Biological and Genomic Characterization of a Novel Tobamovirus Infecting Hoya spp.

Foliar symptoms suggestive of virus infection were observed on the ornamental plant hoya (Hoya spp.; commonly known as waxflower) in Florida. An agent that reacted with commercially available tobamovirus detection reagents was mechanically transmitted to Chenopodium quinoa and Nicotiana benthamiana. Rod-shaped particles ∼300 nm in length and typical of tobamoviruses were observed in partially purified virion preparations by electron microscopy. An experimental host range was determined by mechanical inoculation with virions, and systemic infections were observed in plants in the Asclepiadaceae, Apocynaceae, and Solanaceae families. Some species in the Solanaceae and Chenopodiaceae families allowed virus replication only in inoculated leaves, and were thus only local hosts for the virus. Tested plants in the Amaranthaceae, Apiaceae, Brassicaceae, Cucurbitaceae, Fabaceae, and Malvaceae did not support either local or systemic virus infection. The complete genome for the virus was sequenced and shown to have a typical tobamovirus organization. Comparisons of genome nucleotide sequence and individual gene deduced amino acid sequences indicate that it is a novel tobamovirus sharing the highest level of sequence identity with Streptocarpus flower break virus and members of the Brassicaceae-infecting subgroup of tobamoviruses. The virus, for which the name Hoya chlorotic spot virus (HoCSV) is proposed, was detected in multiple hoya plants from different locations in Florida.

Genomic and Biological Characterization of Tomato necrotic streak virus, a Novel Subgroup 2 Ilarvirus Infecting Tomato in Florida.

Genomic and biological characterization of Tomato necrotic streak virus (TomNSV), a recently described ilarvirus infecting tomato in Florida, was completed. The full genome sequence revealed that TomNSV is a novel subgroup 2 ilarvirus that is distinct from other previously reported tomato-infecting ilarviruses: Tobacco streak virus, Parietaria mottle virus, and Tomato necrotic spot virus included in subgroup 1. In a host range experiment, TomNSV infected members of the Solanaceae and Chenopodiaceae plant families but did not infect sunflower (Helianthus annuus L.) or green bean (Phaseolus vulgaris L.). In tomato plants, the virus moved downward to the roots from the initial point of infection and then upward from the roots to tissues of active growth such as fruit, flowers, and young leaves where symptoms were produced. Thus, young leaves, fruit, and flowers are ideal for sampling for TomNSV. The transmission rate by seed collected from infected tomato plants was determined to be 0.33%. Collectively, the results of these experiments indicated that TomNSV is the causal agent of the necrotic streak disease of tomato observed in Florida since 2013.

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.

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.

Presence of P1b and absence of HC-Pro in Squash vein yellowing virus suggests a general feature of the genus Ipomovirus in the family Potyviridae.

The genus Ipomovirus is one of six currently recognized genera in the family Potyviridae. The complete nucleotide sequence of Squash vein yellowing virus (SqVYV), a putative ipomovirus recently described in Florida, has been determined. The 9836 nt SqVYV genomic RNA [excluding the poly(A) tail] has one large open reading frame encoding a single polyprotein of 3172 amino acids, typical of the genome organization for most members in the family Potyviridae. The 10 mature proteins predicted to be derived from the SqVYV polyprotein include P1a and P1b but no HC-Pro, similar to Cucumber vein yellowing virus (CVYV) but different from Sweet potato mild mottle virus (SPMMV), both recognized members of the genus Ipomovirus. Phylogenetic analysis of these proteins supports classification of SqVYV as a novel species within the genus Ipomovirus. However, the similar genome organization strategy of SqVYV and CVYV, which differs from that of SPMMV, indicates that the taxonomy of the genus Ipomovirus needs to be re-examined and a new genus created within the family Potyviridae to accommodate the observed discrepancies in ipomovirus genome organization.

Squash vein yellowing virus Detection Using Nested Polymerase Chain Reaction Demonstrates that the Cucurbit Weed Momordica charantia Is a Reservoir Host.

