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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.

Both potato psyllid yellows and zebra chip (ZC) potato diseases are associated with the potato psyllid, Bactericera cockerelli (Sulc). Aboveground plant symptoms of both diseases are similar but there is a difference in symptoms in potato tubers. ZC has recently been associated with a new species of the bacterium liberibacter, ‘Candidatus Liberibacter solanacearum’, also known as ‘Ca. Liberibacter psyllaurous’. Mechanisms by which the potato psyllid might cause either ZC or potato psyllid yellows symptoms are not understood. Insect transmission studies were conducted to demonstrate psyllid vectoring of both diseases and to compare symptoms and development of the two diseases. Potato plants were exposed to both liberibacter-free and liberibacter-carrying potato psyllids and later evaluated for plant and tuber symptoms. These plants and tubers were then tested for liberibacter by polymerase chain reaction (PCR). In addition, potato plants exhibiting severe psyllid yellows/ZC-like symptoms were collected from a commercial potato field heavily infested with the potato psyllid and tested for liberibacter. PCR detected ‘Ca. Liberibacter solanacearum’ in ZC symptomatic plants and tubers resulting from exposure to liberibacter-carrying psyllids. Despite development of foliar symptoms that resemble those of ZC in plants exposed to liberibacter-free psyllids, no liberibacter was detected in these plants with psyllid yellows. Moreover, tubers from these plants with psyllid yellows did not exhibit any symptoms of ZC infection and tested negative for the bacterium. No liberibacter was detected in plants or tubers collected from the psyllid-infested potato field, suggesting that the observed symptoms were due to psyllid yellows. Furthermore, potato plants that were infected with liberibacter died sooner than plants that were infected with psyllid yellows. Although an association between liberibacter and ZC has been established, no pathogen is yet associated with potato psyllid yellows and mechanisms by which psyllid yellows symptoms are induced by the potato psyllid remain unclear.

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%.

Viruses in certain genera in family Caulimoviridae were shown to integrate their genomic sequences into their host genomes and exist as endogenous pararetroviral sequences (EPRV). However, members of the genus Caulimovirus remained to be the exception and are known to exist only as episomal elements in the infected cell. We present evidence that the DNA genome of a new and distinct Caulimovirus species, associated with dahlia mosaic, is integrated into its host genome, dahlia (Dahlia variabilis). Using cloned viral genes as probes, Southern blot hybridization of total plant DNA from dahlia seedlings showed the presence of viral DNA in the host DNA. Fluorescent in situ hybridization using labeled DNA probes from the D10 genome localized the viral sequences in dahlia chromosomes. The natural integration of a Caulimovirus genome into its host and its existence as an EPRV suggests the co-evolution of this plant–virus pathosystem.

The genome structure and organization of a new and distinct caulimovirus that is widespread in dahlia (Dahlia variabilis) was determined. The double-stranded DNA genome was ca. 7.0 kb in size and shared many of the features of the members of the genus Caulimovirus, such as the presence of genes potentially coding for the movement protein, the inclusion body protein, and the reverse transcriptase (RT), and an intergenic region consisting of a potential 35S promoter. However, the virus differed from the previously described dahlia mosaic caulimovirus and other known caulimoviruses in that the aphid transmission factor (ATF) was absent and the putative coat protein contained a C-terminal deletion and was fused in-frame with the RT. Sequence identity at the amino acid level with known caulimoviruses including a previously reported caulimovirus from dahlia was low and ranged from 32 to 72%. The absence of an ATF and the highly divergent nature of the genomic sequence are characteristics of this new caulimovirus that is widely associated with dahlia.

Iris yellow spot virus (IYSV) was first observed in sweet onions in Georgia (USA) in 2003 in the Vidalia region. The virus had been reported in the oniongrowing regions in western USA several years before being detected in Georgia in the east. Although symptoms were observed on onions in Peru several years earlier, the presence of IYSV was not confirmed in Peru until after the virus was detected in Georgia. We characterized nine isolates of IYSV recovered from sweet onions in both Georgia (four isolates) and Peru (five isolates) by sequencing the nucleocapsid (N) gene and compared those sequences with sequences available in GenBank. Sequence divergence between IYSV isolates from Georgia and Peru was low with 1.1%, and comparisons with IYSV isolates from other regions showed divergence of up to 11.4%. Bootstrap analysis indicated with a high degree of confidence that the Georgia and Peruvian isolates fell into the same clade and were different from known isolates from western USA that fell into sister clades. The high degree of similarity between Georgia and Peruvian isolates suggests that gene flow occurred from Peru into Georgia.

Dahlia mosaic, caused by Dahlia mosaic virus (DMV), is one of the most important viral diseases of dahlia. Molecular characterization of DMV showed the association of two distinct caulimoviruses (DMV-D10, DMV-Portland) and a D10-like sequence variant (DMV-Holland) with the disease. Using primers specific to these two viruses and the sequence variant, a polymerase chain reaction–based assay was used to determine their relative incidence in several dahlia samples from the United States and the Netherlands. Testing was done on samples collected in 2005 and 2006 in the United States and in 2006 in the Netherlands. Results indicated the predominance of DMV-D10 over DMV-Portland and DMV-Holland in both the United States and the Netherlands. Using conserved regions of the viral genome, primers were designed and used to detect all three sequences. Results suggested that DMV-D10 is predominantly associated with dahlia mosaic, but diagnostics should also include testing for DMV-Portland and DMV-Holland.

