicated mixed infection of GFLV with GLRaV-1, GLRaV-3, and GVA. To confirm the presence of GFLV, the 322-bp sequence representing a portion of the coat protein encoded by RNA-2 genomic segment was cloned into pCR2.1 (Invitrogen Corp., Carlsbad, CA). Amplicons obtained from six individual grapevines in the two vineyards were used for cloning. Three independent clones per amplicon were sequenced from both orientations. Pairwise comparison of these sequences showed 99 to 100% nucleotide sequence identity among themselves, indicating that GFLV isolates from the two vineyards may be identical. A comparison of the consensus sequence (GenBank Accession No. EU573307) with corresponding sequences of other GFLVs deposited in GenBank showed 89 to 91% identity at the nucleotide level and 95 to 99% identity at the amino acid level. However, mixed infection of GFLV with different viruses in the two vineyards suggests separate introduction of the planting material. ELISA with GFLV-specific antibodies further confirmed the presence of the virus in samples that were positive in RT-PCR. To our knowledge, this is the first report of GFLV in grapevines grown in the Pacific Northwest states of the United States. Further investigations are being carried out on the distribution, symptoms, molecular variability, and nematode vector transmission of GFLV. References: (1) P. Andret-Link et al. J. Plant Pathol. 86:183, 2004. (2) R. R. Martin et al. Plant Dis. 89:763, 2005. (3) A. Minafra et al. Arch. Virol. 142:417, 1997 (4) A. Rowhani et al. Phytopathology 83:749, 1993.