Craig G. Webster

Role of the Insect Supervectors Bemisia tabaci and Frankliniella occidentalis in the Emergence and Global Spread of Plant Viruses

Emergence of insect-transmitted plant viruses over the past 10-20 years has been disproportionately driven by two so-called supervectors: the whitefly, Bemisia tabaci, and the Western flower thrips, Frankliniella occidentalis. High rates of reproduction and dispersal, extreme polyphagy, and development of insecticide resistance, together with human activities, have made these insects global pests. These supervectors transmit a diversity of plant viruses by different mechanisms and mediate virus emergence through local evolution, host shifts, mixed infections, and global spread. Associated virus evolution involves reassortment, recombination, and component capture. Emergence of B. tabaci-transmitted geminiviruses (begomoviruses), ipomoviruses, and torradoviruses has led to global disease outbreaks as well as multiple paradigm shifts. Similarly, F. occidentalis has mediated tospovirus host shifts and global dissemination and the emergence of pollen-transmitted ilarviruses. The plant virus-supervector interaction offers exciting opportunities for basic research and global implementation of generalized disease management strategies to reduce economic and environmental impacts.

A natural M RNA reassortant arising from two species of plant- and insect-infecting bunyaviruses and comparison of its sequence and biological properties to parental species

Reassortment allows multicomponent viruses to exchange genome segments, a process well-documented in the vertebrate- and arthropod-infecting members of the family Bunyaviridae but not between distinct species of the plant- and insect-infecting members of the genus Tospovirus. Genome sequence comparisons of a virus causing severe tospovirus-like symptoms in Florida tomato with Groundnut ringspot virus (GRSV) and Tomato chlorotic spot virus (TCSV) demonstrated that reassortment has occurred, with the large (L) and small (S) RNAs coming from GRSV and the medium (M) RNA coming from TCSV (i.e. L(G)M(T)S(G)). Neither parental genotype is known to occur in the U.S. suggesting that L(G)M(T)S(G) was introduced as a reassortant. L(G)M(T)S(G) was transmitted by western flower thrips (Frankliniella occidentalis [Pergande]), and was not able to overcome the Sw5 resistance gene of tomato. Our demonstration of reassortment between GRSV and TCSV suggests caution in defining species within the family Bunyaviridae based on their ability to reassort.

Emergence of Groundnut ringspot virus and Tomato chlorotic spot virus in Vegetables in Florida and the Southeastern United States

Groundnut ringspot virus (GRSV) and Tomato chlorotic spot virus (TCSV) are two emerging tospoviruses in Florida. In a survey of the southeastern United States, GRSV and TCSV were frequently detected in solanaceous crops and weeds with tospovirus-like symptoms in south Florida, and occurred sympatrically with Tomato spotted wilt virus (TSWV) in tomato and pepper in south Florida. TSWV was the only tospovirus detected in other survey locations, with the exceptions of GRSV from tomato (Solanum lycopersicum) in South Carolina and New York, both of which are first reports. Impatiens (Impatiens walleriana) and lettuce (Lactuca sativa) were the only non-solanaceous GRSV and/or TCSV hosts identified in experimental host range studies. Little genetic diversity was observed in GRSV and TCSV sequences, likely due to the recent introductions of both viruses. All GRSV isolates characterized were reassortants with the TCSV M RNA. In laboratory transmission studies, Frankliniella schultzei was a more efficient vector of GRSV than F. occidentalis. TCSV was acquired more efficiently than GRSV by F. occidentalis but upon acquisition, transmission frequencies were similar. Further spread of GRSV and TCSV in the United States is possible and detection of mixed infections highlights the opportunity for additional reassortment of tospovirus genomic RNAs.

