Complete nucleotide sequence of a virus associated with rusty mottle disease of sweet cherry (Prunus avium).

Cherry rusty mottle is a disease of sweet cherries first described in 1940 in western North America. Because of the graft-transmissible nature of the disease, a viral nature of the disease was assumed. Here, the complete genomic nucleotide sequences of virus isolates from two trees expressing cherry rusty mottle disease symptoms are characterized; the virus is designated cherry rusty mottle associated virus (CRMaV). The biological and molecular characteristics of this virus in comparison to those of cherry necrotic rusty mottle virus (CNRMV) and cherry green ring mottle virus (CGRMV) are described. CRMaV was subsequently detected in additional sweet cherry trees expressing symptoms of cherry rusty mottle disease.

Genomic characterization of pararetroviral sequences in wild Dahlia spp. in natural habitats.

The genome structure and organization of endogenous caulimovirus sequences from dahlia (Dahlia spp), dahlia mosaic virus (DMV)-D10 from three wild species, D. coccinea (D10-DC), D. sherffii (D10-DS) and D. tenuicaulis (D10-DT), were determined and compared to those from cultivated species of dahlia, D. variabilis (DvEPRS). The complete ca. 7-kb dsDNA genomes of D10-DC, D10-DS, and D10-DT had a structure and organization typical of a caulimovirus and shared 89.3 to 96.6% amino acid sequence identity in various open reading frames (ORF) when compared to DvEPRS. The absence of the aphid transmission factor and the truncated coat protein fused with the reverse transcriptase ORF were common among these DMV-D10 isolates from wild Dahlia species.

Molecular characterization of allium virus X, a new potexvirus in the family Alphaflexiviridae, infecting ornamental allium.

ABTRACT: A new potexvirus affecting ornamental allium spp in the Netherlands was identified and characterized at the molecular level. The virus had a single-stranded RNA genome of 7100 bp (excluding the 18 bp poly A tail). The genome organization was found to be typical of members of the genus Potexvirus and consisted of five open reading frames (ORF). Nucleotide and amino acid sequence comparisons with those of known potexvirus members showed that this virus is related to Hosta virus X and Hydrangea ringspot virus. Sequence similarities and phylogenetic relationships suggested that the allium virus is a new and distinct species in the genus Potexvirus and the name, Allium virus X (AlVX) is proposed.

Structure and genome organization of the large RNA of iris yellow spot virus (genus Tospovirus, family Bunyaviridae).

The structure and organization of the large (L) RNA of iris yellow spot virus (IYSV) was determined, and with this report, the complete genomic sequence of IYSV of the genus Tospovirus, family Bunyaviridae has been elucidated. The L RNA of IYSV was 8,880 nucleotides in length and contained a single open reading frame in the viral complementary (vc) strand. The primary translation product of 331.17 kDa shared many of the features of the viral RNA-dependent RNA polymerase (RdRp) coded by L RNAs of known tospoviruses. The 5' and 3' termini of IYSV L RNA (vc) contain two untranslated regions of 33 and 226 nucleotides, respectively, and both termini have conserved terminal nucleotides, another common feature of tospovirus genomic RNAs. Conserved motifs characteristic of RdRps of members of the family Bunyaviridae were present in the IYSV RdRp.

Identification and molecular characterization of a new potyvirus infecting Triteleia species.

Plants of Triteleia hyacinthina, Triteleia ixioides Starlight, and Triteleia laxa Corina with severe mosaic and yellow vein-banding were found to be infected with a potyvirus. The 3'-terminal region of the virus was amplified by RT-PCR from total RNA using a potyvirus-specific degenerate primer (poty5P: 5' GGN AAY AAY AGY GGN CAR CC 3') and an oligo-dTprimer. The sequence generated included the 3'-NIb protein coding region (680 nucleotides), the entire coat protein coding region (840 nucleotides), and 3'-untranslated region (UTR) (253 nucleotides). Amino acid identity of the whole CP between the triteleia virus and potyvirus member ranged from 54% Apium virus Y (ApVY) to 67% Auraujia mosaic virus (ArjMV) and Twisted-stalk chlorotic streak virus (TSCSV) and the core ranged from 59% (ApVY) to 75% (ArjMV). The 3-UTR showed no significant homology with other known potyviruses. Phylogenetic relationships suggest this triteleia virus is a new member of the Potyvirus genus and the name of "Triteleia mosaic virus" (TriMV) is proposed. This is the first report of a potyvirus infecting triteleia.

