James W. Moyer

The Role of Weed Hosts and Tobacco Thrips, Frankliniella fusca, in the Epidemiology of Tomato spotted wilt virus

Wild plant species were systematically sampled to characterize reproduction of thrips, the vector of Tomato spotted wilt virus (TSWV), and natural sources TSWV infection. Thrips populations were monitored on 28 common perennial, biennial, and annual plant species over two noncrop seasons at six field locations across North Carolina. Sonchus asper, Stellaria media, and Taraxacum officianale consistently supported the largest populations of immature TSWV vector species. The tobacco thrips, Frankliniella fusca, was the most abundant TSWV vector species collected, comprising over 95% of vector species in each survey season. Perennial plant species (i.e., Plantago rugelii and Taraxacum officianale) were often only locally abundant, and many annual species (Cerastium vulgatum, Sonchus asper, and Stellaria media) were more widely distributed. Perennial species, including P. rugelii and Rumex crispus, remained TSWV infected for 2 years in a small-plot field test. Where these perennial species are locally abundant, they may serve as important and long-lasting TSWV inoculum sources. In random surveys across 12 locations in North Carolina, TSWV infection was documented by double antibody sandwich enzyme-linked immunosorbent assay in 35 of 72 (49%) common perennial (N = 10), biennial (N = 4), and annual (N = 21) plant species across 18 plant families. Estimated rates of TSWV infection were highest in Cerastium vulgatum (4.2%), Lactuca scariola (1.3%), Molluga verticillata (4.3%), Plantago rugelii (3.4%), Ranunculus sardous (3.6%), Sonchus asper (5.1%), Stellaria media (1.4%), and Taraxacum officianale (5.8%). Nine plant species were determined to be new host recordings for TSWV infection, including Cardamine hirsuta, Eupatorium capillifolium, Geranium carolinianum, Gnaphalium purpureum, Linaria canadense, Molluga verticillata, Pyrrhopappus carolinianus, Raphanus raphanistrum, and Triodanis perfoliata. Our findings document the relative potential of a number of common annual, biennial, and perennial plant species to act as important reproductive sites for F. fusca and as acquisition sources of TSWV for spread to susceptible crops.

The molecular population genetics of the Tomato spotted wilt virus (TSWV) genome.

RNA viruses are characterized by high genetic variability resulting in rapid adaptation to new or resistant hosts. Research for plant RNA virus genetic structure and its variability has been relatively scarce compared to abundant research done for human and animal RNA viruses. Here, we utilized a molecular population genetic framework to characterize the evolution of a highly pathogenic plant RNA virus [Tomato spotted wilt virus (TSWV), Tospovirus, Bunyaviridae]. Data from genes encoding five viral proteins were used for phylogenetic analysis, and for estimation of population parameters, subpopulation differentiation, recombination, divergence between Tospovirus species, and selective constraints on the TSWV genome. Our analysis has defined the geographical structure of TSWV, attributed possibly to founder effects. Also, we identify positive selection favouring divergence between Tospovirus species. At the species level, purifying selection has acted to preserve protein function, although certain amino acids appear to be under positive selection. This analysis provides demonstration of population structuring and species‐wide population expansions in a multisegmented plant RNA virus, using sequence‐based molecular population genetic analyses. It also identifies specific amino acid sites subject to selection within Bunyaviridae and estimates the level of genetic heterogeneity of a highly pathogenic plant RNA virus. The study of the variability of TSWV populations lays the foundation in the development of strategies for the control of other viral diseases in floral crops.

Overwintering of Frankliniella fusca (Thysanoptera: Thripidae) on Winter Annual Weeds Infected with Tomato spotted wilt virus and Patterns of Virus Movement Between Susceptible Weed Hosts.

