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PLANT GENETIC RESOURCES

Screening the Watermelon Germplasm Collection for Resistance to Papaya Ringspot Virus Type-W

^a Dep. of Horticultural Science, North Carolina State Univ., Raleigh, NC 27695-7609
^b Dep. of Plant Pathology, North Carolina State Univ., Raleigh, NC 27695-7619

* Corresponding author (todd_wehner{at}ncsu.edu)

	ABSTRACT

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS AND DISCUSSION REFERENCES

 
Papaya ringspot virus watermelon strain (PRSV-W), formerly watermelon mosaic virus-1, is a major disease of watermelon [Citrullus lanatus (Thunb.) Matsum. & Nakai]. The objectives of this study were (i) to screen the USDA watermelon germplasm collection for PRSV-W resistance, (ii) to verify the disease rating for the most resistant and most susceptible accessions, (iii) to determine the number of escapes on the basis of the retest of the germplasm screening test. The experiment was a randomized complete block with five replications and 1275 accessions. ‘Charleston Gray’ susceptible checks were used to verify that the PRSV-W inoculum was virulent. Enzyme-linked immunosorbent assay (ELISA) was performed after the last rating to determine whether the virus was in the plant tissue. The PI accessions with the highest resistance to PRSV-W that also had resistance to other watermelon viruses (ZYMV, zucchini yellow mosaic virus or WMV, watermelon mosaic virus, formerly watermelon mosaic virus-2) were PI 244018, PI 244019, PI 255137, and PI 482299. The first retest of the most resistant 21 PI accessions showed that there were some escapes that were not resistant to PRSV-W. Of the 21 PI accessions in the retest, seven PI accessions were identified for further testing. Of the 60 resistant PI accessions in the final retest, eight had resistance with a rating of 3.6 or less for the best, average, and maximum ratings: PI 244017 (best over all tests), PI 244019, PI 482342, PI 482318, PI 485583, PI 482379, PI 595203, and PI 244018.

Abbreviations: PRSV-W, papaya ringspot virus-watermelon strain • ZYMV, zucchini yellow mosaic virus • WMV, watermelon mosaic virus

	INTRODUCTION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS AND DISCUSSION REFERENCES

 
WATERMELON IS A major crop in the southern USA. The most important virus diseases of watermelon in the USA are papaya ringspot virus-watermelon strain (PRSV-W, formerly watermelon mosaic virus-1), watermelon mosaic virus (WMV, formerly watermelon mosaic virus-2), and zucchini yellow mosaic (ZYMV) (Adlerz and Crall, 1967). Virus diseases are destructive to the watermelon crop and are difficult to control (Sherf and Macnab, 1986).

Major virus control strategies include the use of insecticides to eliminate virus vectors, herbicides to remove alternate hosts for the virus, and genetic resistance (Provvidenti, 1993), which is often pathogen-specific (Grumet, 1989). Of those controls, the most economical method is genetic resistance. Virus resistance may also be accomplished through virus coat proteins transferred into existing cultivars (Namba et al., 1992; Quemada et al., 1990), or by screening of germplasm collections. Either method might be used successfully in watermelon. The watermelon germplasm collection has been screened for resistance to several virus diseases. Boyhan et al. (1992) identified PI accessions resistant to ZYMV and Gillaspie and Wright (1993) identified PI accessions resistant to WMV. Researchers have screened other cucurbit species for resistance to PRSV-W and the inheritance of the resistance has been determined. Provvidenti and Gonsalves (1982) found in Cucumis metuliferus E. Meyer ex Naudin that accessions resistant to WMV were also resistant to PRSV and that the resistance was controlled by a single dominant gene.

PRSV-W virus infects all the agriculturally important species of the Cucurbitaceae (Provvidenti, 1993). PRSV-W was known as watermelon mosaic virus-1 until it was shown to be a strain of papaya ringspot virus (Provvidenti, 1993). This virus is transmitted in a nonpersistent manner by 24 species of aphid in 15 genera. Resistance to the virus has been identified in cucumber (Cucumis sativus L.), melon (Cucumis melo L.), squash (Cucurbita spp.), and gourds (Lagenaria spp. and Luffa spp.) (Provvidenti, 1993).