Molecular characterization of pea enation mosaic virus and bean leafroll virus from the Pacific Northwest, USA.

The family Luteoviridae consists of eight viruses assigned to three different genera, Luteovirus, Polerovirus and Enamovirus. The complete genomic sequences of pea enation mosaic virus (genus Enamovirus) and bean leafroll virus (genus Luteovirus) from the Pacific Northwest, USA, were determined. Annotation, sequence comparisons, and phylogenetic analysis of selected genes together with those of known polero- and enamoviruses were conducted.

First Report of Tomato yellow leaf curl virus in Tomato in Mauritius.

Tomato is a food crop of economic importance in Mauritius. It is grown in open fields and in greenhouses by more than 4,500 small- and large-scale growers throughout the island. Open-field tomatoes are mostly a cooking type, while those produced in greenhouses are salad types. Acreage under production is approximately 900 ha with an annual production of approximately 11,500 tons. In September 2009, plants with reduced leaf size, leaf curling, and yellow margins associated with plant dwarfism were observed in open-field tomato crops in the southern part of the island. Whitefly populations were observed in these fields. These symptoms were suggestive of infection with a leaf curl-causing begomovirus such as Tomato yellow leaf curl virus (TYLCV) (family Geminiviridae, genus Begomovirus). Similar symptoms caused by TYLCV were reported in neighboring Reunion Island in 1997 (1). In October 2009, 3.15 ha of tomato were surveyed in the south at la Flora, Camp diable, L'escalier, Plein Bois, and Plaine Magnien to monitor the disease. Symptomatic plants were observed in all areas surveyed and disease incidence ranged from 5 to 50%. The disease was more prevalent in tomato 'Swaraksha' and 'Epoch', which are widely cultivated. Seventeen symptomatic leaf samples from La flora, Camp Diable, L'escalier, Plein Bois, and Plaine Magnien areas were collected for begomovirus detection by PCR. Total DNA was extracted and tested using AV494 (5'-GCC YAT RTA YAG RAA GCC MAG-3') and AC1048 (5'-GGR TTD GAR GCA TGH GTA CAT G-3') primers from the core region of the coat protein that detect most begomoviruses (2). Seventeen of 17 samples (100%) gave an amplicon of expected size. PCR amplicons from selected samples were cloned and sequenced. The consensus sequence was assembled, and the sequence (GenBank Accession no. HM448447) had 100% identity with nucleotides 458 to 1,036 of the Almeria isolate (GenBank Accession no. AJ489258), an isolate from the Netherlands (FJ439569), Morocco (EF060196), and Spain (AJ519441), and nucleotides 451 to 1,029 of the RE4 isolate from Reunion Island (AM409201). On the basis of the initial sequence obtained, specific primers (RM-TYLCV 583C: 5'-CCA CGA GTA ACA TCA CTA ACA-3' and RM-TYLCV 895F: 5'-GGA ACA GGC ATT AGT TAA GAG-3') were designed to amplify the remainder of the genomic sequence by PCR followed by cloning and sequencing of the amplicons. At least three clones were sequenced to arrive at the consensus sequence. Sequence comparisons showed that the TYLCV isolate from Mauritius had the greatest sequence identity (95 to 100%) with the above isolates. To our knowledge, this is the first report of TYLCV in tomato in Mauritius. In view of the economic importance of leaf curl disease in tomato in many parts of the world, an island-wide survey needs to be carried out to monitor the disease and assess its impact on tomato production. References: (1) M. Peterschmitt et al. Plant Dis. 83:303, 1999. (2) S. D. Wyatt and J. K. Brown. Phytopathology 86:1288, 1996.