Squash vein yellowing virus (SqVYV) is a recently described ipomovirus from cucurbits in Florida that induces the relatively unusual symptoms in watermelon of plant death and fruit rind necrosis and discoloration, commonly known in Florida as watermelon vine decline. In this report, SqVYV infection of Momordica charantia (Balsam-apple), a common cucurbit weed, collected in 2005 and 2007 from within or adjacent to fields of declining watermelon, is demonstrated through the use of nested polymerase chain reaction (PCR). M. charantia plants located in or around fallow watermelon fields between spring and fall 2007 watermelon crops were also infected with SqVYV, indicating that this weed can serve as an oversummering host for this virus. Furthermore, whiteflies were able to acquire SqVYV from infected M. charantia and transmit it to squash and watermelon. Nested PCR was 10 to 1,000 times more sensitive than non-nested PCR for SqVYV detection in several cucurbit hosts, including M. charantia and watermelon. Melothria pendula (creeping cucumber), another common cucurbit weed, was experimentally infected with SqVYV. These results suggest that improved management of M. charantia and other cucurbit weeds needs to be incorporated into watermelon vine decline management plans to reduce sources of SqVYV and other cucurbit viruses.

Identification and characterization of a novel whitefly-transmitted member of the family potyviridae isolated from cucurbits in Florida.

ABSTRACT A novel whitefly-transmitted member of the family Potyviridae was isolated from a squash plant (Cucurbita pepo) with vein yellowing symptoms in Florida. The virus, for which the name Squash vein yellowing virus (SqVYV) is proposed, has flexuous rod-shaped particles of approximately 840 nm in length. The experimental host range was limited to species in the family Cucurbitaceae, with the most dramatic symptoms observed in squash and watermelon, but excluded all tested species in the families Amaranthaceae, Apocynaceae, Asteraceae, Chenopodiaceae, Fabaceae, Malvaceae, and Solanaceae. The virus was transmitted by whiteflies (Bemisia tabaci) but was not transmitted by aphids (Myzus persicae). Infection by SqVYV induced inclusion bodies visible by electron and light microscopy that were characteristic of members of the family Potyviridae. Comparison of the SqVYV coat protein gene and protein sequences with those of recognized members of the family Potyviridae indicate that it is a novel member of the genus Ipomovirus. A limited survey revealed that SqVYV also was present in watermelon plants suffering from a vine decline and fruit rot recently observed in Florida and was sufficient to induce these symptoms in greenhouse-grown watermelon, suggesting that SqVYV is the likely cause of this disease.

Development of a rapid, sensitive TaqMan real-time RT-PCR assay for the detection of Rose rosette virus using multiple gene targets.

Rose rosette virus (RRV), belonging to the genus Emaravirus, is a highly destructive pathogen that causes rose rosette disease. The disease is a major concern for the rose industry in the U.S. due to the lack of highly sensitive methods for early detection of RRV. This is critical, as early identification of the infected plants and eradication is necessary in minimizing the risks associated with the spread of the disease. A highly reliable, specific and sensitive detection assay is thus required to test and confirm the presence of RRV in suspected plant samples. In this study a TaqMan real-time reverse transcription-polymerase chain reaction (RT-PCR) assay was developed for the detection of RRV from infected roses, utilizing multiple gene targets. Four pairs of primers and probes; two of them (RRV_2-1 and RRV_2-2) based on the consensus sequences of the glycoprotein gene (RNA2) and the other two (RRV_3-2 and RRV_3-5) based on the nucleocapsid gene (RNA3) were designed. The specificity of the primers and probes was evaluated against other representative viruses infecting roses, belonging to the genera Alfamovirus, Cucumovirus, Ilarvirus, Nepovirus, Tobamovirus, and Tospovirus and one Emaravirus (Wheat mosaic virus). Dilution assays using the in vitro transcripts (spiked with total RNA from healthy plants, and non-spiked) showed that all the primers and probes are highly sensitive in consistently detecting RRV with a detection limit of 1 fg. Testing of the infected plants over a period of time (three times in monthly intervals) indicated high reproducibility, with the primer/probe RRV_3-5 showing 100% positive detection, while RRV_2-1, RRV_2-2 and RRV_3-2 showed 90% positive detection. The developed real-time RT-PCR assay is reliable, highly sensitive, and can be easily used in diagnostic laboratories for testing and confirmation of RRV.