Zucchini yellow mosaic virus: biological properties, detection procedures and comparison of coat protein gene sequences

Between 2006 and 2010, 5324 samples from at least 34 weed, two cultivated legume and 11 native species were collected from three cucurbit-growing areas in tropical or subtropical Western Australia. Two new alternative hosts of zucchini yellow mosaic virus (ZYMV) were identified, the Australian native cucurbit Cucumis maderaspatanus, and the naturalised legume species Rhyncosia minima. Low-level (0.7%) seed transmission of ZYMV was found in seedlings grown from seed collected from zucchini (Cucurbita pepo) fruit infected with isolate Cvn-1. Seed transmission was absent in >9500 pumpkin (C.maxima and C. moschata) seedlings from fruit infected with isolate Knx-1. Leaf samples from symptomatic cucurbit plants collected from fields in five cucurbit-growing areas in four Australian states were tested for the presence of ZYMV. When 42 complete coat protein (CP) nucleotide (nt) sequences from the new ZYMV isolates obtained were compared to those of 101 complete CP nt sequences from five other continents, phylogenetic analysis of the 143 ZYMV sequences revealed three distinct groups (A, B and C), with four subgroups in A (I-IV) and two in B (I-II). The new Australian sequences grouped according to collection location, fitting within A-I, A-II and B-II. The 16 new sequences from one isolated location in tropical northern Western Australia all grouped into subgroup B-II, which contained no other isolates. In contrast, the three sequences from the Northern Territory fitted into A-II with 94.6-99.0% nt identities with isolates from the United States, Iran, China and Japan. The 23 new sequences from the central west coast and two east coast locations all fitted into A-I, with 95.9-98.9% nt identities to sequences from Europe and Japan. These findings suggest that (i) there have been at least three separate ZYMV introductions into Australia and (ii) there are few changes to local isolate CP sequences following their establishment in remote growing areas. Isolates from A-I and B-II induced chlorotic symptoms in inoculated leaves of Chenopodium quinoa, but an isolate from A-II caused symptomless infection. One of three commercial ZYMV-specific antibodies did not detect all Australian isolates reliably by ELISA. A multiplex real-time PCR using dual-labelled probes was developed, which distinguished between Australian ZYMV isolates belonging to phylogenetic groups A-I, A-II and B-II.

Indigenous and introduced potyviruses of legumes and Passiflora spp. from Australia: biological properties and comparison of coat protein nucleotide sequences

Five Australian potyviruses, passion fruit woodiness virus (PWV), passiflora mosaic virus (PaMV), passiflora virus Y, clitoria chlorosis virus (ClCV) and hardenbergia mosaic virus (HarMV), and two introduced potyviruses, bean common mosaic virus (BCMV) and cowpea aphid-borne mosaic virus (CAbMV), were detected in nine wild or cultivated Passiflora and legume species growing in tropical, subtropical or Mediterranean climatic regions of Western Australia. When ClCV (1), PaMV (1), PaVY (8) and PWV (5) isolates were inoculated to 15 plant species, PWV and two PaVY P. foetida isolates infected P. edulis and P. caerulea readily but legumes only occasionally. Another PaVY P. foetida isolate resembled five PaVY legume isolates in infecting legumes readily but not infecting P. edulis. PaMV resembled PaVY legume isolates in legumes but also infected P. edulis. ClCV did not infect P. edulis or P. caerulea and behaved differently from PaVY legume isolates and PaMV when inoculated to two legume species. When complete coat protein (CP) nucleotide (nt) sequences of 33 new isolates were compared with 41 others, PWV (8), HarMV (4), PaMV (1) and ClCV (1) were within a large group of Australian isolates, while PaVY (14), CAbMV (1) and BCMV (3) isolates were in three other groups. Variation among PWV and PaVY isolates was sufficient for division into four clades each (I-IV). A variable block of 56 amino acid residues at the N-terminal region of the CPs of PaMV and ClCV distinguished them from PWV. Comparison of PWV, PaMV and ClCV CP sequences showed that nt identities were both above and below the 76-77% potyvirus species threshold level. This research gives insights into invasion of new hosts by potyviruses at the natural vegetation and cultivated area interface, and illustrates the potential of indigenous viruses to emerge to infect introduced plants.