ABSTRACT Overwintering of tobacco thrips, Frankliniella fusca, was investigated on common winter annual host plants infected with Tomato spotted wilt virus (TSWV). Populations of tobacco thrips produced on TSWV-infected plants did not differ from those produced on healthy plants, whereas populations varied greatly among host plant species. The mean per plant populations of F. fusca averaged 401, 162, and 10 thrips per plant on Stellaria media, Scleranthus annuus, and Sonchus asper, respectively, during peak abundance in May. Adult F. fusca collected from plant hosts were predominately brachypterous throughout the winter and early spring, but macropterous forms predominated in late spring. Weed hosts varied in their ability to serve as overwintering sources of TSWV inoculum. Following the initial infection by TSWV in October 1997, 75% of Scleranthus annuus and Stellaria media retained infection over the winter and spring season, whereas only 17% of Sonchus asper plants remained infected throughout the same interval. Mortality of TSWV-infected Sonchus asper plants exceeded 25%, but mortality of infected Stellaria media and Scleranthus annuus did not exceed 8%. TSWV transmission by thrips produced on infected plants was greatest on Stellaria media (18%), intermediate on Scleranthus annuus (6%), and lowest on Sonchus asper (2%). Very few viruliferous F. fusca were recovered from soil samples collected below infected wild host plants. Vegetative growth stages of Stellaria media, Sonchus asper, and Ranunculus sardous were more susceptible to F. fusca transmission of TSWV than flowering growth stages, whereas both growth stages of Scleranthus annuus were equally susceptible. In a field study to monitor the spatial and temporal patterns of virus movement from a central source of TSWV-infected Stellaria media to adjacent plots of R. sardous, the incidence of infection in R. sardous plots increased from <1% in March to >42% in June 1999. Infection levels in the Stellaria media inoculum source remained high throughout the experiment, averaging nearly 80% until June 1999 when all Stellaria media plants had senesced. Dispersal of TSWV from the inoculum source extended to the limits of the experimental plot (>37 m). Significant directional patterns of TSWV spread to the R. sardous plots were detected in April and May but not in June. R. sardous infections were detected as early as March and April, suggesting that overwintering inoculum levels in an area can increase rapidly during the spring in susceptible weed hosts prior to planting of susceptible crops. This increase in the abundance of TSWV inoculum sources occurs at a time when vector populations are increasing rapidly. The spread of TSWV among weeds in the spring serves to bridge the period when overwintered inoculum sources decline and susceptible crops are planted.

High frequency shoot regeneration and Agrobacterium-mediated transformation of chrysanthemum (Dendranthema grandiflora)

Abstract An efficient, high-frequency transformation protocol was developed for the commercial chrysanthemum cultivar ‘Iridon’. Regeneration protocols were also developed for two other commercial cultivars, ‘Hekla’ and ‘Polaris’. All protocols utilized embryogenesis media composed of the basal medium of Murashige and Skoog (MS) supplemented with 11.5 μM indoleacetic acid and 0.5 or 1.0 μM benzyladenine. Regenerationof shoots from cultivar ‘Iridon’ was obtained with continuous culture on these media until shoots were transferred to rooting medium at approximately 6 weeks. By contrast, shoot regeneration from ‘Hekla’ explants required transfer to hormone-free medium 2 weeks after initial culturing. ‘Polaris’ regeneration required transfer of explants to hormone-free medium when shoot primordia first developed as well as continuous culture of explants in the absence of blue wavelengths of light. Shoots from all cultivars were rooted on 25% MS medium with 0.5 μM naphthaleneacetic acid. Three wild type strains of Agrobacterium tumefaciens (Ach5, A281, Chry5) were evaluated for tumor production on the three cultivars. Chry5 and A281 were significantly more virulent on all three cultivars than was Ach5. Transformed plants of ‘Iridon’ were obtained using Agrobacterium strain EHA105, a disarmed version of A281. Explants were transformed with two plasmids, pBI121, which encodes kanamycin resistance and β-glucuronidase (GUS) activity, and pBI121 containing the nucleocapsid gene of a highly virulent isolate of tomato spotted wilt virus (TSWV). Transformed shoots regenerated and rooted on medium containing 50 μg/ml kanamycin. Vegetatively propagated progeny of transformed plants were identified which expressed GUS activity and which contained multiple copies of the TSWV nucleocapsid gene.

