Gary W. Knox

A rapid assay for detection of Rose rosette virus using reverse transcription-recombinase polymerase amplification using multiple gene targets

Rose rosette disease caused by Rose rosette virus (RRV; genus Emaravirus) is the most economically relevant disease of Knock Out® series roses in the U.S. As there are no effective chemical control options for the disease, the most critical disease management strategies include the use of virus free clean plants for propagation and early detection and destruction of infected plants. The current diagnostic techniques for RRV including end-point reverse transcription-polymerase chain reaction (RT-PCR) and real-time PCR (RT-qPCR) are highly sensitive, but limited to diagnostic labs with the equipment and expertise; and is time consuming. To address this limitation, an isothermal reverse transcription-recombinase polymerase amplification (RT-RPA) assay based on multiple gene targets for specific detection of RRV was developed. The assay is highly specific and did not cross react with other viruses belonging to the inclusive and exclusive genus. Dilution assays using the in vitro transcripts showed that the primer sets designed (RPA-267, RPA-131, and RPA-321) are highly sensitive, consistently detecting RRV with a detection limit of 1fg/μL. Testing of the infected plants using the primer sets indicated that the virus could be detected from leaves, stems and petals of roses. The primer pair RPA-267 produced 100% positive detection of the virus from infected leaf tissues, while primer set RPA-131 produced 100% detection from stems and petals. The primer set RPA-321 produced 83%, 87.5% and 75% positive detection from leaves, petals and stem tissues, respectively. In addition, the assay has been efficiently used in the detection of RRV infecting Knock Out® roses, collected from different states in the U.S. The assay can be completed in 20min as compared to the end-point RT-PCR assay (3-4h) and RT-qPCR (1.5h). The RT-RPA assay is reliable, rapid, highly sensitive, and can be easily used in diagnostic laboratories for detection of RRV with no need for any special equipment.

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.

Induction, Identification, and Characterization of Tetraploids in Japanese Privet (Ligustrum japonicum)

A number of privet species (Ligustrum spp.) that are important to the nursery and landscape industry have escaped cultivation and become invasive or weedy in the United States and other countries. Induced tetraploids in these species may produce new selections or cultivars with reduced or eliminated invasive potential. Applying drops of semisolid agar containing 0.1% to 0.3% colchicine and 0.2% dimethyl sulfoxide (DMSO) to newly emerged seedlings of japanese privet (Ligustrum japonicum Thunb.) resulted in 15.6% to 22.6% tetraploid induction. The nuclear DNA content of tetraploids was 5.31 pg/2C, 101.9% higher than that of diploids. Compared with diploid plants, tetraploids were more compact, with an average of 31.0% shorter plant height and 33.1% smaller canopy width. Tetraploids had 29.2% thicker internodes, and their leaves were 39.5% larger and 33.8% thicker, resulting in 42.1% to 24.1% greater fresh or dry leaf weights (per leaf) in tetraploids compared with diploids. Without indole-3-butyric acid (IBA) treatment, cuttings from tetraploids showed 28% lower rooting than diploids. IBA treatments improved the rooting of tetraploid cuttings, resulting in 65% rooting success. These results indicate that tetraploids can be readily induced in japanese privet and induced tetraploids show significant changes in plant growth and size, shoot growth, leaf morphology, and rooting of cuttings. The modified tetraploid induction method and the induced tetraploids are expected to be useful for producing new selections or cultivars with reduced invasive potential in japanese and other privets. Exotic plant invasions are considered one of the main causes of the degradation of ecosystems and the loss of biodiversity globally (Theoharides and Dukes, 2007). Ornamental horticulture has been recognized as the main source of plant invaders (Bell et al., 2003; Niemiera and Von Holle, 2009; Reichard andWhite, 2001; Rejm anek, 2014). The economic impacts of invasive plant species in the United States are estimated at nearly

A field based detection method for Rose rosette virus using isothermal probe-based Reverse transcription-recombinase polymerase amplification assay

