Survival of Xanthomonas fragariae on Common Materials.

Xanthomonas fragariae causes strawberry angular leaf spot (ALS), an important disease for the strawberry nursery industry in North America. To identify potential inoculum sources, the survival of X. fragariae was examined on the surfaces of 11 common materials found in nurseries: corrugated cardboard, cotton balls, cotton cloth (t-shirt), strawberry leaf, sheet metal, plastic, rubber, Tyvek, wood (balsa), glass (microscope slide), and latex (latex glove). Prefabricated rectangular samples (7.62 by 2.54 cm) of each material were immersed in a bacterial suspension for 15 min, after which the samples were stored at approximately 20°C (room temperature) or -4°C (the cold storage temperature for dormant plants in strawberry nurseries) for 1, 3, 7, 14, 30, 60, 90, 180, 270, and 365 days after inoculation (DAI). After the storage period elapsed, bacteria were recovered from the surfaces of each of the samples with phosphate-buffered saline (PBS)-soaked cotton balls. Survival rate was determined with a viability real-time quantitative PCR procedure and in a plant bioassay that involved rub inoculation of strawberry leaflets with the PBS-soaked cotton balls used to recover bacteria from the samples. Results showed that X. fragariae could survive on all surfaces but that survival rate differed among materials and storage temperature. All materials were capable of harboring viable bacteria up to 7 DAI when stored at -4°C based on the formation of lesions on inoculated leaves in the plant bioassay. The longest survival observed was 270 DAI on cardboard stored at -4°C. At room temperature, cardboard, cotton balls, cotton t-shirt, and strawberry leaf tissue supported small bacterial populations up to 14 DAI. The information from this study can be used to improve sanitation practices for ALS management in strawberry nurseries.

A Long-Amplicon Viability-qPCR Test for Quantifying Living Pathogens that Cause Bacterial Spot in Tomato Seed.

Bacterial spot is one of the most serious diseases of tomato. It is caused by four species of Xanthomonas: X. euvesicatoria, X. gardneri, X. perforans, and X. vesicatoria. Contaminated or infected seed can be a major source of inoculum for this disease. The use of certified pathogen-free seed is one of the primary management practices to reduce the inoculum load in commercial production. Current seed testing protocols rely mainly on plating the seed extract and conventional PCR; however, the plating method cannot detect viable but nonculturable cells, and the conventional PCR assay has limited capability to differentiate DNA extracted from viable or dead bacterial cells. To improve the sensitivity and specificity of the tomato seed testing method for bacterial spot pathogens, a long-amplicon quantitative PCR (qPCR) assay coupled with propidium monoazide (PMA-qPCR) was developed to quantify selectively the four pathogenic Xanthomonas species in tomato seed. The optimized PMA-qPCR procedure was evaluated on pure bacterial suspensions, bacteria-spiked seed extracts, and seed extracts of inoculated and naturally infected seed. A crude DNA extraction protocol also was developed, and PMA-qPCR with crude bacterial DNA extracts resulted in accurate quantification of 104 to 108 CFU/ml of viable bacteria when mixed with dead cells at concentrations as high as 107 CFU/ml in the seed extracts. With DNA purified from concentrated seed extracts, the PMA-qPCR assay was able to detect DNA of the target pathogens in seed samples spiked with ≥75 CFU/ml (about 0.5 CFU/seed) of the viable pathogens. Latent class analysis of the inoculated and naturally infected seed samples showed that the PMA-qPCR assay had greater sensitivity than plating the seed extracts on the semiselective modified Tween Medium B and CKTM media for all four target species. Being much faster and more sensitive than dilution plating, the PMA-qPCR assay has potential to be used as a standalone tool or in combination with the plating method to improve tomato seed testing and advance the production of clean seed.

Complete genome sequence of Xanthomonas phage RiverRider, a novel N4-like bacteriophage that infects the strawberry pathogen Xanthomonas fragariae.