Low genetic diversity of Squash vein yellowing virus in wild and cultivated cucurbits in the U.S. suggests a recent introduction.

Squash vein yellowing virus (SqVYV) isolates were collected from cultivated and weedy cucurbits representing major hosts and locations in the U.S. and analyzed to better understand the diversity and population structure. No differences in symptoms were observed in field-collected isolate source plants or subsequently inoculated greenhouse plants, and the complete genome of an SqVYV isolate from a wild cucurbit host (smellmelon, Cucumis melo var. dudaim) was highly similar (99.4% nucleotide identity, 99.3% amino acid identity) to the previously published type isolate from squash. Although analysis of the coat protein (CP) and two serine proteases (P1a and P1b) sequences for 41 isolates showed little diversity across seven years of sampling, it revealed two distinct groups of SqVYV isolates with low intra-group diversity. Our analyses also suggested that recombination had occurred between SqVYV isolates, similar to other ipomoviruses. Selection pressures on the genome regions analyzed were negative indicating purifying selection was occurring. The magnitude of negative selection in SqVYV was consistent with what has been reported for other ipomoviruses, and was greatest for the CP and least for the P1b. The observed genetic diversity was similar to that reported for Cucumber vein yellowing virus but less than that reported for Sweet potato mild mottle virus, Cassava brown streak virus and Ugandan cassava brown streak virus. Collectively, these results indicate that the current U.S. population of SqVYV has undergone a recent genetic bottleneck and was introduced from elsewhere.

Widespread Occurrence and Low Genetic Diversity of Colombian datura virus in Brugmansia Suggest an Anthropogenic Role in Virus Selection and Spread

Brugmansia (Brugmansia spp.) is a perennial shrub in the Solanaceae, originating from South America, that is a popular landscape plant in the tropics and subtropics and container plant in temperate regions. Virus-like symptoms including mosaic, rugosity, and faint chlorotic spots were first observed on leaves of Brugmansia plants in a south Florida nursery in November 2003. Colombian datura virus (CDV) was identified in these initial plants and subsequent Brugmansia and Datura metel (a Brugmansia relative also grown as an ornamental) plants obtained from Florida, Connecticut, Wisconsin, and California. Overall, 77.5% of Brugmansia and two of four D. metel plants tested were infected with CDV. Partial NIb/CP sequences of 28 Brugmansia CDV isolates from this study were compared with all 16 CDV isolates in GenBank and found to share high levels of nucleotide and amino acid identity, with negative selection estimated to be occurring. A single Brugmansia plant was also infected with a recently described tobamovirus. The low genetic diversity of CDV observed, along with negative selection pressure on NIb/CP, suggests a recent ancestry (<400 years) of the worldwide population of CDV, coinciding with anthropogenic collection and dissemination of Brugmansia plants from their center of origin.

Control of Beet western yellows virus in Brassica napus crops: infection resistance in Australian genotypes and effectiveness of imidacloprid seed dressing