Use of conserved genomic regions and degenerate primers in a PCR-based assay for the detection of members of the genus Caulimovirus.

The genus Caulimovirus consists of several distinct virus species with a double-stranded DNA genome that infect diverse plant species. A comparative analysis of the sequences of known Caulimovirus species revealed two regions that are conserved in all Caulimovirus species with the exception of Strawberry vein banding virus. Degenerate primers based on these two regions were designed and tested in a polymerase chain reaction-based assay for broad spectrum detection of members of this genus. Cauliflower mosaic virus, Figwort mosaic virus and three distinct caulimoviruses associated with dahlia (Dahlia variabilis) were used to show the utility of this test in detecting diverse caulimoviruses. The primer pair gave an amplicon of expected size (840bp). Amplicons from each virus were cloned and sequenced to verify their identity. The primer pair and the PCR assay provide approach for the broad spectrum detection of several members of the genus Caulimovirus.

Nucleotide sequence and genome organization of the medium RNA of Iris yellow spot virus from the United States.

Iris yellow spot tospovirus (IYSV) of the family Bunyaviridae causes a serious disease in onion in the USA and other parts of the world. Inspite of its economic importance, the complete genomic sequence of IYSV from the USA is not available. The genome structure and organization of the medium (M) RNA of a Washington (WA) isolate of IYSV were determined and compared to the corresponding region of two isolates previously described from Brazil and The Netherlands. Sequence analysis showed that the M-RNA was 4,817 nucleotides long and potentially coded for the movement protein (NSm) in the viral sense and the glycoprotein precursor (Gn and Gc) in the viral complementary sense. The predicted sizes of NSm and Gn/Gc precursor were 34.7 and 128.84 kDa, respectively. The two open reading frames are separated by a 380 nucleotide intergenic region. Phylogenetic analysis of the NSm and Gn/Gc genes from the WA isolate showed grouping that reflected their respective serogroups. The WA isolate formed a close cluster with the two previously reported IYSV isolates and the IYSV cluster was distinguishable from other tospovirus species. This is the first report of complete genomic sequence of the M-RNA of IYSV from the US.

Helleborus net necrosis virus: A New Carlavirus Associated with 'Black Death' of Helleborus spp.

Symptoms of 'black death' were observed on Helleborus spp. in each of three independent nurseries from across the United States. A new virus of the genus Carlavirus was identified in association with this disease. Symptomatic plants contained curved, rod-shaped particles averaging 800 by 17 nm, and yielded predominant bands of double-stranded (ds)RNA corresponding to approximately 9.0, 2.6, and 1.7 kbp. Amplification with degenerate primers for carlaviruses yielded a product of approximately 3,000 bp from diseased plants. Complete genomic sequences of two virus isolates were determined. Particle size, dsRNA patterns, genome organization, and sequence were consistent with members of the family Flexiviridae, genus Carlavirus. The name Helleborus net necrosis virus (HeNNV) is proposed for the virus associated with black death of Helleborus spp. in the United States. The sequence of the 3' terminus of Helleborus mosaic virus (HeMV) (genus Carlavirus) was also determined. Nucleotide sequences of HeNNV and HeMV were only 49% identical, revealing the distinct nature of these viruses. Assays for other viruses failed to reveal a consistent association of any other virus with black death symptoms. Cucumber mosaic virus was detected in hellebore specimens both with and without distinct black death symptoms.

First Report of Natural Infection of Penstemon acuminatus with Cucumber mosaic virus in the Treasure Valley Region of Idaho and Oregon.