Xanthomonas phage RiverRider is a novel N4-like bacteriophage and the first phage isolated from the plant pathogen Xanthomonas fragariae. Electron microscopy revealed a Podoviridae morphology consisting of isometric heads and short noncontractile tails. The complete genome of RiverRider is 76,355 bp in length, with 90 open reading frames and seven tRNAs. The genome is characteristic of N4-like bacteriophages in both content and organization, having predicted proteins characterized into the functional groups of transcription, DNA metabolism, DNA replication, lysis, lysis inhibition, structure and DNA packaging. Amino acid sequence comparisons for proteins in these categories showed highest similarities to well-characterized N4-like bacteriophages isolated from Achromobacter xylosoxidans and Erwinia amylovora. However, the tail fiber proteins of RiverRider are clearly distinct from those of other N4-like phages. RiverRider was able to infect seven different strains of X. fragariae and none of the other species of Xanthomonas tested.

Detection of Viable Xanthomonas fragariae Cells in Strawberry Using Propidium Monoazide and Long-Amplicon Quantitative PCR.

Xanthomonas fragariae causes angular leaf spot in strawberry. The pathogen's association with its host tissue is thought to be a condition for its survival. Consequently, transmission of the pathogen to field production sites occurs almost exclusively through the movement of contaminated planting stock. The aim of this study was to develop a propidium monoazide (PMA)-quantitative PCR (qPCR) protocol for specific detection of viable X. fragariae cells. The qPCR procedure was developed for two different primer pairs: one producing a long amplicon (863 bp) and the other a short amplicon (61 bp). Both pairs were tested on mixtures of viable and heat-killed bacteria cells, bacteria-spiked strawberry petiole samples, and petioles collected from symptomatic, inoculated plants. The results showed that long-amplicon PMA-qPCR enabled specific and sensitive detection of X. fragariae with a detection limit of 103 CFU/ml, and it significantly improved PMA efficiency in differentiating viable from dead bacterial cells relative to short-amplicon PMA-qPCR. Based on the delta threshold cycle (Ct) values (i.e., the difference in Ct values between PMA-treated and nontreated samples), the long-amplicon PMA-qPCR was able to suppress the detection of dead X. fragariae cells 1.9- to 3.1-fold across all petiole samples tested. The quantification results from PMA-qPCR for mixtures of viable and dead cells were highly correlated with the predicted bacterial concentrations in a linear relationship (R2 = 0.981). This assay can be useful for identifying inoculum sources in the strawberry production cycle, which may lead to improved disease management strategies.

Spatial and Temporal Physiognomies of Whitefly and Tomato Yellow Leaf Curl Virus Epidemics in Southwestern Florida Tomato Fields.

Epidemics of tomato yellow leaf curl virus (TYLCV; species Tomato yellow leaf curl begomovirus) have been problematic to tomato production in the southeastern United States since the first detection of the virus in Florida in the late 1990s. Current strategies for management focus on farm-centric tactics that have had limited success for controlling either TYLCV or its whitefly vector. Areawide pest management (AWPM)-loosely defined as a coordinated effort to implement management strategies on a regional scale-may be a viable management alternative. A prerequisite for development of an AWPM program is an understanding of the spatial and temporal dynamics of the target pathogen and pest populations. The objective of this study was to characterize populations of whitefly and TYLCV in commercial tomato production fields in southwestern Florida and utilize this information to develop predictors of whitefly density and TYLCV disease incidence as a function of environmental and geographical factors. Scouting reports were submitted by cooperating growers located across approximately 20,000 acres in southwestern Florida from 2006 to 2012. Daily weather data were obtained from several local weather stations. Moran's I was used to assess spatial relationships and polynomial distributed lag regression was used to determine the relationship between weather variables, whitefly, and TYLCV. Analyses showed that the incidence of TYLCV increased proportionally with mean whitefly density as the season progressed. Nearest-neighbor analyses showed a strong linear relationship between the logarithms of whitefly densities in neighboring fields. A similar relationship was found with TYLCV incidences. Correlograms based on Moran's I showed that these relationships extended beyond neighboring fields and out to approximately 2.5 km for TYLCV and up to 5 km for whitefly, and that values of I were generally higher during the latter half of the production season for TYLCV. Weather was better at predicting whitefly density than at predicting TYLCV incidence. Whitefly density was best predicted by the number of days with an average temperature between 16 and 24°C (T16to24), relative humidity (RH) over the previous 31 days, and vapor pressure deficit over the last 8 days. TYLCV incidence was best predicted by T16to24, RH, and maximum wind speed over the previous 31 days. Results of this study helped to identify the extent to which populations of whitefly and TYLCV exist over the agricultural landscape of southwestern Florida, and the environmental conditions that favor epidemic growth. This information was used to propose an approach to AWPM for timing control measures for managing TYLCV epidemics.