Tomato spotted wilt virus (TSWV) is an economically important viral pathogen of flue-cured tobacco, Nicotiana tabacum. Disease development and in planta distribution of TSWV were studied following mechanical inoculation of cv. K326 at various stages of growth. The effect of plant age on the disease development, distribution of symptoms and TSWV were studied by inoculating plants in five age groups, 40, 60, 75, 95 and 100 days after sowing (DAS). The plant age at the time of infection had no significant influence on the incidence of localised infection; however, it had a significant effect on the development of systemic symptoms and distribution of TSWV in the plant. In a higher proportion of plants (89.2%), no systemic symptoms developed when plants were inoculated at 60-100 DAS. However, 90% of plants became systemically infected when plants were inoculated at 40 DAS. The systemic symptom expression was severe and distributed in all the leaves in 40-DAS plants, whereas in 60- to 100-DAS plants, it was erratic and restricted only to a few upper leaves. Results show that plant age is an important factor for TSWV infection of tobacco and mature tobacco plants significantly reduced the systemic development of the disease.

Viral small RNAs (vsRNAs) are one of the key elements involved in RNA silencing-based defense against viruses in plants. We analyzed the vsRNA profiles in Nicotiana benthamiana and Solanum lycopersicum infected by polygonum ringspot virus (PolRSV) (Tospovirus, Bunyaviridae). VsRNAs were abundant in both hosts, but a different size profile was observed, with an abundance peak at 21 in N. benthamiana and at 22. nt in tomato. VsRNAs mapping to the PolRSV L genomic segment were under-represented in both hosts, while S and M segments were differentially and highly targeted in N. benthamiana and tomato, respectively. Differences in preferential targeting of single ORFs were observed, with over-representation of NSs ORF derived reads in N. benthamiana. Intergenic regions (IGRs)-mapping vsRNAs were under represented, while enrichment of vsRNAs reads mapping to the NSs positive sense strand was observed in both hosts. Comparison with a previous study on tomato spotted wilt virus (TSWV) under the same experimental conditions, showed that the relative accumulation of PolRSV specific and endogenous sRNAs was similar to the one observed for silencing suppressor deficient TSWV strains, suggesting possible different properties of PolRSV NSs silencing suppressor compared to that of TSWV.

Tomato spotted wilt virus (TSWV) is an economically important viral pathogen of a wide range of field and horticultural crops. We developed an artificial microRNA (amiRNA) strategy against TSWV, targeting the nucleoprotein (N) and silencing suppressor (NSs) genes. The amiRNA constructs replaced the natural miRNA in a shortened Arabidopsis 173-nucleotide (nt) miR159a precursor backbone (athmiR159a) without the stem base extending beyond the miR/miR* duplex. Further, each amiRNA was modified to contain a mismatch (wobble) sequence at nucleotide position 12 and 13 on the complementary strand amiRNA*, mimicking the endogenous miR159a sequence structure. Transient expression in Nicotiana benthamiana demonstrated that the introduction of a wobble sequence did not alter amiRNA expression levels. Following challenge inoculation with TSWV, plants expressing N-specific amiRNAs with or without the wobble remained asymptomatic and were negative for TSWV by ELISA. In contrast, plants expressing the NSs-specific amiRNAs were symptomatic and accumulated high levels of TSWV. Similar findings were obtained in stably transformed Nicotiana tabacum plants. Our results show that a shortened 173-nt athmiR159a backbone is sufficient to express amiRNAs and that the presence of mismatch at position 12-13 does not influence amiRNA expression or conferring of resistance. We also show that selection of target gene and positional effect are critical in amiRNA-based approach for introducing resistance. These findings open the possibility of employing the amiRNA approach for broad-spectrum resistance to tospoviruses as well as other viruses.

Tospoviruses are plant-infecting viruses belonging to the family Bunyaviridae . We used a collection of wild-type, phylogenetically distinct tomato spotted wilt virus isolates and related silencing-suppressor defective mutants to study the effects on the small RNA (sRNA) accumulation during infection of Nicotiana benthamiana . Our data showed that absence of a functional silencing suppressor determined a marked increase of the total amount of viral sRNAs (vsRNAs), and specifically of the 21 nt class. We observed a common under-representation of vsRNAs mapping to the intergenic region of S and M genomic segments, and preferential mapping of the reads against the viral sense open reading frames, with the exception of the NSs gene. The NSs-mutant strains showed enrichment of NSm-derived vsRNA compared to the expected amount based on gene size. Analysis of 5′ terminal nucleotide preference evidenced a significant enrichment in U for the 21 nt- and in A for 24 nt-long endogenous sRNAs in all the samples. Hotspot analysis revealed a common abundant accumulation of reads at the 5′ end of the L segment, mostly in the antiviral sense, for the NSs-defective isolates, suggesting that absence of the silencing suppressor can influence preferential targeting of the viral genome.