Field resistance to Tomato spotted wilt virus in transgenic peanut (Arachis hypogaea L.) expressing an antisense nucleocapsid gene sequence

Peanut (Arachis hypogaea L.) lines transgenic for the antisense nucleocapsid (N) gene of a Tomato spotted wilt virus (TSWV) strain isolated from peanut were generated by microprojectile-mediated transformation of repetitive somatic embryos of cultivars VC1 and AT120. The selectable marker (hygromycin resistance) and the N gene were on separate plasmids. A total of 207 VC1 and 120 AT120 hygromycin-resistant lines were produced. Of all the VC1 plants recovered 71% were cotransformed with the N gene (N+), but all plants were sterile. For AT120, 48 of the transgenic cell lines converted into plants. Polymerase chain reaction (PCR) screening showed 15 of the lines were transgenic for the N gene (N+), and two of these lines were fertile. A field test was conducted in 1998 at Ashburn, GA, using seeds from each fertile line, along with segregated and non-transgenic controls. Plants from four randomly selected field plots were examined for symptoms and analyzed by double-antibody sandwich enzyme-linked immunoabsorbent assay and PCR at 10 and 14 weeks after planting. At 14 weeks, 76% of the N+ plants were symptomless, while 2% were severely symptomatic or dead. In contrast, only 42% of the plants lacking the N gene were symptomless and 50% were severely symptomatic or dead. Northern blot analysis of selected field-resistant plants detected transgene RNA, and the transcript level appeared undiminished after viral exposure.

Variation in thrips species composition in field crops and implications for tomato spotted wilt epidemiology in North Carolina

Thrips were surveyed in tomato spotted wilt‐susceptible crops in five areas across North Carolina. Tomato, pepper, and tobacco plants in commercial fields were sampled and 30 species of thrips were collected over a 3‐year period. The most common species overall was Frankliniella tritici (Fitch). The most common thrips species that are known to vector Tomato Spotted Wilt Virus (TSWV) were F. fusca (Hinds), and F. occidentalis (Pergande). Relatively low numbers of Thrips tabaci Lindeman, another reported vector, were collected. The spatial and temporal occurrence of vectors varied with sampling method, crop species, region of North Carolina, and localized areas within each region. In a laboratory experiment, no difference was detected between the ability of F. fusca and F. occidentalis to acquire and transmit a local isolate of TSWV. Based on vector efficiency and occurrence, F. fusca is considered the most important vector of TSWV in tobacco, whereas both F. fusca and F. occidentalis are important vectors of TSWV in tomato and pepper.

Tomato spotted wilt virus resistance in chrysanthemum expressing the viral nucleocapsid gene

Three tomato spotted wilt virus (TSWV) nucleocapsid (N) gene constructs were employed for Agrobacterium-mediated transformation of chrysanthemum (Dendranthema grandiflora) cv. Polaris. These constructs contained either a full-length N gene (pTSWVN+), a full-length N gene encoding a truncated N protein (pTSWVNt), or an antisense version of the full-length N gene (pTSWVN-), all derived from a dahlia isolate of TSWV (TSWV-D). Initial resistance screens were conducted on cuttings made from 152 pTSWVN+, 37 pTSWVNt, and 47 pTSWVN- transformed plants employing a highly virulent, heterologous strain of TSWV (TSWV-GB) isolated from chrysanthemum and vectored by thrips. This screening served to eliminate the majority of TSWV-susceptible transgenic lines. More rigorous resistance tests with three rounds of mechanical inoculation with TSWV-GB identified one pTSWVNt and two pTSWVN- transformed lines that exhibited a total lack of systemic symptoms and no virus accumulation. Six other lines, including some pTSWVN+, exhibited a lack of one or more of the destructive necrotic TSWV symptoms (stem canker and apical bud death) and a delay in symptom expression. Both sense and antisense constructs, therefore, were found to be effective at yielding TSWV resistance in chrysanthemum. Molecular analysis revealed that the highly TSWV-resistant pTSWVNt line had no detectable levels of N protein. All three resistant lines had low levels of N gene transcript and at least three transgene insertion sites within their genomes, although susceptible lines often had a similar number of insertion sites. The generation of Polaris lines resistant to TSWV transmitted either mechanically or by thrips represents the first time a major ornamental crop has been genetically engineered for disease resistance.