Risk Factors for Bud Perennation of Podosphaera macularis on Hop.

The hop powdery mildew fungus Podosphaera macularis persists from season to season in the Pacific Northwestern United States through infection of crown buds because only one of the mating types needed to produce the ascigerous stage is presently found in this region. Bud infection and successful overwintering of the fungus leads to the emergence of heavily infected shoots in early spring (termed flag shoots). Historical data of flag shoot occurrence and incidence in Oregon and Washington State during 2000 to 2017 were analyzed to identify their association with the incidence of powdery mildew, growers' use of fungicides, autumn and winter temperature, and other production factors. During this period, flag shoots were found on 0.05% of plants evaluated in Oregon and 0.57% in Washington. In Oregon, the incidence of powdery mildew on leaves was most severe and the number of fungicide applications made by growers greatest in yards where flag shoots were found in spring. Similarly, the incidence of plants with powdery mildew in Washington was significantly associated with the number of flag shoots present in early spring, although the number of fungicide applications made was independent of flag shoot occurrence. The occurrence of flag shoots was associated with prior occurrence of flag shoots in a yard, the incidence of foliar powdery mildew in the previous year, grower pruning method, and, in Washington, winter temperature. A census of hop yards in the eastern extent of the Oregon production region during 2014 to 2017 found flag shoots in 27 of 489 yards evaluated. In yards without flag shoots, 338 yards (73.2%) were chemically pruning or not pruned, whereas the remaining 124 (26.8%) were mechanically pruned. Of the 27 yards with flag shoots, 22 were either chemically pruned or not pruned and 4 were mechanically pruned in mid-April, well after the initial emergence of flag shoots. The prevalence of yards with flag shoots also was related to thoroughness of pruning in spring (8.1% of yards with incomplete pruning versus 1.9% of yards with thorough pruning). A Bayesian logistic regression model was fit to the data from the intensively assessed yards in Oregon, with binary risk factors for occurrence of a flag shoot in the previous year, occurrence of foliar mildew in the previous year, and thoroughness of pruning in spring. The model indicated that the median and 95% highest posterior density interval of the probability of flag shoot occurrence was 0.0008 (0.0000 to 0.0053) when a yard had no risk factors but risk increased to 0.0065 (0.0000 to 0.0283) to 0.43 (0.175 to 0.709) when one to all three of the risk factors were present. The entirety of this research indicates that P. macularis appears to persist in a subset of chronically affected hop yards, particularly yards where spring pruning is conducted poorly. Targeted management of the disease in a subset of fields most at risk for producing flag shoots could potentially influence powdery mildew development regionwide.

Colonization and Movement of Xanthomonas fragariae in Strawberry Tissues.

Xanthomonas fragariae causes angular leaf spot of strawberry, an important disease in strawberry growing regions worldwide. To better understand how X. fragariae multiplies and moves in strawberry plants, a green fluorescent protein (GFP)-labeled strain was constructed and used to monitor the pathogen's presence in leaf, petiole, and crown tissue with fluorescence microscopy following natural and wound inoculation in three strawberry cultivars. Taqman PCR was used to quantify bacterial densities in these same tissues regardless of the presence of GFP signal. Results showed X. fragariae colonized leaf mesophyll, the top 1 cm portion of the petiole adjacent to the leaf blade, and was occasionally found colonizing xylem vessels down to the middle of the petioles. The colonization of vascular bundles and the limited systemic movement that was observed appeared to be a passive process, of which the frequency increased with wounding and direct infiltration of bacteria into leaf veins. X. fragariae was able to directly enter petioles and colonize the space under the epidermis. Systemic movement of the bacteria into crown and other uninoculated tissues was not detected visually by GFP. However, X. fragariae was occasionally detected in these tissues by qPCR, but at quantities very near the qPCR detection limit. Petiole tissue harboring bacteria introduced either by direct entry through natural openings or wounds, or by systemic movement from infected foliar tissue, likely serves as a main source of initial inoculum in field plantings.