35 billion (Pimentel et al., 2005). To help mitigate this huge impact and meet the nursery and landscape industry’s need for plant materials, horticulturists have been searching for cultivars with reduced invasive potential (Knox and Wilson, 2006; Trueblood et al., 2010; Wilson and Mecca, 2003; Wilson et al., 2004, 2012). Multiple ornamental breeding programs in the United States have initiated breeding projects to develop new cultivars with reduced or eliminated invasive potential in major invasive ornamental shrubs or trees (Anderson, 2007; Czarnecki et al., 2012; Freyre et al., 2012; Leonhardt and Shi, 2009; Olsen, 2007; Phillips et al., 2015; Ranney et al., 2007, 2010; Thammina et al., 2011; Vining et al., 2012). Developing genetic tools and generating germplasm resources that can be used for this objective have become essential and important for current and future ornamental plant breeding (Anderson, 2007; Li et al., 2004; Olsen, 2007; Thammina et al., 2011; Vining et al., 2012). Several species of Ligustrum L. (privet) are commonly used as ornamentals in many parts of the world (Dirr, 1998; Wilson et al., 2014). In the landscape, these plants are valued for their evergreen leaves, white flowers, adaptability to a range of landscape conditions, tolerance to pruning, resistance to diseases, and wide availability (Dirr, 1998). Some Ligustrum species have escaped cultivation and become naturalized in natural areas (Munger, 2003). For example, 16 countries report naturalization of chinese privet (Ligustrum sinense Lour.) (Morris et al.; 2002; Munger, 2003). In the United States, chinese privet has escaped in 20 states and is considered invasive in many states (FLEPPC, 2015; Munger, 2003). Glossy privet (Ligustrum lucidum W.T. Aiton) has escaped cultivation in 10 states in the United States (Munger, 2003; Wilson et al., 2014) and is also a weed in Australia (Panetta, 2000), New Zealand (Miller and Henzell, 2000), and Argentina (Hoyos et al., 2010). Japanese privet (L. japonicum Thunb.) is native to Japan and eastern Asia and was introduced to the United States from Japan and Korea in 1845 as an ornamental landscape plant (Munger, 2003; Wilson et al., 2014). Since then, this species has been widely used in the landscape in the southeastern, southern, and western United States. It has escaped cultivation and become naturalized in 12 southeastern states in the United States (Munger, 2003). Japanese privet commonly forms dense thickets in the field or forest understories, shading and displacing many native species. Once established, it is difficult to eradicate. The invasiveness of many ornamentals is attributed to a number of factors including prolific production of viable seeds. Thus, making plants sterile or seedless can reduce, even eliminate, their invasive potential (Anderson, 2007; Ranney, 2004, 2006). Several genetic tools have been used to reduce seed production, viability, and/or germination, including natural mutations, artificial mutagenesis, interspecific hybridization, ploidy manipulation (Czarnecki et al., 2012; Freyre et al., 2012; Trueblood et al., 2010), endosperm culture (Thammina et al., 2011), and transgenics (Li et al., 2004; Vining et al., 2012). So far, ploidy manipulation has resulted in considerable success and yielded multiple sterile, noninvasive cultivars or breeding lines in several important ornamental plants, such as HORTSCIENCE VOL. 51(11) NOVEMBER 2016 1371 Hypericum androsaemum L. (Trueblood et al., 2010), Lantana camara L. (Czarnecki et al., 2012), and Ruellia simplex Wright (Freyre et al., 2012). The key step in ploidy manipulation is chromosome doubling and induction of stable tetraploids, which are the gateway to obtain other ploidy levels (triploids, pentaploids, hexaploids, octoploids, etc.) through interploid crosses. Several antimitotic agents have been used to inhibit the separation of chromosomes at the anaphase of cell division and achieve chromosome doubling in