International audience

Tomato spotted wilt virus (TSWV) represents a major constraint to the production of important vegetable and ornamental crops in several countries around the world, including those in Europe. In spite of their economic importance, European TSWV isolates have only been partially characterized, and a complete genome sequence has not been determined yet. In this study, we completed the whole genome sequence of two distinct TSWV isolates from Italy, p105 and p202/3WT. The sequences of the L and M segments of p105 and of the L segment of p202/3WT were determined using a combined approach of RT-PCR and small RNA (sRNAs) contig assembly. Phylogenetic analysis based on RNA-dependent RNA polymerase and G(N/)G(C) protein sequences grouped the two isolates in two different clades, showing that different evolutive lineages are present among Italian TSWV isolates. Analysis of possible recombination/reassortment events among our isolates and other available full-length genome TSWV sequences showed a likely reassortment event involving the L segment.

Spotted wilt disease, caused by Tomato spotted wilt virus (TSWV), is an economically important disease in peanut, pepper, tobacco, and tomato in the southeastern United States. However, very little is known about the biological variability existent in the virus population. Fourteen isolates of TSWV collected in Georgia were evaluated for symptom severity. The majority of the isolates produced severe systemic necrosis. One mild (GATb-1) and one severe (GAL) isolate were further examined because of the distinct differences in their virulence and symptomatology on tobacco. GATb-1 caused a few chlorotic spots and mild systemic symptoms, whereas GAL produced a large number of local lesions and severe systemic necrosis. Distinct differences in the response of selected commercial cultivars of peanut, tobacco, and tomato to GATb-1 and GAL infection were observed. GAL was lethal to a widely grown tobacco cultivar, K326. Georgia Green, a field resistant peanut cultivar, and C11-2-39, a breeding line with the highest level of known resistance to TSWV, were more susceptible to GAL than to GATb-1. BHN 444, a newly released TSWV-resistant tomato cultivar, showed a resistant reaction, whereas Stiletto, a newly released TSWV-resistant pepper cultivar, was susceptible to both GATb-1 and GAL isolates. Information on the biological diversity of TSWV may be useful in developing more durable TSWV-resistant crops.

Four studies were conducted in Georgia during spring 1999, 2000, 2001, and 2002 to evaluate various management tactics for reducing thrips and thrips-vectored tomato spotted wilt virus (TSWV) in tomato and their interactions relative to fruit yield. Populations of thrips vectors of TSWV, Frankliniella occidentalis (Pergande) and Frankliniella fusca (Hinds), were determined using flower and sticky trap samples. The management practices evaluated were host plant resistance, insecticide treatments, and silver or metallic reflective mulch. Averaged over all tests, the TSWV-resistant tomato ‘BHN444′ on silver mulch treatment had the largest effect in terms of reducing thrips and spotted wilt and increasing marketable yield. Of the insecticide treatments tested, the imidacloprid soil treatment followed by early applications of a thrips-effective foliar insecticide treatment provided significant increase in yield over other treatments. Tomato yield was negatively correlated with the number of F. fusca and percentage of TSWV incidence. F. occidentalis per blossom was positively correlated with percentage of TSWV incidence, but not with yield. No significant interactions were observed between cultivar reflective mulch main plot treatments and insecticide subplot treatments; thus, treatment seemed to be additive in reducing the economic impact of thrips-vectored TSWV. Control tactics that manage thrips early in the growing season significantly increased tomato yield in years when the incidence of TSWV was high (>17%).

A survey of Potato virus Y (PVY) was conducted in farmers' fields in 9 Korean provinces, Gyeonggi-do, Gangwon-do, Gyeongsangnam-do, Gyeongsangbuk-do, Jeollanam-do, Jeollabuk~io, Chungchcongnam-do, Chungcheongbuk-do and Jeju-do from 1998 to 2000. One hundred and fifty nine samples from potato and tobacco were collected and analyzed. Based on ELISA results, three samples PVY-tob, PVY-potlii and PVY-pot-chw representing PVYN and PVYo, respectively, were further subjected to biological, serological and molecular analysis. Host range studies showed that PVY-pot-chw is symptomatically distinguishable from PVY-tob and PVY-pot-j isolates, both of which were symptomatically indistinguishable. However, all the three isolates failed to infect 10 capsicum varieties systemically. Isolates PVY-tob and PVY-pot-j reacted strongly wlth monoclonal antibody (MAb), anti-PVYN while PVY-pot-chw failed to react wlth the MAb but reacted strongly wlth anti-PVYo'c. Interestingly, both PVY-tob and PVY-pot-j isolates did not react with anti-Pvyo,C indicating that isolate PVY-pot-chw is serologically distinguishable from both PVY-tob and PVY-potlii. In order to ascertain the taxonomic status of the three isolates, the 3' untranslated terminal regions (3'-UTR) were sequenced. Alignment of the sequences revealed 990/0 sequence identity between PVY-tob and PVY-pot-j, both of which had 780/0 nt sequences identical to PVY-pot-chw. Phylogenetic analysis of the aligned 3'-UTRs clustered PVY-tob and PVY-pot-j in the PVY~ subgroup, and PVY-pot~:hw in the PVYO subgroup.