Development of Phytophthora Fruit Rot Caused by Phytophthora capsici on Resistant and Susceptible Watermelon Fruit of Different Ages.

Watermelon is an important crop grown in 44 states in the United States. Phytophthora fruit rot caused by Phytophthora capsici is a serious disease in the southeastern U.S.A., where over 50% of the watermelons are produced. The disease has resulted in severe losses to watermelon growers, especially in Georgia, South Carolina, and North Carolina during the past few years. Several fruit rot-resistant watermelon germplasm lines have been developed for use in breeding programs. To evaluate the development of Phytophthora fruit rot on fruit of different ages, plants of fruit rot-resistant and susceptible lines were planted at weekly intervals for five consecutive weeks in experiments conducted over three years (2011 to 2013). Flowers were routinely inspected and hand pollinated to ensure having fruit of different ages. In each year, different aged fruit were harvested on the same day and inoculated with a 5-mm agar plug from an actively growing colony of P. capsici. Inoculated fruit were maintained in a room set to conditions conducive for disease development (>95% relative humidity, 26 ± 2°C). After 5 days, lesion diameter and intensity of sporulation was recorded for each fruit. Lesion diameter and sporulation intensity were significantly greater on fruit of susceptible lines compared with resistant lines. Fruit age did not have an effect on either measurement on susceptible (Sugar Baby) or resistant lines (PI 560020 and PI 595203). Our results showed that resistance to Phytophthora fruit rot in watermelon was not correlated with fruit age.

Development and Evaluation of ELISA and qRT-PCR for Identification of Squash vein yellowing virus in Cucurbits.

Squash vein yellowing virus (SqVYV) causes viral watermelon vine decline. To facilitate detection of SqVYV, enzyme linked-immunosorbent assay (ELISA) and quantitative reverse-transcription polymerase chain reaction (qRT-PCR) diagnostic methods were developed. Both methods were capable of detecting SqVYV in a wide range of cucurbit hosts. ELISA was able to detect virus in infected host tissue diluted to at least 1:2,560, which was sufficient for detection in symptomatic squash and watermelon plants. The qRT-PCR method was capable of reliably detecting as few as 3.4 copies of a cloned fragment of SqVYV genomic RNA with an average cycle threshold (Ct) value of 36.4. The sensitivities and specificities for each detection method were estimated by latent class analysis for a set of inoculated squash and watermelon plants at two sampling scales. The scales were hierarchical, with individual plants representing the upper scale and samples from the plant representing the lower scale. The number of samples per plant varied from 1 to 8, and a plant was diagnosed positive if any of its samples tested positive. For all analyses, a cutoff Ct of 35 was chosen for qRT-PCR, which is approximately 2.5 cycles lower than the lowest Ct value achieved for mock-inoculated plants (presumed to be a false positive). qRT-PCR showed high sensitivities (≥0.99) at both sampling scales for squash and watermelon, whereas the sensitivities for ELISA ranged from 0.58 to 0.76. The specificities for both tests were very similar (≥0.94), with ELISA sometimes outperforming qRT-PCR. These diagnostic methods provide additional tools for the identification of SqVYV and management of SqVYV-induced watermelon vine decline.

A Loop-Mediated Isothermal Amplification Assay and Sample Preparation Procedure for Sensitive Detection of Xanthomonas fragariae in Strawberry.