Eighteen Brassica napus (canola) genotypes were examined for their responses to infection with Beet western yellows virus (BWYV) and infestation by Myzus persicae (green peach aphid) in a field experiment and in a series of pot experiments under controlled-environment conditions. When exposed to infection with BWYV in the field, plants of cvv. Tranby and Trigold remained uninfected with BWYV. Only 2–5% of plants of cvv. Stubby and Banjo became infected, but infection incidence was 14–23% in cvv. Tanami and Jade, and reached 45–65% in 12 other commercial cultivars or advanced breeding lines of B. napus. Once plants became infected, the sensitivity rankings for most genotypes were 2–3: mild to moderate symptoms consisting of plant stunting and reddening of lower leaves. When plants of cvv. Tranby, Trigold, Stubby, and susceptible control cv. Pinnacle growing in pots were exposed to spread of BWYV by viruliferous winged M. persicae flying from an infested cv. Pinnacle plant infected with BWYV, similar numbers of aphids colonised each of the different cultivars. Thus, no aphid feeding preference was apparent among the different B. napus cultivars. However, all 18 plants of cv. Pinnacle became infected with BWYV, but only 1, 2, and 5 plants of cvv. Trigold, Tranby, and Stubby became infected, respectively. When 68 plants each of cvv. Tranby, Trigold, and Stubby were each inoculated with 1–10 viruliferous aphids/plant, only 1 of cv. Trigold, 3 of cv. Tranby, and 6 of cv. Stubby became infected with BWYV despite infection of 45 plants of cv. Pinnacle. This shows that cvv. Tranby, Trigold, and Stubby have resistance to infection with BWYV by aphid transmission. In 2 experiments when viruliferous M. persicae were placed on plants of B. napus grown from seed treated with imidacloprid (240 g a.i./100 kg seed), they infested 72% of plants grown from treated seed and transmitted BWYV to 62% of them regardless of the growth stage inoculated. Aphids colonised 100% of plants grown from untreated seed but 0% of plants sprayed with imidacloprid (2 g a.i./L water), and infection with BWYV was diminished markedly by the foliar spray. This suggests that insufficient insecticide adhered to most of the dressed seeds to kill the aphids and prevent BWYV transmission. B. napus cultivars found to have infection resistance to BWYV can be used in conjunction with imidacloprid seed dressings (if applied effectively) as components of an integrated disease management strategy for control of BWYV in B. napus crops.

Squash vein yellowing virus Infection of Vining Cucurbits and the Vine Decline Response

The responses of a diverse group of vining cucurbits to inoculation with Squash vein yellowing virus (SqVYV) were determined. For the first time, Cucurbita maxima, Cucumis dipsaceus, and Cucumis metuliferus were observed to develop necrosis and plant death similar to the SqVYV-induced vine decline in watermelon (Citrullus lanatus var. lanatus). The majority of cucurbits inoculated, however, either exhibited no symptoms of infection, or developed relatively mild symptoms such as vein yellowing of upper, noninoculated leaves. All inoculated plants were sectioned and tested for the presence of SqVYV. The virus was widely distributed in mature, fruit-bearing cucurbits with over 72% of plant sections testing positive for SqVYV by tissue-blot and/or reverse transcription-polymerase chain reaction. Plants of several cucurbits, including a wild citron (Citrullus lanatus var. citroides), were symptomless and had a decreased frequency of virus infection of vine segments compared to susceptible vining cucurbits, indicating a higher level of resistance. However, no significant relationship between the frequency of infection or virus distribution within plants and the symptom response was observed. These results demonstrate that a diverse group of cucurbits may decline when infected with SqVYV, and suggest that widespread distribution of virus within the plant is not the sole cause of decline.

Physiological Effects of Squash vein yellowing virus Infection on Watermelon

Squash vein yellowing virus (SqVYV) is the cause of viral watermelon vine decline. The virus is whitefly-transmitted, induces a systemic wilt of watermelon plants, and causes necrosis and discoloration of the fruit rind. In the field, SqVYV is often detected in watermelon in mixed infections with other viruses including the aphid-transmitted Papaya ringspot virus type W (PRSV-W). In this study, watermelon plants of different ages were inoculated with SqVYV or SqVYV+PRSV-W in the greenhouse or SqVYV in the field to characterize the physiological response to infection. Symptoms of vine decline appeared about 12 to 16 days after inoculation with SqVYV regardless of plant age at time of inoculation, plant growth habit (trellised or nontrellised), and location (greenhouse or field). However, the presence of PRSV-W delayed the appearance of vine decline symptoms by 2 to 4 days, and vine decline did not develop on plants with no fruit. For all inoculation treatments, more severe symptoms were observed in younger watermelon plants. Physiological responses to SqVYV infection included reduction in plant and fruit weights, alterations in fruit rind and flesh color, reduction in fruit sucrose content, increase in fruit acid content, and changes in plant nutrient composition, particularly increases in Ca, Mg, B, Mn, and Zn and decreases in K and N. These results demonstrate wide-ranging physiological effects of SqVYV infection and provide new insights into watermelon vine decline.