Tomato spotted wilt virus (TSWV) is a serious constraint to tomato production worldwide. Losses are significant because the disease is difficult to control and most of the commercially available tomato cultivars are susceptible to TSWV. This study was intended to provide information that could be used to design more appropriate disease management strategies. The objective was to determine the relationship of tomato plant growth and fruit yield with the time of TSWV symptom appearance. Experiments were carried out during Spring 1999 and 2000, using drip irrigation and plastic film mulched beds with black plastic mulch alone (1999) or different colored mulches (2000). The mulches used were black, black-on-silver, gray-on-black, red, silver-on-black, silver (painted) and white-on-black, and bare soil. The 1999 experiment included a single TSWV-susceptible cultivar (Florida-47), while the 2000-experiment included two TSWV-susceptible (Florida-91 and Sun Chaser) and one TSWV-resistant cultivars (BHN-444). Colored mulches and tomato cultivars affected the time between transplanting and appearance of first symptoms of TSWV. For all tomato cultivars, vegetative top fresh weight (FW), fruit number and total fruit yield increased linearly with the time the plants remained free from TSWV symptoms. Marketable fruit yield also increased as the time from transplanting to the first appearance of symptoms increased. When data for cultivars were pooled, vegetative top FW and total fruit yield were reduced by 2.1% and 2.3%, respectively, for each day prior to harvesting that plants showed TSWV symptoms.

Deep sequencing technology has enabled the analysis of small RNA profiles of virus-infected plants and could provide insights into virus-host interactions. Potato virus Y is an economically important viral pathogen of potato worldwide. In this study, we investigated the nature and relative levels of virus-derived small interfering RNAs (vsiRNAs) in potato cv. Russet Burbank infected with three biologically distinct and economically important strains of PVY, the ordinary strain (PVY-O), tobacco veinal-necrotic strain (PVY-N) and tuber necrotic strain (PVY-NTN). The analysis showed an overall abundance of vsiRNAs of 20-24nt in PVY-infected plants. Considerable differences were present in the distribution of vsiRNAs as well as total small RNAs. The 21nt class was the most prevalent in PVY-infected plants irrespective of the virus strain, whereas in healthy potato plants, the 24nt class was the most dominant. vsiRNAs were derived from every position in the PVY genome, though certain hotspots were identified for each of the PVY strains. Among the three strains used, the population of vsiRNAs of different size classes was relatively different with PVY-NTN accumulating the highest level of vsiRNAs, while PVY-N infected plants had the least population of vsiRNAs. Unique vsiRNAs mapping to PVY genome in PVY-infected plants amounted to 3.13, 1.93 and 1.70% for NTN, N and O, respectively. There was a bias in the generation of vsiRNAs from the plus strand of the genome in comparison to the negative strand. The highest number of total vsiRNAs was from the cytoplasmic inclusion protein gene (CI) in PVY-O and PVY-NTN strains, whereas from PVY-N, the NIb gene produced maximum total vsiRNAs. These findings indicate that the three PVY strains interact differently in the same host genetic background and provided insights into virus-host interactions in an important food crop.

Thrips-transmitted Iris yellow spot virus (IYSV) is an important economic constraint to the production of bulb and seed onion crops in the United States and many other parts of the world. Because the virus is exclusively spread by thrips, the ability to rapidly detect the virus in thrips vectors would facilitate studies on the role of thrips in virus epidemiology, and thus formulation of better vector management strategies. Using a polyclonal antiserum produced against the recombinant, Escherichia coli-expressed nonstructural protein coded by the small (S) RNA of IYSV, an enzyme linked immunosorbent assay was developed for detecting IYSV in individual as well as groups of adult thrips. The approach enabled estimating the proportion of potential thrips transmitters in a large number of field-collected thrips collected from field-grown onion plants. Availability of a practical and inexpensive test to identify viruliferous thrips would be useful in epidemiological studies to better understand the role of thrips vectors in outbreaks of this economically important virus of onion.