Xanthomonas fragariae is a bacterium that causes angular leaf spot of strawberry. Asymptomatic infection is common and contributes to the difficulties in disease management. The aim of this study was to develop a loop-mediated isothermal amplification (LAMP) assay as an efficient method for detection of asymptomatic infections of X. fragariae. In addition, a new method of sample preparation was developed that allows sampling of a larger amount of plant tissue, hence increasing the detection rate in real-life samples. The sample preparation procedure includes an overnight incubation of strawberry tissues in phosphate-buffered saline (PBS), followed by a quick sample concentration and a boiling step to extract DNA for amplification. The detection limit of the LAMP assay was approximately 2×10(3) CFU/mL for pure bacteria culture and 300 CFU/mL for bacteria spiked strawberry leaf and petiole samples. LAMP provided a 2-3 fold lower detection limit than the standard qPCR assay but was faster, and more user-friendly. The LAMP assay should serve as a rapid, sensitive and cost-effective tool for detecting asymptomatic infections of X. fragariae in strawberry nursery stock and contribute to improved disease management.

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.

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.

Spatial and Temporal Analysis of Squash vein yellowing virus Infections in Watermelon.

Squash vein yellowing virus (SqVYV) is a whitefly-transmitted ipomovirus infecting watermelon and other cucurbits that was recently introduced to Florida. Effects on watermelon are devastating, with total vine collapse, often near harvest, and fruit rendered unmarketable by brown, discolored flesh. The epidemiology of SqVYV was studied in a 1-ha field of 'Fiesta' watermelon over six growing seasons (I to VI) to characterize the spatial patterning of disease and temporal rate of disease progress, as well as its association with Cucurbit leaf crumple virus (CuLCrV) and Cucurbit yellow stunting disorder virus (CYSDV), two additional whitefly-transmitted viruses that often occur with SqVYV. The field was scouted at regular intervals for the length of the season for incidence of virus and number of whiteflies. Incidence of SqVYV reached 100% during seasons I, II, and V and 20% during season III. SqVYV did not occur during seasons IV and VI. SqVYV progressed in a characteristic logistic fashion in seasons I, II, and V but less so in season III. The rate of disease progress was similar for the three seasons with high disease incidence, with an average value of 0.18. A positive correlation between the area under the disease progress curve and whitefly-days was found, where both progress curves were calculated as a function of thermal time (degree days, base 0°C). SqVYV displayed significant but variable levels of aggregation, as indicated by its fit to the ß-binomial distribution, the binary power law, and ordinary runs analysis. Association analysis indicated that the viruses were largely transmitted independently. Results of this study provide epidemiological information that will be useful in the development of management strategies for SqVYV-induced vine decline, and provide new information for CuLCrV and CYSDV.

The use of latent class analysis to estimate the sensitivities and specificities of diagnostic tests for Squash vein yellowing virus in cucurbit species when there is no gold standard.

Squash vein yellowing virus (SqVYV) is the causal agent of viral watermelon vine decline, one of the most serious diseases in watermelon (Citrullus lanatus L.) production in the southeastern United States. At present, there is not a gold standard diagnostic test for determining the true status of SqVYV infection in plants. Current diagnostic methods for identification of SqVYV-infected plants or tissues are based on the reverse-transcription polymerase chain reaction (RT-PCR), tissue blot nucleic acid hybridization assays (TB), and expression of visual symptoms. A quantitative assessment of the performance of these diagnostic tests is lacking, which may lead to an incorrect interpretation of results. In this study, latent class analysis (LCA) was used to estimate the sensitivities and specificities of RT-PCR, TB, and visual assessment of symptoms as diagnostic tests for SqVYV. The LCA model assumes that the observed diagnostic test responses are linked to an underlying latent (nonobserved) disease status of the population, and can be used to estimate sensitivity and specificity of the individual tests, as well as to derive an estimate of the incidence of disease when a gold standard test does not exist. LCA can also be expanded to evaluate the effect of factors and was done here to determine whether diagnostic test performances varied among the type of plant tissue being tested (crown versus vine tissue), where plant samples were taken relative to the position of the crown (i.e., distance from the crown), host (i.e., genus), and habitat (field-grown versus greenhouse-grown plants). Results showed that RT-PCR had the highest sensitivity (0.94) and specificity (0.98) of the three tests. TB had better sensitivity than symptoms for detection of SqVYV infection (0.70 versus 0.32), while the visual assessment of symptoms was more specific than TB and, thus, a better indicator of noninfection (0.98 versus 0.65). With respect to the grouping variables, RT-PCR and TB had better sensitivity but poorer specificity for diagnosing SqVYV infection in crown tissue than it did in vine tissue, whereas symptoms had very poor sensitivity but excellent specificity in both tissues for all cucurbits analyzed in this study. Test performance also varied with habitat and genus but not with distance from the crown. The results given here provide quantitative measurements of test performance for a range of conditions and provide the information needed to interpret test results when tests are used in parallel or serial combination for a diagnosis.

