Chandrasekar S. Kousik

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.

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.

Genetic diversity among Lagenaria siceraria accessions containing resistance to root-knot nematodes, whiteflies, ZYMV or powdery mildew.

In recent years, there has been an increased interest in Europe and in the USA in grafting watermelon onto bottle gourd, Lagenaria siceraria (Mol.) Standl. In this study, genetic diversity and relationships were examined [using 236 sequence-related amplified polymorphism markers] among 56 United States plant introductions (PIs) of L. siceraria and PIs of important cucurbit crops [including Cucurbita maxima Duchesne (winter squash), Cucurbita pepo L. (squash and pumpkin), Citrullus spp. (watermelon), Cucumis melo L. (melon) and Cucumis sativus L. (cucumber)]. The analysis showed that L. siceraria is distinct and has similar genetic distances to the cucurbit species examined herein. The L. siceraria PIs were assembled into two major clusters. One cluster includes groups of PIs collected mostly in South Asia (India) and a few PIs collected in the Mediterranean region and in Northeast Africa. The second cluster includes groups of PIs collected mainly in Southern Africa and in North, Central and South America, and PIs collected in China, Indonesia and Cyprus. All L. siceraria PIs in this study were susceptible to the southern root-knot nematode (RKN) [ Meloidogyne incognita (Kofoid and White) Sandground]. However, several PIs, among them a group of closely related PIs collected in Mexico and Florida, were less infected with southern RKNs. All L. siceraria PIs were infested with whiteflies [ Bemisia tabaci (Gennadius)], while several PIs were less infested than others and need further evaluation and selection for developing breeding lines that may be less appealing to this pest. Most of the PIs that showed resistance to zucchini yellow mosaic virus and tolerance to powdery mildew were collected in India and belong to the same phylogenetic groups (PGs). Experiments with L. siceraria PIs representing different PGs showed similar grafting compatibility with watermelon. Findings from this study should be useful for the development of superior L. siceraria rootstock lines with enhanced resistance to diseases and insect pests of cucurbit crops.

Sensitivity of Isolates of Phytophthora capsici from the Eastern United States to Fluopicolide

Fluopicolide, a pyridinylmethyl-benzamide fungicide, was registered in the United States in 2008 to control diseases caused by Oomycete pathogens, such as Phytophthora capsici, on cucurbit and solanaceous vegetables. The main objective of this study was to determine baseline sensitivity to fluopicolide in isolates of P. capsici from the southeastern and midwestern United States. A total of 69 isolates from Florida (14 isolates), Georgia (14 isolates), Michigan (24 isolates), North Carolina (3 isolates), and South Carolina (17 isolates) that had not been previously exposed to fluopicolide were grown on fungicide-amended medium to determine sensitivity of mycelia, sporangia, and zoospores to the fungicide. All isolates of P. capsici tested (range of 54 to 69 isolates per assay) were sensitive to fluopicolide in all four assays. The median EC50 fluopicolide concentration was 0.22, 2.08, 0.048, and 0.10 mg/liter in the mycelial growth, zoospore germination, sporangia production, and zoospore production assays, respectively. For mycelial growth and zoospore germination, isolates from Michigan had a higher mean EC50 value than isolates from the four southeastern states. This is the first report of variation in baseline sensitivity to a fungicide by P. capsici isolates from different regions of the United States. In the sporangia production and zoospore production assays, isolates from different states did not differ in sensitivity. Single rates of fluopicolide were tested with additional isolates to validate discriminatory rates for monitoring sensitivity. A concentration of 0.3 or 1.0 mg/liter is recommended for mycelial growth, and 0.1 mg/liter is recommended for sporangia and zoospore production.

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.

Development and Field Evaluation of Multiple Virus-Resistant Bottle Gourd (Lagenaria siceraria)

In an effort to develop bottle gourd (Lagenaria siceraria) as a widely adapted rootstock for watermelon grafting, we sought to identify lines with broad resistance to several cucurbit viruses that are economically important in the United States. Preliminary analysis under greenhouse conditions indicated that the currently available commercial watermelon rootstocks were either highly susceptible or somewhat tolerant to one or more viruses. However, in greenhouse screening, several breeding lines of bottle gourd displayed broad-spectrum resistance to four viruses tested, including Zucchini yellow mosaic virus, Watermelon mosaic virus (WMV), Papaya ringspot virus watermelon strain (PRSV-W), and Squash vein yellowing virus. Resistance to PRSV-W and WMV was confirmed through field trials in two consecutive years at two different locations in South Carolina. Two breeding lines (USVL#1-8 and USVL#5-5) with broad-spectrum virus resistance could be useful materials for watermelon rootstock development.