Thrips-transmitted Iris yellow spot virus is an economically important viral pathogen of Allium crops worldwide. A global analysis of known IYSV nucleocapsid gene (N gene) sequences was carried out to determine the comparative population structure, spatial and temporal dynamics with reference to its genetic diversity and evolution. A total of 98 complete N gene sequences (including 8 sequences reported in this study) available in GenBank and reported from 23 countries were characterized by in-silico RFLP analysis. Based on RFLP, 94% of the isolates could be grouped into NL or BR types while the rest belonged to neither group. The relative proportion of NL and BR types was 46% and 48%, respectively. A temporal shift in the IYSV genotypes with a greater incremental incidence of IYSVBR was found over IYSVNL before 2005 compared to after 2005. The virus population had at least one evolutionarily significant recombination event, involving IYSVBR and IYSVNL. Codon substitution studies did not identify any significant differences among the genotypes of IYSV. However, N gene codons were minimally positively selected, moderately negatively selected denoting the action of purifying selection, thus rejecting the theory of neutral mutation in IYSV population. However, one codon position (139) was found to be positively selected in all the genotypes. Population selection statistics in the IYSVBR, IYSVNL genotypes and in the population as a whole also revealed the action of purifying selection or population expansion, whereas IYSVother displayed a decrease in population size. Genetic differentiation studies showed inherent differentiation and infrequent gene flow between IYSVBR and IYSVNL genotypes corroborating the geographical confinement of these genotypes. Taken together the study suggests that the observed diversity in IYSV population and temporal shift in IYSVBR genotype is attributable to genetic recombination, abundance of purifying selection, insignificant positive selection and population expansion. Restricted gene flow between the two major IYSV genotypes further emphasizes the role of genetic drift in modeling the population architecture, evolutionary lineage and epidemiology of IYSV.

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.

Dahlia mosaic is a serious disease affecting dahlias. In addition to the Dahlia mosaic virus (DMV) reported previously, we characterized two putative new caulimoviruses, tentatively designated as DMV-D10 and Dahlia common mosaic virus (DCMV), from dahlia. To better understand their relative incidence in dahlia, a total of 213 samples were collected during 2007 and 2008 from several varieties of cultivated dahlia (D. variabilis) in the United States. Samples were tested for the three caulimoviruses using virus-specific primers in a polymerase chain reaction. Amplicons were cloned and sequenced to confirm the infection of dahlia with these viruses. Results showed that DMV-D10 was the most prevalent (94%) followed by DCMV (48.5%) and DMV (23%). Mixed infections were common and viruses were detected irrespective of symptom expression at the time of sampling. Two percent of the samples were not infected by any of the three tested caulimoviruses. Results suggest that caulimovirus infections are widespread in dahlia and highlight the need for testing and production of virus-free material to reduce their spread.

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 (840 bp). 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.

Dahlia is an important ornamental crop in the U.S. The economic value of the crop is often affected by viral diseases. Of several viruses that infect dahlia, dahlia mosaic virus (DMV) is of most concern. However, little or no information is available about its distribution. A survey of dahlias in several states in the U.S. was carried out during 2003 and 2004. Samples from CA, GA, MD, ME, MT, NM, PA, OR, and WA were tested for DMV. To develop a molecular detection assay, the viral genome was cloned and sequenced and based on the sequence information, DMV-specific primers were used in a PCR-based assay. DMV was detected in >90% of the samples tested. Based on the detection of DMV, a wide range of symptoms were found to be associated with DMV infection. A real-time PCR assay was adapted for rapid detection of DMV. Considering its widespread occurrence, steps are needed to limit its further spread. An effective intervention program would include use of virus-free material to minimize its impact. Availability of a rapid and sensitive detection method such as the once described should facilitate not only production of virus-free dahlias but elimination of virus infected material from breeding and propagating stocks. This is the first report of a survey to determine the extent of DMV incidence in dahlias.

The potato psyllid, Bactericera cockerelli (Sulc) (Hemiptera: Triozidae) is a serious pest of potato and other solanaceous crops. B. cockerelli has been associated with the bacterium "Candidatus Liberibacter solanacearum" (Lso), the causal agent of zebra chip, a new and economically important disease of potato in the United States, Mexico, Central America, and New Zealand. The biology of liberibacter transmission to potato and other host plants by the potato psyllid is largely unknown. The current study determined Lso acquisition by adult psyllids following different acquisition access periods (AAP) on potato and tomato, quantified Lso titer over time in postacquisition psyllids, determined Lso-acquisition rate in psyllids at each AAP on each source of inoculum, and determined influence of host plant Lso titer on Lso acquisition rates and postacquisition titer in psyllids over time. Results showed that Lso detection rates and titer increased over time in psyllids following AAPs of 8, 24, and 72 h on tomato and potato and Lso titer was highest when psyllids acquired Lso from tomato versus potato. Lso titer ranged from 200- to 400-fold higher in tomato leaves, petioles, and stems than those of potato. The increase of Lso titer in the insects reached a plateau after an average of 15 d following 24 and 72 h AAP on potato or tomato. At this 15-d plateau, Lso titer in postacquisition psyllids was comparable with that of infective psyllids from the Lso-infected laboratory colony. Lso-acquisition rate in psyllids fed on potato and tomato increased up to 5 and 20, 15 and 35, 35 and 75, and 80 and 100%, respectively, when the insects were allowed access to plants for 4, 8, 24, and 72 h, respectively.