ornamental plants (Contreras, 2012; Contreras et al., 2010; Nadler et al., 2012; Vining et al., 2012). Colchicine has been one of the widely used agents to induce tetraploids in numerous ornamental plants (Abdoli et al., 2013; Henny et al., 2009; Lehrer et al., 2008; Leonhardt and Shi, 2009). Several types of plant tissues or organs, such as seeds, seedlings, axillary buds or shoot tips, embryos, and cultured cells or tissues, have been used as the target for colchicine treatment with various rates of successful tetraploid induction and mixoploidy (Habbard et al., 2016; Henny et al., 2009; Jones et al., 2007; Lehrer et al., 2008; Leonhardt and Shi, 2009; Vining et al., 2012). The availability of an effective and efficient chromosome doubling and tetraploid induction protocol is critical for successful ploidy manipulation and development of sterile, noninvasive cultivars in invasive ornamental plants. Our literature searches and quick surveys of privet cultivars indicated a lack of tetraploids in glossy, chinese, and japanese privet. Preliminary work on treating axillary buds of privet plants with colchicine resulted in few solid tetraploids. Thus, the main objectives of this study were to use japanese privet as a model to evaluate the effectiveness and efficiency of the semisolid agar method for induction of stable tetraploids in privet and to characterize the effects of chromosome doubling and tetraploidy on shoot growth and leaf morphology. Materials and Methods Plant materials.Open-pollinated seedswere collected from mature shrubs of L. japonicum ‘Texanum’ grown at the University of Florida (UF) North Florida Research and Education Center, Quincy, FL, in Feb. 2010 and shipped to the UF Gulf Coast Research and Education Center (GCREC), Wimauma, FL. Dry seeds were sown onto a commercial potting mix (Metro Mix 200; Sun Gro Horticulture, Agawam, MA) in plastic containers (15 cm in diameter) in Feb. 2011. Seeds were covered with a layer of horticultural grade vermiculite ( 1 cm thick) and germinated in a growth room at 24 C with 16 h of light (120 to 150 mmol·m·s) and 8 h of dark. About 5weeks after sowing, seedlings emerged with fully expanded cotyledons. Induction, identification, and confirmation of tetraploids. The semisolid agar method of Jones et al. (2007) was used with some modifications. Colchicine was applied to the growing points of newly emerged seedlings at the cotyledonary stage to induce tetraploids. A 1% colchicine stock solution was made by dissolving colchicine power (Sigma-Aldrich, St. Louis, MO) in water. The colchicine stock solution was added to melted semisolid agar containing one-fourth-strength Murashige and Skoog (MS) basal salts, 0.55%agar, andDMSO (Sigma-Aldrich; final concentration 0.2%) to a final concentration. Three colchicine concentrations were used: 0.1%, 0.2%, and 0.3%. The colchicine-containing agar solution was kept at 40 C and then 30 mL were pipetted onto the growing point of each privet seedling. Treated seedlings were placed in a high humidity ( 100% relative humidity) growth chamber at 24 C in dark to minimize colchicine degradation by light. Control seedlings received semisolid agar without colchicine. All treatments were repeated during three consecutive days. After treatment, seedlings were grown in a greenhouse, and fertigated with 50 to 100 ppm nitrogen as needed. Seedlings were kept in community containers until they produced shoots with several true leaves and later transplanted individually into new containers (10 to 15 cm in diameter). Seedlings were grown in these containers for 1 year, and then transplanted into ground beds at the UF’s GCREC in Mar. 2013. All transplanted plants were grown in full sun, irrigated through drip tapes, and fertilized with 15 g of 15N–3.9P–12K controlled-release fertilizer (Osmocote; Scotts, Marysville, OH). To identify tetraploids, multiple fully expanded young leaveswere collected from each plant, and each leaf was analyzed separat