Previous research has demonstrated that screening watermelon for resistance to PRSV-W should be effective. Munger et al. (1984) used an unidentified isolate of PRSV-W to find genetic differences among seven watermelon PI accessions. Hojo et al. (1991a) used an aggressive Brazilian isolate, Ab-081, to screen watermelon for virus resistance. They identified one resistant accession, BT-8501, a wild, bitter-fruited watermelon from Africa (Hojo et al., 1991b). Additionally, there may be field tolerance available in some land races of watermelon (Provvidenti, 1986).

The objectives of this study were (i) to screen the USDA watermelon germplasm collection for PRSV-W resistance; (ii) to verify the disease rating for the most resistant and most susceptible accessions; and (iii) to determine the number of escapes on the basis of the retest of the germplasm screening test.

	MATERIALS AND METHODS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS AND DISCUSSION REFERENCES

 
Three large experiments were performed: a germplasm screening, an early retest of the screening results, and a final retest. All experiments were performed in the North Carolina State University plant pathology greenhouses. Greenhouse temperatures ranged from 23 to 43°C (day) and 12 to 24°C (night). The virus isolate was obtained from D.E. Purcifull, University of Florida, Gainesville. The PRSV-W isolate used was 2052 described by Baker et al. (1991) and was maintained on ‘Gray Zucchini’ squash (Cucurbita pepo L.) from Seminis Vegetable Seeds (Woodland, CA). All Citrullus Plant Introduction (PI) accessions were obtained from the Southern Regional Plant Introduction Station at Griffin, GA. PI accessions originated in 68 different countries, with 46 countries having fewer than 10 accessions each. Countries with the most accessions in the collection of 1275 were Turkey (296), Yugoslavia (163), Zimbabwe (122), India (120), Spain (71), Zambia (55), South Africa (36), Syria (28), Iran (27), China (26), and Nigeria (23).

Inoculum was produced by grinding infected Gray Zucchini squash leaves with mortar and pestle in 0.02 M phosphate buffer, pH 7.0. Leaf to buffer ratio was 1:5 (1 g infected leaf to 5 mL buffer). The inoculation procedure was the same for increasing on squash and for screening watermelon. Inoculation consisted of dusting one leaf on each 3-wk-old plant with an 800-mesh carborundum, then applying the inoculum to the leaf with a pestle which was rotated in a circular motion 8 to 10 times as if painting the leaf with inoculum. After inoculation, carborundum was rinsed off the leaves to improve light interception, and the plants were maintained in aphid-proof cages. All Gray Zucchini squash plants were seeded in metromix 200 (Scotts-Sierra Horticultural Products Company, Marysville, OH) in 160-mm diameter (1550-mL volume) clay pots. Plants were fertilized weekly with 150 mg kg^-1 Peters Professional 20-20-20 N-P-K (Scotts-Sierra Horticultural Products Company, Marysville, OH).

The germplasm screening test was a randomized complete block with five replications of 1275 accessions. Each plot was a 100 x 100 mm square pot (600-mL volume) planted with two seeds and thinned to one plant before inoculation. In addition to the accessions tested, there were 50 check plants per replication of ‘Charleston Gray’ that were inoculated with the virus, and 50 plants of Charleston Gray that were not inoculated. The inoculated checks served as verification of viral infection and the uninoculated checks served as an indicator of other disease in the greenhouse that might confound symptom expression.

To screen large numbers of PI accessions for resistance to PRSV-W, a rating system was used that took into account the different growth habits and leaf morphologies of the different accessions. The rating system was general enough to allow for the differences in the PI accessions but specific enough to distinguish resistant plants. Plants were inoculated at the first true leaf stage, and rated weekly for 6 wk on a scale of 1 to 9 on the basis of severity of viral symptoms, where 1 = none, 2 = tendrils absent, 3 = tendrils absent, slightly stunted growth, 4 = mosaic patches and/or necrotic spots on leaves, 5 = leaves near apical meristem deformed, meristem yellow and reduced in size, 6 = apical meristem withered and brown, 7 = apical meristem dead with more basal leaves dying, 8 = most leaves dead, main stem green/yellow, 9 = plant dead. After the second rating, plants which had not emerged at the time of inoculation and those plants which were inoculated and had a rating under 4 were reinoculated to reduce the number of escapes. After the sixth rating, all plants that were not dead were tested by ELISA (Agdia Incorporated, Elkhart, IN) to determine if there was virus present in the leaf tissue. Tissue used for testing was taken from a sample of the top five leaves of the plant. Those plants which did not have virus in their system and which had a low rating were considered resistant.