BACKGROUND:Viral small RNAs (vsiRNAs) in the infected host can be generated from viral double-stranded RNA replicative intermediates, self-complementary regions of the viral genome or from the action of host RNA-dependent RNA polymerases on viral templates. The vsiRNA abundance and profile as well as the endogenous small RNA population can vary between different hosts infected by the same virus influencing viral pathogenicity and host response. There are no reports on the analysis of vsiRNAs of Tomato spotted wilt virus (TSWV), a segmented negative stranded RNA virus in the family Bunyaviridae, with two of its gene segments showing ambisense gene arrangement. The virus causes significant economic losses to numerous field and horticultural crops worldwide. PRINCIPAL FINDINGS:Tomato spotted wilt virus (TSWV)-specific vsiRNAs were characterized by deep sequencing in virus-infected experimental host Nicotiana benthamiana and a commercial, susceptible host tomato. The total small (s) RNA reads in TSWV-infected tomato sample showed relatively equal distribution of 21, 22 and 24 nt, whereas N. benthamiana sample was dominated by 24 nt total sRNAs. The number of vsiRNA reads detected in tomato was many a magnitude (~350:1) higher than those found in N. benthamiana, however the profile of vsiRNAs in terms of relative abundance 21, 22 and 24 nt class size was similar in both the hosts. Maximum vsiRNA reads were obtained for the M RNA segment of TSWV while the largest L RNA segment had the least number of vsiRNAs in both tomato and N. benthamiana. Only the silencing suppressor, NSs, of TSWV recorded higher antisense vsiRNA with respect to the coding frame among all the genes of TSWV. SIGNIFICANCE:Details of the origin, distribution and abundance of TSWV vsiRNAs could be useful in designing efficient targets for exploiting RNA interference for virus resistance. It also has major implications toward our understanding of the differential processing of vsiRNAs in antiviral defense and viral pathogenicity.

Thrips-transmitted Iris yellow spot virus (IYSV) (Family Bunyaviridae, Genus Tospovirus) affects onion production in the United States and worldwide. The presence of IYSV in Georgia was confirmed in 2003. Two important thrips species that transmit tospoviruses, the onion thrips (Thrips tabaci (Lindeman)) and the tobacco thrips (Frankliniella fusca (Hinds)) are known to infest onion in Georgia. However, T. tabaci is the only confirmed vector of IYSV. Experiments were conducted to test the vector status of F. fusca in comparison with T. tabaci. F. fusca and T. tabaci larvae and adults reared on IYSV-infected hosts were tested with antiserum specific to the nonstructural protein of IYSV through an antigen coated plate ELISA. The detection rates for F. fusca larvae and adults were 4.5 and 5.1%, respectively, and for T. tabaci larvae and adults they were 20.0 and 24.0%, respectively, indicating that both F. fusca and T. tabaci can transmit IYSV. Further, transmission efficiencies of F. fusca and T. tabaci were evaluated by using an indicator host, lisianthus (Eustoma russellianum (Salisbury)). Both F. fusca and T. tabaci transmitted IYSV at 18.3 and 76.6%, respectively. Results confirmed that F. fusca also can transmit IYSV but at a lower efficiency than T. tabaci. To attest if low vector competency of our laboratory-reared F. fusca population affected its IYSV transmission capability, a Tomato spotted wilt virus (Family Bunyaviridae, Genus Tospovirus) transmission experiment was conducted. F. fusca transmitted Tomato spotted wilt virus at a competent rate (90%) suggesting that the transmission efficiency of a competent thrips vector can widely vary between two closely related viruses.

In the Mediterranean region, pea, bean, and faba bean production is affected by around 17 major viruses. These viruses do not have the same ecology and consequently require a variety of different preventive measures to control them. Some of these viruses have a narrow host range, such as Faba bean necrotic yellows virus (FBNYV), and others, such as Alfalfa mosaic virus (AMV) and Cucumber mosaic virus (CMV), a very wide host range. Such features are important when identifying sources of virus inoculum in a region, and the vectors can transmit viruses from natural reservoirs to the crop plants. Some of these viruses are seed borne and, consequently, can be disseminated long distances through infected seeds. Crop losses caused by these viruses are variable, depending on the sensitivity and susceptibility of the crop to infection. Host resistance genes have been identified for some of these viruses, but in others, such as FBNYV, no resistance genes in faba bean have been identified yet. Significant progress was made in developing precise methods for the identification of these viruses, and new virus problems are being identified every year. This chapter is not intended to be a review for pea, bean, and faba bean viruses, but rather focuses on the major viruses which affect these crops in the Mediterranean basin with focus on the progress made over the past two decades.

Iris yellow spot virus (IYSV; genus Tospovirus, family Bunyaviridae) is a serious constraint for onion production in the United States and several other parts of the world. A few experimental indicator hosts were reported for IYSV but there is no report of infection of Arabidopsis by IYSV. Establishing IYSV infection in onion by mechanical inoculation has so far proven to be inefficient resulting in numerous escapes. Using Arabidopsis thaliana as a host for studies on the biology and tospovirus-host interactions has an advantage of the plant's small genome size, short life cycle, and a genome that has been sequenced. Several ecotypes were evaluated for their susceptibility to IYSV and identified some where IYSV produces localized infection. Together with wild type and various mutants, the IYSV-Arabidopsis system could be useful for studying host-tospovirus interactions. Accepted for publication 12 June 2012. Published 14 July 2012.

