Iris Yellow Spot Virus Prolongs the Adult Lifespan of Its Primary Vector, Onion Thrips (Thrips tabaci) (Thysanoptera: Thripidae).

Iris yellow spot virus (IYSV) from the genus Tospovirus, family Peribunyaviridae, reduces yield in several crops, especially Allium spp. IYSV is primarily transmitted by onion thrips (Thrips tabaci), but little is known about how IYSV impacts the biology of its principal vector. In a controlled experiment, the effect of IYSV on the lifespan and fecundity of onion thrips was examined. Larvae were reared on IYSV-infected onions until pupation. Individual pupae were confined until adults eclosed, and the lifespan and total progeny produced per adult were monitored daily. Thrips were tested for the virus in reverse-transcriptase polymerase chain reaction using specific primers to confirm the presence of IYSV. Results indicated that 114 and 35 out of 149 eclosing adults tested positive (viruliferous) and negative (nonviruliferous) for IYSV, respectively. The viruliferous adults lived 1.1-6.1 d longer (average of 3.6 d) than nonviruliferous adults. Fecundity of viruliferous and nonviruliferous onion thrips was similar with 2.0 ± 0.1 and 2.3 ± 0.3 offspring produced per female per day, respectively. Fecundity for both viruliferous and nonviruliferous thrips also was significantly positively correlated with lifespan. These findings suggest that the longer lifespan of viruliferous onion thrips adults may allow this primary vector of IYSV to infect more plants, thereby exacerbating IYSV epidemics.

Importance of Transplanted Onions Contributing to Late-Season Iris yellow spot virus Epidemics in New York.

Iris yellow spot virus (IYSV) is an economically significant tospovirus of onion transmitted by onion thrips (Thrips tabaci Lindeman). IYSV epidemics in onion fields are common in New York; however, the role of various habitats contributing to viruliferous onion thrips populations and IYSV epidemics is not known. In a 2-year field study in New York, the abundance of dispersing onion thrips, including those determined to be viruliferous via reverse-transcriptase polymerase chain reaction, was recorded in habitats known to harbor both IYSV and its vector. Results showed that viruliferous thrips were encountered in all habitats; however, transplanted onion sites accounted for 49 to 51% of the total estimated numbers of viruliferous thrips. During early to midseason, transplanted onion sites had 9 to 11 times more viruliferous thrips than the other habitats. These results indicate that transplanted onion fields are the most important habitat for generating IYSV epidemics in all onion fields (transplanted and direct-seeded) in New York. Our findings suggest that onion growers should control onion thrips in transplanted fields early in the season to minimize risk of IYSV epidemics later in the season.

Biochemical analysis of NSs from different tospoviruses.

Tospoviruses suppress antiviral RNA interference by coding for an RNA silencing suppressor (NSs) protein. Previously, using NSs-containing crude plant and insect cell extracts, the affinity of NSs for double-stranded (ds)RNA molecules was demonstrated by electrophoretic mobility shifts assays (EMSAs). While NSs from tomato spotted wilt virus (TSWV) and groundnut ringspot virus (GRSV) were able to bind small and long dsRNA molecules, the one from tomato yellow ring virus (TYRV), a distinct Asian tospovirus, only bound small dsRNA. Here, using bacterially expressed and purified NSs from GRSV and TYRV, it is shown that they are both able to bind to small and long dsRNA. Binding of siRNAs by NSs revealed two consecutive shifts, i.e. a first shift at low NSs concentrations followed by a second larger one at higher concentrations. When NSs of TSWV resistance inducing (RI) and resistance breaking (RB) isolates were analyzed using extracts from infected plants only a major siRNA shift was observed. In contrast, plant extracts containing the respective transiently expressed NSs proteins showed only the lower shift with NSsRI but no shift with NSsRB. The observed affinity for RNA duplexes, as well as the two-stepwise shift pattern, is discussed in light of NSs as a suppressor of silencing and its importance for tospovirus infection.