Susceptibility of Cultivated Native Wildflowers to Deer Damage

Rose rosette disease, caused by Rose rosette virus (RRV; genus Emaravirus) is a major threat to the rose industry in the U.S. The only strategy currently available for disease management is early detection and eradication of the infected plants, thereby limiting its potential spread. Current RT-PCR based diagnostic methods for RRV are time consuming and are inconsistent in detecting the virus from symptomatic plants. Real-time RT-qPCR assay is highly sensitive for detection of RRV, but it is expensive and requires well-equipped laboratories. Both the RT-PCR and RT-qPCR cannot be used in a field-based testing for RRV. Hence a novel probe based, isothermal reverse transcription-recombinase polymerase amplification (RT-exoRPA) assay, using primer/probe designed based on the nucleocapsid gene of the RRV has been developed. The assay is highly specific and did not give a positive reaction to other viruses infecting roses belonging to both inclusive and exclusive genus. Dilution assays using the in vitro transcript showed that the primer/probe set is highly sensitive, with a detection limit of 1 fg/μl. In addition, a rapid technique for the extraction of viral RNA (<5min) has been standardized from RRV infected tissue sources, using PBS-T buffer (pH 7.4), which facilitates the virus adsorption onto the PCR tubes at 4°C for 2min, followed by denaturation to release the RNA. RT-exoRPA analysis of the infected plants using the primer/probe indicated that the virus could be detected from leaves, stems, petals, pollen, primary roots and secondary roots. In addition, the assay was efficiently used in the diagnosis of RRV from different rose varieties, collected from different states in the U.S. The entire process, including the extraction can be completed in 25min, with less sophisticated equipments. The developed assay can be used with high efficiency in large scale field testing for rapid detection of RRV in commercial nurseries and landscapes.

Experiential Nursery Integrated Pest Management Workshop Series to Enhance Grower Practice Adoption

Abstract Foraging preference of Odocoileus virginianus (White-tailed Deer) at ornamental plantings was compared amongst 11 wildflower species native to north Florida and south Georgia. Deer exhibited strong preference for Coreopsis floridana (Florida Tickseed), C. gladiata (Coastalplain Tickseed), C. integrifolia (Fringeleaf Tickseed), and Rudbeckia fulgida (Orange Coneflower). Browsing significantly reduced the height of Florida, Coastalplain, and Fringeleaf Tickseeds, and reduced the number of Florida and Fringeleaf Tickseed flowers. Browsing pressure remained high throughout the growing season; therefore, temporary exclosures are unlikely to offer a viable solution to damage caused by deer. Information on variation in deer preference between species and across seasons should help private landowners and public land managers make strategic decisions regarding which species to establish at residences, food plots, or roadside beautification projects.

New Host Records for Caliothrips striatus (Thysanoptera: Thripidae) on Magnolia spp. with the Description of the Second Instar

Three, 2-day hands-on experiential learningworkshopswere presented in three southeastern United States cities in June 2014, by the Southern Nursery Integrated Pest Management (SNIPM) working group. Attendees were provided 4 hours of instruction including hands-on demonstrations in horticultural management, arthropods, plant diseases, and weeds. Participants completed initial surveys for gains in knowledge, skills, and abilities as well as their intentions to adopt various integrated pest management (IPM) practices after the workshop. After 3 years, participants were again surveyed to determine practice adoption. Respondents changed their IPM practice behavior because of attending the workshops. Those returning the survey set aside more time to scout deliberately for pests, plant diseases, and weeds; used a standardized sampling plan when scouting; and adopted more sanitation practices to prevent plant disease. Fewer horticultural management practices were adopted than respondents originally intended. Future emphasis should be placed on using monitoring techniques to estimate pest emergence, for example, traps and pheromone lures, as well as plant phenology and record keeping. However, more work is needed to highlight both the immediate and longterm economic benefits of IPM practice adoption in southeastern U.S. nursery production.

Evaluation of a Light-activated Nanoparticle Formulation of Titanium Dioxide with Zinc for Management of Bacterial Leaf Spot on Rosa ‘Noare’

In the current literature, apart from brief descriptions of feeding hosts, little is known regarding the habits of Caliothrips striatus Hood. This species is native to North America with a range of the eastern United States; and Wilson (1975) described it as mainly using tulip trees, Liriodendron tulipifera L., as a host plant and only occasionally being found on magnolias. The host association information for C. striatus is limited. The feeding behavior is known only on L. tulipifera, and the plant hosts utilized for reproduction have not been documented. The current study addresses this issue. In the present study observations were conducted at arboretums in South Carolina (N 34° 53’ 52.22’’, W 82° 49’ 43.90’’, 315 m asl), Georgia (N. 34° 16’ 08.61’’, 83° 26’ 58.82’’, 222 m asl) and Tennessee (N 35° 52’ 21.85’’, W 88° 50’ 40.08’’, 125 m asl) from Sep to Nov in 2010 and 2011. In the arboretums the thrips were found in large numbers causing severe feeding damage to the leaves (Fig. 1). Damage caused by C. striatus on species