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.

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.

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.

The diseases caused by thrips-transmitted tospoviruses (genus Tospovirus, family Bunyaviridae) are a major constraint to production of important vegetable, legume and ornamental crops in different parts of the world. Tospoviruses are characterized by having tripartite RNA genomes and utilizing both negative and ambisense genome expression strategies. Their often wide and overlapping host ranges, emergence of resistance-breaking strains, circulative and propagative relationship with polyphagous thrips vectors, and difficulties in predicting their outbreaks pose challenges to development and implementation of effective management programmes. Despite these challenges, for a few tospoviruses, considerable progress has been made in successful development and deployment of practical and effective integrated disease management programmes. This has been due to increased understanding of their molecular biology, plant-virus and virus-vector interactions and epidemiology, and to identification of risk factors that contribute to increased disease incidence and of tactics to mitigate those risk factors. However, challenges remain as resistance-breaking or other new strains of known tospoviruses and completely new tospovirus species continue to be described from various parts of the world and have the potential to cause damaging epidemics. To protect crops from the losses caused by severe tospovirus outbreaks, continued vigilance is required to identify and characterize these emerging tospoviruses, determine their impact on crop production, understand their epidemiologies and develop, evaluate and implement control measures to reduce their impact on crop production.

SBWMV was recently also reported from an adjacent county in Oregon. Confirmation of SBWMV in Washington highlights the need for increased awareness of the disease and implementing measures to contain its further spread in the region. Accepted for publication 18 September 2009. Published 4 December 2009.

) is an economically important pathogen of onion bulb and seed crops in the USA and other parts of the world (2,4). Studies on biological characteristics of the virus have been limited due to difficulties in obtaining consistent and reproducible mechanical transmission and lack of indicator hosts. The existence of strains of IYSV is not known though differences in symptomatology, disease severity, and yield losses could be attributed to the presence of different IYSV strains. To better understand the biological diversity of IYSV, several plant species were evaluated for their response to mechanical inoculation with IYSV under controlled greenhouse conditions. Seeds of

In recent years, field floriculture development in Lithuania has been given increased attention particularly for the benefit of small family farms that produce seedlings of perennial ornamentals for the domestic market and for export to the neighboring countries. To better understand the incidence and diversity of caulimoviruses recently reported in dahlia, the authors further investigated new Lithuanian dahlia cultivars for the presence of various caulimoviruses using species-specific primers and polymerase chain reaction. Accepted for publication 16 December 2009. Published 2 March 2009.

Herrero, S., Culbreath, A. K., Csinos, A. S., Pappu, H. R., Rufty, R. B., and Daub, M. E. 2000. Nucleocapsid gene-mediated transgenic resistance provides protection against Tomato spotted wilt virus epidemics in the field. Phytopathology 90:139-147. Transformation of plants with the nucleocapsid (N) gene of Tomato spotted wilt tospovirus (TSWV) provides resistance to disease development; however, information is lacking on the response of plants to natural inoculum in the field. Three tobacco cultivars were transformed with the N gene of a dahlia isolate of TSWV (TSWV-D), and plants were evaluated over several generations in the greenhouse. The resistant phenotype was more frequently observed in ‘Burley 21’ than in ‘KY-14’ or ‘K-326’, but highly resistant ‘Burley 21’ transgenic lines were resistant to only 44% of the heterologous TSWV isolates tested. Advanced generation (R 3 and R4) transgenic resistant lines of ‘Burley 21’ and a ‘K-326’ F 1 hybrid containing the N genes of two TSWV isolates were evaluated in the field near Tifton, GA, where TSWV is endemic. Disease development was monitored by symptom expression and enzyme-linked immunosorbent assay (ELISA) analysis. Whereas incidence of TSWV infection in ‘Burley 21’ susceptible controls was 20% in 1996 and 62% in 1997, the mean incidence in transgenic lines was reduced to 4 and 31%, respectively. Three transgenic ‘Burley 21’ lines were identified that had significantly lower incidence of disease than susceptible controls over the two years of the study. In addition, the rate of disease increase at the onset of the 1997 epidemic was reduced for all the ‘Burley 21’ transgenic lines compared with the susceptible controls. The ‘K-326’ F 1 hybrid was as susceptible as the ‘K326’ nontransformed control. ELISA analysis demonstrated that symptomless plants from the most resistant ‘Burley 21’ transgenic lines accumulated detectable nucleocapsid protein, whereas symptomless plants from more susceptible lines did not. We conclude that transgenic resistance to TSWV is effective in reducing incidence of the disease in the field, and that accumulation of transgene protein may be important in broad-spectrum resistance.