Considerations of scale in the analysis of spatial pattern of plant disease epidemics.

Scale is an important but somewhat neglected subject in plant pathology. Scale serves as an abstract concept, providing a framework for organizing observations and theoretical models, and plays a functional role in the organization of ecological communities and physical processes. Rich methodological resources are available to plant pathologists interested in considering either or both aspects of scale in their research. We summarize important concepts in both areas of the literature, particularly as they apply to the spatial pattern of plant disease, and highlight some new results that emphasize the importance of scaling on the emergence of different types of probability distribution in empirical observation. We also highlight the important links between heterogeneity and scale, which are of central importance in plant disease epidemiology and the analysis of spatial pattern. We consider statistical approaches that are available, where actual physical scale is known, and for more conceptual research on hierarchies, where scale plays a more abstract role, particularly for field-based research. For the latter, we highlight methods that plant pathologists could consider to account for the effect of scale in the design of field studies.

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.

Association of Spring Pruning Practices with Severity of Powdery Mildew and Downy Mildew on Hop.

Downy mildew (caused by Pseudoperonospora humuli) and powdery mildew (caused by Podosphaera macularis) are important diseases of hop in the Pacific Northwest United States, and cultural practices may affect the severity of both diseases. The association of spring pruning quality and timing with severity of downy mildew and powdery mildew was assessed through analysis of survey data collected from commercial hop yards in Oregon and Washington. Among 149 hop yards surveyed, the most common pruning method was chemical desiccation (48% of yards), mechanical pruning (23%), or a combination of these practices (15%). The quality of pruning was assessed using a three-category ordinal scale ("excellent", "moderate", or "poor") based on the amount of foliage remaining on plants following pruning. Excellent pruning quality was attained more often in yards pruned twice (74.6 to 82.1% of yards) versus once (33.8% of yards), independent of pruning method. Seasonal severity of downy mildew in Oregon increased approximately twofold with reduction in pruning quality from excellent to moderate to poor. Pruning quality was not significantly related to levels of powdery mildew on leaves or cones in Oregon. Under more severe disease pressure in Washington, however, seasonal severity of powdery mildew on leaves and the incidence of cones with powdery mildew were significantly greater in yards that had poor pruning compared with excellent pruning. Moreover, yards that had excellent pruning quality received, on average, 1.1 to 1.5 fewer fungicide applications per season for downy mildew or powdery mildew compared with yards that had moderate or poor pruning quality. This savings was associated with delayed initiation of the first application by 7.5 to 14.2 days in yards with excellent pruning quality. Replicated experiments in commercial yards in Oregon quantified the effect of delaying pruning timing 5 to 21 days compared with growers' standard practices on the diseases and yield. Downy mildew suppression by delayed pruning was dependent on cultivar and year of sampling, being significantly reduced fivefold only in 'Willamette' in 2007. Severity of powdery mildew and cone yield was similar between plots that received the delayed or standard pruning timing treatments. Collectively, these studies emphasize that early spring sanitation measures are associated with reduced primary inoculum and are critically important for managing both downy mildew and powdery mildew. A savings of at least one fungicide application per year appears achievable when spring pruning is conducted thoroughly and slightly delayed compared with growers' current practices.

Ecology and management of whitefly-transmitted viruses of vegetable crops in Florida.