Penstemons are perennials that are grown for their attractive flowers in the United States. Penstemon species (P. acuminatus, P. deustus, and P. speciosus) are among the native forbs considered as a high priority for restoration of great basin rangelands. During the summer of 2008, symptoms of red spots and rings were observed on leaves of P. acuminatus (family Scrophulariaceae) in an experimental trial in Malheur County, Oregon where the seeds from several native forbs were multiplied for restoration of range plants in intermountain areas. These plants were cultivated as part of the Great Basin Native Plant Selection and Increase Project. Several native wildflower species are grown for seed production in these experimental plots. Plants showed red foliar ringspots and streaks late in the season. Fungal or bacterial infection was ruled out. Two tospoviruses, Impatiens necrotic spot virus and Tomato spotted wilt virus, and one nepovirus, Tomato ring spot virus, are known to infect penstemon (2,3). Recently, a strain of Turnip vein-clearing virus, referred to as Penstemon ringspot virus, was reported in penstemon from Minnesota (1). Symptomatic leaves from the penstemon plants were negative for these viruses when tested by ELISA or reverse transcription (RT)-PCR. However, samples were found to be positive for Cucumber mosaic virus (CMV) when tested by a commercially available kit (Agdia Inc., Elkhart, IN). To verify CMV infection, total nucleic acid extracts from the symptomatic areas of the leaves were prepared and used in RT-PCR. Primers specific to the RNA-3 of CMV were designed on the basis of CMV sequences available in GenBank. The primer pair consisted of CMV V166: 5' CCA ACC TTT GTA GGG AGT GA 3' and CMV C563: 5' TAC ACG AGG ACG GCG TAC TT 3'. An amplicon of the expected size (400 bp) was obtained and cloned and sequenced. BLAST search of the GenBank for related sequences showed that the sequence obtained from penstemon was highly identical to several CMV sequences, with the highest identity (98%) with that of a sequence from Taiwan (GenBank No. D49496). CMV from infected penstemon was successfully transmitted by mechanical inoculation to cucumber seedlings. Infection of cucumber plants was confirmed by ELISA and RT-PCR. To our knowledge, this is the first report of CMV infection of P. acuminatus. With the ongoing efforts to revegetate the intermountain west with native forbs, there is a need for a comprehensive survey of pests and diseases affecting these plants. References: (1) B. E. Lockhart et al. Plant Dis. 92:725, 2008. (2) D. Louro. Acta Hortic. 431:99, 1996. (3) M. Navalinskiene et al. Trans. Estonian Agric. Univ. 209:140, 2000.

Nucleotide sequence and genome organization of a member of a new and distinct Caulimovirus species from dahlia.

A distinct caulimovirus, associated with dahlia mosaic, was cloned and sequenced. The caulimovirus, tentatively designated as dahlia common mosaic virus (DCMV), had a double-stranded DNA genome of ca. 8 kb. The genome organization of DCMV was found to be typical of members of the genus Caulimovirus and consisted of six major open reading frames (ORFs), ORFs I-VI, and one minor ORF, ORF VII. Sequence comparisons with the DNA genomes of two known caulimoviruses isolated from dahlia, Dahlia mosaic virus (DMV) and an endogenous caulimovirus, DMV-D10, showed that DCMV is a member of a distinct caulimovirus species, with sequence identities among various ORFs ranging from 25 to 80%.

Serological and Molecular Assays for Rapid and Sensitive Detection of Iris yellow spot virus Infection of Bulb and Seed Onion Crops.

Iris yellow spot virus (IYSV) has spread rapidly in the United States and has become an important economic constraint to the production of both bulb and seed onion crops. Symptoms caused by IYSV may be confused with those caused by other fungal and bacterial pathogens and virus-specific, reliable, sensitive, and rapid detection methods would improve the diagnosis. Antiserum was produced to Escherichia coli-expressed nucleocapsid protein of IYSV and an indirect format of the enzyme-linked immunosorbent assay (ELISA) was developed. IYSV could be detected in onion tissue at dilutions of up to 1:1,000. An IYSV-specific primer pair was designed and used in a real-time reverse-transcription polymerase chain reaction (RT-PCR) assay for the rapid detection of IYSV. Compared with standard RT-PCR, real-time RT-PCR was more rapid and sensitive. A commercially available RNA extraction kit and a total nucleic acid extraction method were compared for the quality of the templates obtained for use in real-time RT-PCR and there was no difference in limits of detection. Availability of ELISA- and PCR-based rapid and sensitive detection methods would facilitate accurate virus diagnosis and aid in better understanding of the epidemiology of the disease and in development of management strategies.