Diallel analysis of sweetpotatoes for resistance to sweetpotato virus disease

Sweetpotato virus disease (SPVD) is due to the dual infection and synergistic interaction of Sweetpotato feathery mottle potyvirus (SPFMV) and Sweetpotato chlorotic stunt crinivirus(SPCSV), and causes up to 98% yield loss in sweetpotato in East Africa. This study was conducted to determine the inheritance of resistance to SPVD in sweetpotato and to estimate the nature of genetic variance. Ten parental clones varying in reaction to SPVD were crossed in a half diallel mating design to generate 45 full-sib families. The families were graft-inoculated with SPCSV and SPFMV to induce SPVD and evaluated for resistance in a randomized complete block design at two sites in Namulonge, Uganda during 1998–2000. In serological assays for SPFMV and SPCSV,resistance to symptom development and recovery from initial systemic SPVD symptoms, characterised resistant genotypes. Genetic component analysis showed significant effects for both general combining ability (GCA) and specific combining ability (SCA) for resistance to SPVD. GCA to SCA variance component ratios were large (0.51–0.87), hence GCA effects were more important than SCA effects. Resistant parents exhibited high GCA indicating that additive gene effects were predominant in the inheritance of resistance to SPVD and recovery. Narrow-sense heritability (31–41%) and broad-sense heritability (73–98%) were moderate to high, indicating that rapid genetic gains for SPVD resistance could be accomplished by mass selection breeding techniques. Two genotypes, New Kawogo and Sowola, had high negative GCA effects and had several families in specific crosses,which exhibited rapid recovery from SPVD,and are promising parents for enhancement of SPVD resistance and recovery.

Population Genetic Analysis of Tomato spotted wilt virus on Peanut in North Carolina and Virginia

Exploring the genetic diversity and evolutionary history of plant viruses is critical to understanding their ecology and epidemiology. In this study, maximum-likelihood and population genetics-based methods were used to investigate the population structure, genetic diversity, and sources of genetic variation in field isolates of Tomato spotted wilt virus (TSWV) from peanut in North Carolina and Virginia. Selected regions of the nucleocapsid, movement, and RNA-dependent RNA polymerase genes were amplified and sequenced to identify haplotypes and infer genetic relationships between isolates of TSWV with heuristic methods. The haplotype structure of each locus consisted of 1 or 2 predominant haplotypes and >100 haplotypes represented by a single isolate. No specific haplotypes were associated with geographic area, peanut cultivar, or year of isolation. The population was panmictic at the regional level and high levels of genetic diversity were observed among isolates. There was evidence for positive selection on single amino acids in each gene on a background of predominant purifying selection acting upon each locus. The results of compatibility analyses and the persistence of specific gene sequences in isolates collected over three field seasons suggest that recombination was occurring in the population. Estimates of the population mutation rate suggest that mutation has had a significant effect on the shaping of this population and, together with purifying selection, these forces have been the predominant evolutionary forces influencing the TSWV population in peanut in North Carolina and Virginia.

Tomato spotted wilt virus on potato in eastern North Carolina

Leaf, stem, and tuber samples collected from diseased potato plants exhibiting tospovirus-like symptoms in eastern North Carolina (NC) were assayed for the presence ofTomato spotted wilt virus (TSWV) by serology (ImmunoStrips) and reverse transcription-polymerase chain reaction (RT-PCR) methods. All symptomatic samples tested positive for TSWV with serology and RT-PCR. Mechanical inoculation of the indicator hostNicotiana benthamiana with symptomatic leaf, stem, and tuber tissue resulted in plants with symptoms of systemic virus infection. The presence of TSWV in infected tissue ofN. benthamiana was confirmed with serology, RT-PCR, and sequence analysis of the nucleocapsid (N) protein gene. Phylogenetic analysis of the predicted N protein of three isolates of TSWV from potato in NC and 11 isolates obtained from peanut, pepper, and tomato in Georgia and NC suggested that the isolates were related. One isolate of TSWV from potato in NC was selected for vector transmission tests, and the virus was transmitted to potato with tobacco thrips (Frankliniella fusca), but no vertical transmission from infected seed tubers to actively developing potato sprouts, stems or leaves was observed. This is the first documentation of the occurrence, isolation and characterization of TSWV from field-grown potato in North America.