Iris yellow spot virus is an economically important pathogen affecting both bulb and seed onion crops in the US and several parts of the world. In January 2008, two commercial onion fields in Yuma County, Arizona, had plants with symptoms suggestive of IYSV infection. IYSV infection was confirmed by ELISA, and the virus infection was further verified by reverse transcription-polymerase chain reaction. Accepted for publication 29 May 2008. Published 11 July 2008.

Iris yellow spot virus is an economically important viral pathogen of onion. The virus has been reported from several countries in the world in recent years and, in Europe, the virus was reported from the Netherlands, Slovenia, Italy, and more recently from Spain. This is the first report of the virus in France, which ranks fifth in onion production in the European Union. The impact of the IYSV on onion and shallot in the different production areas of France remains to be seen. Accepted for publication 7 March 2008. Published 10 June 2008.

Garlic (Allium sativum L. and A. longicuspis Regel.) germplasm maintained by the USDA-ARS Western Regional Plant Introduction Station (WRPIS) was screened for four viruses commonly infecting garlic [Garlic common latent carlavirus (GCLV), Leek yellow stripe potyvirus (LYSV), Onion yellow dwarf potyvirus (OYDV), and Shallot latent carlavirus (SLV)] plus three others [Iris yellow spot tospovirus (IYSV), Tobacco rattle tobravirus (TRV), and Tomato spotted wilt tospovirus (TSWV)]. Over 200 accessions were rated for the incidence of symptomatic plants during two production seasons, in July 2005 and June 2006. In both years, incidence of symptomatic plants ranged from less than 10% to 100% in individual accessions. Laboratory testing of selected accessions showed that the accessions were predominantly infected with OYDV (60% of infections), followed by GCLV (25%) and LYSV (15%). Accepted for publication 26 June 2008. Published 19 September 2008.

A semi-automated, rapid and sensitive method that combines magnetic capture-hybridization and reverse-transcription polymerase chain reaction (MCH/RT-PCR) for the detection of plant viruses is described. The assay uses a target specific biotin-labelled oligoprobe for RNA capture and streptavidin-coated magnetic beads for subsequent RNA-oligoprobe hybrid isolation from plant lysate. Detection and specific identification was accomplished by RT-PCR. This approach was investigated for the specific detection of Tobacco rattle virus and for the detection of viruses within the Potexvirus group in leaves, dormant bulbs and corms of flower bulbs of different species. Dilution series of TRV-infected tulip leaf sap showed that MCH/RT-PCR was 70,588 times more sensitive than enzyme-linked immunosorbent assay (ELISA) and was similar to that of RT-PCR. ELISA underestimated the infection levels of TRV in field samples compared to MCH/RT-PCR. The ability of MCH/RT-PCR to be performed in a microtiter plate on an automatic nucleic acid isolation station facilitates high throughput virus diagnostics. RNA isolation and purification was rapid, specific, sensitive, contamination-free and reproducible making this method amenable for routine indexing of stock plants as part of a management plan to reduce the propagation of virus-infected plants.

Outbreaks of IYSV were first observed in May 2003 in two Imperial County onion seed fields. In August, 2005, symptomatic onion plants were widespread in four fields in the Antelope Valley, Los Angeles County, CA. IYSV infection was confirmed by ELISA and RT-PCR. This was the first known recording of IYSV in Antelope Valley. Increasing incidence and impact of IYSV in a major onion-growing area highlights the need for research into developing managing options for this important disease of onion. Accepted for publication 22 January 2007. Published 8 May 2007.

The potato psyllid, Bactericera cockerelli (Šulc) (Hemiptera: Triozidae), is a vector of the phloem-limited bacterium 'Candidatus Liberibacter solanacearum' (Lso), the putative causal agent of zebra chip disease of potato. Little is known about how potato psyllid transmits Lso to potato. We used electrical penetration graph (EPG) technology to compare stylet probing behaviors and efficiency of Lso transmission of three haplotypes of potato psyllid (Central, Western, Northwestern). All haplotypes exhibited the full suite of stylet behaviors identified in previous studies with this psyllid, including intercellular penetration and secretion of the stylet pathway, xylem ingestion, and phloem activities, the latter comprising salivation and ingestion. The three haplotypes exhibited similar frequency and duration of probing behaviors, with the exception of salivation into phloem, which was of higher duration by psyllids of the Western haplotype. We manipulated how long psyllids were allowed access to potato ("inoculation access period", or IAP) to examine the relationship between phloem activities and Lso transmission. Between 25 and 30% of psyllids reached and salivated into phloem at an IAP of 1 hr, increasing to almost 80% of psyllids as IAP was increased to 24 h. Probability of Lso-transmission was lower across all IAP levels than probability of phloem salivation, indicating that a percentage of infected psyllids which salivated into the phloem failed to transmit Lso. Logistic regression showed that probability of transmission increased as a function of time spent salivating into the phloem; transmission occurred as quickly as 5 min following onset of salivation. A small percentage of infected psyllids showed extremely long salivation events but nonetheless failed to transmit Lso, for unknown reasons. Information from these studies increases our understanding of Lso transmission by potato psyllid, and demonstrates the value of EPG technology in exploring questions of vector efficiency.