Receiver Operating Characteristic curve analysis determines association of individual potato foliage volatiles with onion thrips preference, cultivar and plant age.

Tomato spotted wilt virus (TSWV) causes sporadic but serious disease in Australian potato crops. TSWV is naturally spread to potato by thrips of which Thrips tabaci is the most important. Prior studies indicated possible non-preference of potato cultivars to T. tabaci. Select potato cultivars were assessed for non-preference to T. tabaci in paired and group choice trials. Cultivars 'Bismark', 'Tasman' and 'King Edward' were less preferred than 'Atlantic', 'Russet Burbank' and 'Shepody'. Green leaf volatiles were sampled using solid-phase microextraction from the headspace of potato cultivars of two ages that differed in T. tabaci preference. Analysis of headspace volatile data using Receiver Operating Characteristic curves identified individual volatiles associated with T. tabaci preference and non-preference, young and old plants and individual cultivars. These data could be used to inform breeding programs for selection of T. tabaci resistance to assist with TSWV management, and biological testing of novel thrips management compounds.

Thrips Settling, Oviposition and IYSV Distribution on Onion Foliage.

Thrips tabaci Lindeman (Thysanoptera: Thripidae) adult and larval settling and oviposition on onion (Allium cepa L.) foliage were investigated in relation to leaf position and leaf length at prebulb plant growth stages under controlled conditions. In the laboratory, four and six adult females of T. tabaci were released on onion plants at three-leaf stage and six- to eight-leaf stage, respectively, and thrips egg, nymph, and adult count data were collected on each of the three inner most leaves at every 2-cm leaf segment. Thrips settling and oviposition parameters were quantified during the light period on the above ground portion of onion plants from the distal end of the bulb or leaf sheath "neck" through the tips of the foliage. Results from studies confirmed that distribution of thrips adults, nymphs, and eggs were skewed toward the base of the plant. The settling distributions of thrips adults and nymphs differed slightly from the egg distribution in that oviposition occurred all the way to the tip of the leaf while adults and nymphs were typically not observed near the tip. In a field study, the foliage was divided into three equal partitions, i.e., top, middle, basal thirds, and thrips adults by species, primarily Frankliniella fusca (Hinds) and T. tabaci, were collected from each partition to determine if there was a similar bias of all adult thrips toward the base of the plant. The results suggested that adults of different species appear to segregate along leaf length. Finally, thrips oviposition on 2-cm segments and Iris yellow spot virus positive leaf segments were quantified in the field, irrespective of thrips species. Both variables demonstrated a very similar pattern of bias toward the base of the plant and were significantly correlated.

Long-Distance Dispersal Potential for Onion Thrips (Thysanoptera: Thripidae) and Iris yellow spot virus (Bunyaviridae: Tospovirus) in an Onion Ecosystem.

Onion thrips, Thrips tabaci Lindeman, is a worldwide pest of onion whose feeding damage and transmission of Iris yellow spot virus (IYSV) may reduce onion yields. Little is known about the seasonal dynamics of T. tabaci dispersal, the distance of dispersal, or the movement of thrips infected with IYSV during the onion-growing season. To address these questions, T. tabaci adults were collected using transparent sticky card traps in commercial onion fields three times during the onion-growing season (June, July, and late August) at varying heights above the canopy (0.5-6 m above soil surface) and with trap-equipped unmanned aircraft (UAVs) flying 50-60 m above onion fields during August sampling periods in 2012 and 2013. Randomly selected subsamples of captured T. tabaci were tested for IYSV using RT-PCR. Most T. tabaci adults were captured in late August and near the onion canopy (<2 m) throughout the season. However, 4% of T. tabaci adults captured on sticky cards were at altitudes ≥2 m, and T. tabaci were also captured on UAV-mounted traps. These data strongly suggest that long-distance dispersal occurs. More T. tabaci captured on sticky cards tested positive for IYSV in August (53.6%) than earlier in the season (2.3 to 21.5% in June and July, respectively), and 20 and 15% of T. tabaci captured on UAV-mounted traps tested positive for IYSV in 2012 and 2013, respectively. Our results indicate that T. tabaci adults, including viruliferous individuals, engage in long-distance dispersal late in the season and likely contribute to the spread of IYSV.