Occurrence of Two Little Cherry Viruses in Sweet Cherry in Washington State.

Little cherry disease, one of the major viral diseases of sweet cherry (Prunus avium) worldwide, is associated with either of two closteroviruses, Little cherry virus 1 (LChV-1) and Little cherry virus 2 (LChV-2). Two sets of primers corresponding to a portion of the replicase gene of LChV-1 and LChV-2 were used in one-tube reverse-transcription polymerase chain reactions to detect these viruses in total RNA extracts of field-collected sweet cherry tissues. LChV-1 and LChV-2 were detected both alone and in combination in five sweet cherry orchards in Washington State. Sequence analysis of a 240-nucleotide (nt) fragment of the replicase open reading frame (ORF)1b and a 232-nt fragment from a portion of ORF8 and the 3' untranslated region (UTR) of LChV-1 indicated that North American (NA) isolates shared 90 to 99% nucleotide identity in both genome segments analyzed. In contrast, comparisons of NA isolates to two Eurasian isolates of LChV-1 indicated shared nucleotide identities of 79 to 82% in the replicase fragment and 89 to 90% in the ORF8/3'UTR fragment. Sequence variation in the replicase region did not affect detection of LChV-1 in 12 isolates using the replicase-specific primers reported here. This article represents the first report of LChV-1 and LChV-2 in sweet cherry in Washington.

Dahlia mosaic virus and Tobacco streak virus in Dahlia (Dahlia variabilis) in New Zealand.

Dahlia (Dahlia variabilis Hort.) is a significant ornamental plant in New Zealand. Symptoms such as mosaic, ring spots, mottling, and veinal chlorosis, suggestive of a viral infection, are often seen in various dahlia collections. To better understand the incidence of viruses in dahlia in New Zealand, several popularly grown cultivars were evaluated for viruses that are known to infect dahlia. Viruses that were tested included Cucumber mosaic virus (CMV), Dahlia mosaic virus (DMV), Impatiens necrotic spot virus (INSV), Tobacco streak virus (TSV), and Tomato spotted wilt virus (TSWV). At least one symptomatic plant was tested from each of the following cultivars: Akito Dawn, Cincinnati Dancer, Hamari Accord, Hamari Rose, LeBatts Prime, LeVonne Splinter, Riverlea Tropicana, Spartacus, Tartan, Tui Connie, and Wandas Antartica. Except for DMV, initial testing was done by ELISA with commercially available kits for the above viruses. In the case of dahlia mosaic, samples were tested for DMV that was described previously (4) and two additional and distinct caulimoviruses (DMV-D10 and DMV-Holland) that were found to be associated with dahlia (1,2). Primer pairs, ORF6st: ATG GAA GAA ATT AAG GCG T and ORF6end: TTG TCT TCA TCC ATA AAG CAG; DenF1: CAG CAA GAA ACA GGA ATT GA and DenR: TTA CAG TCG AAG CTG CTA AA; and Kapht-F: ATG AGT AAT GCT TCA GCA A and Kapht-R: TGA CCA TGG CTT CTA ACT GT were used for the specific detection of DMV-D10, DMV-Holland, and DMV, respectively (1). None of the samples tested were ELISA positive for CMV, INSV, or TSWV. To verify the TSV infection, TSV-specific primers (5'-GTC CAG ACC ATC CAT CCA AC-3' and 5'-TTG ATT CAC CAG GAA ATC TT-3'), designed based on sequences available in GenBank, were used in reverse transcription (RT)-PCR. For DMV, the diagnostic tests used were electron microscopy and PCR followed by amplicon cloning and sequencing. Electron microscopic observation of leaf-dip preparations showed near isometric virions, approximately 50 to 60 nm in all samples tested. PCR showed that all samples tested were positive for DMV-Holland and DMV-D10. While DMV-Holland is a typical caulimovirus, DMV-D10 was found to exist as an endogenous plant pararetroviral sequence in dahlia (3). One sample each from two cultivars, Spartacus and Tui Connie, were positive for TSV by ELISA, RT-PCR, followed by the sequence analysis of the cloned amplicon. The impact of TSV-infected dahlias as a potential source of inoculum remains to be seen. Our results suggested the prevalence of dahlia mosaic-associated caulimoviruses in several dahlia cultivars and the presence of TSV in New Zealand dahlias. Dahlia mosaic continues to be prevalent in several parts of the world (1), and with the current findings in New Zealand, testing for these viruses should be conducted to ensure virus-free status of the propagating material. References: (1) V. Pahalawatta et al. Plant Dis. 91:1194, 2007. (2) V. Pahalawatta et al. Arch. Virol.153:733, 2008. (3) V. Pahalawatta et al. Virology 376:253, 2008. (4) R. D. Richins and R. J. Shepherd. Virology 124:208, 1983.