A variety of fresh market vegetables, including watermelon and tomato are economically important crops in Florida. Whitefly-transmitted Squash vein yellowing virus (SqVYV) was first identified in squash and watermelon in Florida in 2005 and shown to cause a severe decline of watermelon vines as crops approach harvest. Florida is most economically impacted by SqVYV, although the virus has been detected more recently in Indiana and South Carolina. The origin and evolutionary history of SqVYV, one of the few members of the genus Ipomovirus within the family Potyviridae, are not known. Sequence diversity of SqVYV isolates collected at different times, from different locations and from different plant species is being analyzed for insights into the origin of the virus. More recently, Cucurbit leaf crumple virus (CuLCrV) and Cucurbit yellow stunting disorder virus (CYSDV), also whitefly-transmitted, have been detected in watermelon in Florida. Tomato yellow leaf curl virus (TYLCV) was first detected in south Florida tomato crops in 1997. Several surveys have been conducted in the region to identify alternative hosts for these four viruses. Cucurbit weeds including Balsam-apple (Momordica charantia), creeping cucumber (Melothria pendula) and smellmelon (Cucumis melo var. dudaim) provide reservoirs for SqVYV, CuLCrV and/or CYSDV. Green bean (Phaseolus vulgaris) also can be a reservoir for CuLCrV. No wild hosts of TYLCV have been reported in Florida. The effectiveness of insecticides and silver plastic mulch to manage whiteflies and mitigate TYLCV has been demonstrated and is currently being evaluated for SqVYV, CuLCrV and CYSDV. In addition, potential sources of SqVYV resistance have been identified in greenhouse and field screening of watermelon germplasm. Further studies to refine these sources of resistance are underway. Lastly, a comprehensive map of 33,560 hectares (82,928 acres) of vegetable fields in the three counties comprising the majority of the southwest Florida vegetable production area has been developed to identify 'hot spots' and reservoir crops for viruses and whiteflies, and will be useful in evaluation of management strategies to decrease virus incidence in commercial fields.

Distribution of four viruses in single and mixed infections within infected watermelon plants in Florida.

Whitefly-transmitted Squash vein yellowing virus (SqVYV) and Cucurbit leaf crumple virus (CuLCrV) and aphid-transmitted Papaya ringspot virus type W (PRSV-W) have had serious impact on watermelon production in southwest and west-central Florida in the past 5 years. Tissue-blot nucleic acid hybridization assays were developed for simple, high-throughput detection of these three viruses as well as Cucurbit yellow stunting disorder virus (CYSDV), which was first reported in Florida in 2008. To determine virus distribution within plants, we collected 80 entire plants just before or during the harvest period in a systematic sample, 20 each on 11 April, 18 April, 26 April, and 3 May 2007, from a fruiting commercial watermelon field near Immokalee, FL showing symptoms of infection by SqVYV, CuLCrV, and PRSV-W and, possibly, CYSDV. This was followed by a sampling of five plants collected at harvest showing symptoms of virus infection on 11 October 2007 in a different commercial planting located in Duette, FL. Tissue prints were made from cross sections of watermelon plants from the crowns through the tips at 0.6-m intervals on nylon membranes and nucleic acid hybridization assays were used for virus detection. Results from testing crown tissue showed that SqVYV, CuLCrV, and PRSV-W were present in ≈37, 44, and 54%, respectively, of the 80 plants collected over the four sampling dates from the first field. For individual vines diagnosed with SqVYV, the distribution of SqVYV in vine tissue decreased proportionately with distance from the crown. The probability of detecting SqVYV was 70% at the base of the vine compared with 23% at the tip of the vine. In contrast, CuLCrV tended to be more evenly distributed throughout the plant, with ≈10% higher probability of detection at the growing tip relative to the crown of the plant. The distribution of PRSV-W resembled that of SqVYV but with ≈20% higher probability of detection at the tip of the vine. Similar trends were detected in the smaller sampling; however, CYSDV was also detected in three of the plants. Overall, the results indicated that SqVYV and PRSV-W were distributed differently than CuLCrV in watermelon plants, and this difference has implications on how samples should be collected and may affect vector acquisition and transmission of these viruses.