Genetic and host-associated differentiation within Thrips tabaci Lindeman (Thysanoptera: Thripidae) and its links to Tomato spotted wilt virus-vector competence.

Of eight thelytokous populations of onion thrips (Thrips tabaci) collected from potato (three populations), onion (four) or Chrysanthemum (one) hosts from various regions of Australia, only those from potato were capable of transmitting Tomato spotted wilt virus (TSWV) in controlled transmission experiments. Genetic differentiation of seven of these eight populations, and nine others not tested for TSWV vector competence, was examined by comparison of the DNA sequences of mitochondrial cytochrome oxidase subunit 1 (COI) gene. All Australian populations of T. tabaci grouped within the European 'L2' clade of Brunner et al. (2004). Within this clade the seven populations from potato, the three from onion, and the four from other hosts (Chrysanthemum, Impatiens, lucerne, blackberry nightshade) clustered as three distinct sub-groupings characterised by source host. Geographical source of thrips populations had no influence on genetic diversity. These results link genetic differentiation of thelytokous T. tabaci to source host and to TSWV vector capacity for the first time.

Transmission of Iris yellow spot virus by Frankliniella fusca and Thrips tabaci (Thysanoptera: Thripidae).

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.

Elimination of TSWV from impatiens hawkerii Bull. and regeneration of virus-free plant

The possibility for obtaining virus free plants from Impatiens hawkerii Bull. shoots infected with Tomato spotted wilt virus (TSWV) through meristem-tip culture was examined. TSWV presence in I. hawkerii plants was detected by DAS-ELISA and RT-PCR and identification of the virus was confirmed by sequencing one of the chosen isolate (GenBank Accesion CQ132190). Meristem-tip explants (0.3-1.5 mm) from virus-infected shoots are cultured on MS media supplemented with different concentrations of the cytokinins, CPPU or TDZ (0.01-1.0 uM), respectively. Using this system, a large number of in vitro shoots could be produced from a single explant. Also, cytokinins showed a stimulatory effect on the length, fresh and dry weights of the newly formed shoots. Plant pigments content in I. hawkerii shoots increased significantly in the presence of cytokinins. Rooting of shoots was spontaneous on the same media. Rooted plantlets were transferred to soil where 97 percent successfully acclimatized. By DAS-ELISA and RT-PCR, 80 percent of the in vitro plantlets were shown to be a virus-free. Considering these, the present protocol seems to be an efficient method for in vitro generation of virus-free I. hawkerii plantlets by meristem tip cultures.

Evaluation of onion cultivars for resistance to onion thrips (Thysanoptera: Thripidae) and Iris yellow spot virus.

Onion thrips, Thrips tabaci Lindeman (Thysanoptera: Thripidae), a worldwide pest of onion, Allium cepa L., can reduce onion yield by > 50% and be even more problematic when it transmits Iris yellow spot virus (family Bunyaviridae, genus Tospovirus, IYSV). Because T. tabaci is difficult to control with insecticides and other strategies, field studies on onion, Allium cepa L., resistance to T. tabaci and IYSV were conducted in 2007 and 2008 in two locations in New York state. Forty-nine cultivars were evaluated for resistance by counting the number of larvae weekly and recording leaf damage. In another experiment, the impact of T. tabaci and IYSV on plant growth and yield was examined by spraying half of the plants with an insecticide. Eleven of the 49 cultivars had very little leaf damage and were considered resistant to T. tabaci. Visual assessment indicated that all resistant cultivars had yellow-green- colored foliage, whereas the other 38 had blue-green- colored foliage. The visual assessment of color agreed with data on color taken with a HunterLab Ultra Scan XE colorimeter. The onions 'Colorado 6' and 'NMSU 03-52-1' had the lowest numbers of T. tabaci, suggesting strong antibiosis and/or antixenosis. The other nine cultivars had variable numbers of T. tabaci, indicating a possible combination of categories of resistance. In the nonprotected treatments there were significant reductions in plant height and plant weight in most of the resistant cultivars, but there were reductions in bulb weight only in a few of them. The average of plants infected with IYSV was 10% in 2007 and 60% in 2008. Our findings indicate potential for developing onion resistance to T. tabaci as part of an overall integrated pest management strategy but suggest difficulties in identifying resistance to IYSV.