Iris Yellow Spot Virus in Onion Bulb and Seed Crops in Washington.

The geographic distribution of Iris yellow spot virus (IYSV, Genus Tospovirus, Family Bunyaviridae) in onion (Allium cepa L.) crops in the western United States has increased with the most recent report in Colorado (1,4). Furthermore, the incidence of IYSV has increased significantly in onion crops in the Treasure Valley of southern Idaho and eastern Oregon, where the disease was first detected in the United States (1,2). Surveys of onion seed crops in Washington during the past 2 years showed the presence of plants with symptoms characteristic of IYSV infection, including distinct diamond-shaped chlorotic or necrotic lesions, as well as indistinct circular to irregular, chlorotic or necrotic lesions of various sizes on the scapes of flowering plants. To date, symptomatic plants have been observed in five seed crops in Washington, at incidences ranging from <1% to approximately 20% in individual seed crops. Enzyme-linked immunosorbent assays carried out directly on symptomatic onion samples collected in July 2002, and on Nicotiana benthamiana plants mechanically inoculated with sap from these symptomatic plants, did not detect the presence of IYSV. In late July 2003, symptomatic plants were collected from an onion seed crop in Grant County and tested for IYSV infection by reverse transcription-polymerase chain reaction (RT-PCR). Total nucleic acid was extracted from symptomatic areas of the scapes with the procedure described by Presting et al. (3). Primers specific to the nucleocapsid (NP) gene of IYSV were designed based on sequences in GenBank: 5'-TCA GAA ATC GAG AAA CTT-3' and 5'-TAA TTA TAT CTA TCT TTC TTG G-3' (sense and antisense polarity, respectively). The RT-PCR assay produced an amplicon of the expected size (approximately 700 bp) that was cloned and sequenced. Comparison with the GenBank IYSV gene sequences showed 98% sequence identity of the NP gene. In August 2003, symptoms of IYSV infection were observed in two onion bulb crops, each located within 2 miles of the symptomatic onion seed crop in Grant County. The presence of IYSV in these crops was confirmed by RT-PCR with cloning and sequencing of the amplicon, as described for the seed crop samples. To our knowledge, this is the first confirmation of IYSV in onion bulb and seed crops in Washington, where 16,000 to 18,000 acres of onion bulb crops and 700 to 900 acres of onion seed crops are grown annually (USDA National Agricultural Statistics Service). The increase in prevalence of IYSV in the Pacific Northwest highlights the need for additional research to clarify the epidemiology of this potentially significant pathogen and to develop a regional management program for iris yellow spot. References: (1) J. M. Hall et al. Plant Dis. 77:952, 1993. (2) J. W. Moyer et al. (Abstr.) Phytopathology 93(suppl.):S115, 2003. (3) G. G. Presting et al. Phytopathology 85:436, 1995. (4) H. F. Schwartz et al. Plant Dis. 86:560, 2002.