NMR Spectroscopy Identifies Metabolites Translocated from Powdery Mildew Resistant Rootstocks to Susceptible Watermelon Scions

Powdery mildew (PM) disease causes significant loss in watermelon. Due to the unavailability of a commercial watermelon variety that is resistant to PM, grafting susceptible cultivars on wild resistant rootstocks is being explored as a short-term management strategy to combat this disease. Nuclear magnetic resonance-based metabolic profiles of susceptible and resistant rootstocks of watermelon and their corresponding susceptible scions (Mickey Lee) were compared to screen for potential metabolites related to PM resistance using multivariate principal component analysis. Significant score plot differences between the susceptible and resistant groups were revealed through Mahalanobis distance analysis. Significantly different spectral buckets and their corresponding metabolites (including choline, fumarate, 5-hydroxyindole-3-acetate, and melatonin) have been identified quantitatively using multivariate loading plots and verified by volcano plot analyses. The data suggest that these metabolites were translocated from the powdery mildew resistant rootstocks to their corresponding powdery mildew susceptible scions and can be related to PM disease resistance.

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.

Cucurbit yellow stunting disorder virus detected in pigweed in Florida.

Pigweeds (genus Amaranthus) are problematic weeds in crop production throughout the world and are responsible for significant yield losses in many crops (2). Members of this genus can produce hundreds of thousands of seeds per plant and are also capable of supporting populations of important crop pathogens including viruses, nematodes, fungi, and oomycetes. Thirty-one pigweed samples (tentatively identified as Amaranthus lividus L. based on leaf notch and growth habit) were collected in November and December of 2009 from a watermelon field near Immokalee, FL, previously found to contain watermelon plants infected with three whitefly-transmitted viruses: Cucurbit yellow stunting disorder virus (CYSDV), Cucurbit leaf crumple virus (CuLCrV), and Squash vein yellowing virus (SqVYV). Although no obvious virus symptoms were observed on any of the pigweed plants, whiteflies (Bemisia tabaci), a known vector of CYSDV, CuLCrV, and SqVYV, were observed on leaves. Consequently, replica tissue blots were made from all pigweed samples and tested independently by tissue blot nucleic acid hybridization assay for CYSDV, CuLCrV, or SqVYV (3). Tissue blots indicated CYSDV infection in six pigweed samples. Neither CuLCrV nor SqVYV was detected. Three of the tissue blot-positive pigweed samples were further tested by reverse transcription (RT)-PCR amplification from total RNA (extracted from leaf tissue with TRIzol Reagent [Invitrogen, Carlsbad, CA]) with HSP70 and coat protein (CP) gene primers (1). HSP70 and CP gene RT-PCR products of the expected sizes (175 and 707 nt, respectively) were amplified, sequenced, and found to be 100% identical for all three pigweed samples. The partial HSP70 gene sequence from pigweed shared 98.3 to 100% nucleotide identity with CYSDV isolates from Arizona, California, and Spain (GenBank Accession Nos. FJ492808, EU596530, and NC_004810, respectively). The partial CP gene sequence from pigweed shared 88.8 to 100% nucleotide identity with CYSDV isolates from Arizona, Saudi Arabia, Texas, and Spain (GenBank Accession Nos. EF210558, AF312811, AF312806, and AF312808, respectively). To our knowledge, this is the first report of CYSDV infection of pigweed in Florida. Infection of redroot pigweed (A. retroflexus) was recently reported in California (4). These results collectively indicate that control of noncucurbit weeds may be important for effective management of CYSDV in cucurbit crops. References: (1) S. Adkins et al. Online publication. doi:10.1094/PHP-2009-1118-01-BR. Plant Health Progress, 2009. (2) L. Holm et al. Worlds Weeds: Natural Histories and Distributions. John Wiley and Sons, Inc. New York, NY, 1997. (3) W. W. Turechek et al. Phytopathology 100:1194, 2010. (4) W. M. Wintermantel et al. Plant Dis. 93:685, 2009.