Temporal dynamics of iris yellow spot virus and its vector, Thrips tabaci (Thysanoptera: Thripidae), in seeded and transplanted onion fields.

Onion thrips, Thrips tabaci (Lindeman) (Thysanoptera: Thripidae), can reduce onion bulb yield and transmit iris yellow spot virus (IYSV) (Bunyaviridae: Tospovirus), which can cause additional yield losses. In New York, onions are planted using seeds and imported transplants. IYSV is not seed transmitted, but infected transplants have been found in other U.S. states. Transplants are also larger than seeded onions early in the season, and thrips, some of which may be viruliferous, may preferentially colonize larger plants. Limited information is available on the temporal dynamics of IYSV and its vector in onion fields. In 2007 and 2008, T. tabaci and IYSV levels were monitored in six seeded and six transplanted fields. We found significantly more thrips in transplanted fields early in the season, but by the end of the season seeded fields had higher levels of IYSV. The percentage of sample sites with IYSV-infected plants remained low (<12%) until August, when infection levels increased dramatically in some fields. The densities of adult and larval thrips in August and September were better predictors of final IYSV levels than early season thrips densities. For 2007 and 2008, the time onions were harvested may have been more important in determining IYSV levels than whether the onions were seeded or transplanted. Viruliferous thrips emigrating from harvested onion fields into nonharvested ones may be increasing the primary spread of IYSV in late-harvested onions. Managing T. tabaci populations before harvest, and manipulating the spatial arrangement of fields based on harvest date could mitigate the spread of IYSV.

A CAPS marker to assist selection of tomato spotted wilt virus (TSWV) resistance in pepper.

The hypersensitive resistance to tomato spotted wilt virus (TSWV) in pepper is determined by a single dominant gene (resistant allele: Tsw) in several Capsicum chinense genotypes. In order to facilitate the selection for this resistance, four RAPD (among 250 10-mer primers tested) were found linked to the Tsw locus using the bulked segregant analysis and 153 F2 individuals. A close RAPD marker was converted into a codominant cleaved amplified polymorphic sequence (CAPS) using specific PCR primers and restriction enzymes. This CAPS marker is tightly linked to Tsw (0.9 +/- 0.6 cM) and is helpful for marker-assisted selection in a wide range of genetic intercrosses.

A protoplast system for studying tomato spotted wilt virus infection.

A plant protoplast system for studying tomato spotted wilt tospovirus (TSWV) infection was established and tested. Using polyethylene glycol-mediated inoculation with highly infectious TSWV particles, generally 50% or more of Nicotiana rustica protoplasts were infected. In these cells viral RNA and viral protein synthesis became detectable at 16 h post-inoculation (p.i.) and continued at least until 90 h p.i. Both the structural viral proteins [nucleoprotein (N) and the envelope glycoproteins G1 and G2] and the nonstructural viral proteins NSs and NSm accumulated to amounts sufficient for detection and immunocytological analysis. Local lesion tests on petunia leaves and electron microscopical analysis confirmed the production of mature, infectious virus particles, underlining the conclusion that a full infection cycle was completed in this system. Upon inoculation of Vigna unguiculata (cowpea) protoplasts with TSWV particles, comparable proportions of infected cells and amounts of NSs, NSm and N protein were obtained, but much lower amounts of viral glycoproteins were detected than in N. rustica protoplasts, and progeny virus particles were less abundant. With the N. rustica-based protoplast system, a powerful synchronized single-cell infection system has now become available for more precise in vivo studies of the processes occurring during tospovirus infection.