Originally, we used Chenopodium amaranticolor Coste & A. Reynier to check inoculation efficacy. However, that was not a reliable indicator, so checks of Charleston Gray were inoculated and observed for viral symptoms. In the reinoculation stage, three check plants that were the same age as the test plants to be reinoculated and three 2-wk-old checks were inoculated periodically during the reinoculation to assure that the inoculum was virulent. All PI accessions were maintained in a screened greenhouse which contained no other cucurbits and in which no other viral experiments were being performed. The germplasm screening started in the summer of 1998 and ended in the fall of 1999.

Data were summarized as the average, the maximum, and the best of the six ratings. The best rating was the one with the greatest range over the 1275 cultigens, which was rating 3 in this experiment. Data were analyzed by means of the MEANS, ANOVA, CORRELATION, and GLM procedures of the SAS statistical package (SAS Institute, Cary, NC). Data were based on ratings from single-plant plots, and each rating date was analyzed separately.

An early retest was performed after the completion of the first two replications of the germplasm screening to determine variability within the resistant accessions. This also provided a method for determining the number of escapes and errors in the germplasm screening before continuing with the next replications. Results were used to plan the next studies, and to begin seed increases of cultigens having resistance to PRSV-W. The early retest was performed with one replication of 21 plants per accession from the 21 most resistant accessions along with the susceptible check, Charleston Gray. Inoculation and rating procedures were the same as for the germplasm screening.

A final retest was performed after the completion of the germplasm screening to verify the reaction of the most resistant and susceptible accessions. We were also interested in measuring the variability over replications. The experiment was a randomized complete block with four replications of 72 cultigens along with two susceptible checks (Charleston Gray and ‘Crimson Sweet’). Plots consisted of two 100- x 100-mm square pots (600-mL volume). Extra pots of each accession were planted to assure that all plots would have the same number of plants even with differences in germination. The cultigens were inoculated with four isolates of PRSV-W, which were 2052, W-1A, 1870, and 2040. Plants were inoculated by the rub method at the first true leaf stage, and rated three times weekly on a 1-to-9 scale starting 2 wk after inoculation.

	RESULTS AND DISCUSSION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS AND DISCUSSION REFERENCES

 
Germplasm Screening
Not all of the 1275 accessions germinated in all five replications, and data were obtained for 1248 accessions. The complete dataset was submitted to the Germplasm Resources Information Network (http://www.ars-grin.gov/, 5 Feb. 2002) for those interested in particular cultigens. The most resistant and most susceptible cultigens are presented here, along with checks and cultigens included in the retests (Table 1). The ANOVA indicated that there were highly significant differences (P = 0.01) among accessions for all rating dates. Since the best and average ratings were highly correlated (r = 0.90), and the maximum rating had a smaller F ratio than the other ratings, only the best rating was given in Table 1 to save space. In a study such as this, where most of the 1275 accessions were susceptible and had ratings of 8 to 9, there was the possibility that the few resistant accessions identified were overwhelmed in the analysis of variance by the large number of susceptible accessions. To evaluate the importance of that effect, the ANOVA was rerun with only the most resistant 52 and the most susceptible 52 accessions. That effect was large in this study, with the F ratio changing from 1.5 for the 1248 cultigens to 9.3 for the 104 cultigens. Therefore, both analyses showed significant differences among PI accessions, but the smaller data set had a larger accession F ratio because of the smaller variances for replication and error, and the larger accession variance.

Our results paralleled that of previous researchers in that variation for virus resistance was identified in the watermelon germplasm collection. Boyhan et al. (1992) working with ZYMV, and Gillaspie and Wright (1993) working with WMV were able to identify resistant accessions in their watermelon research. PI 482299, PI 482261, PI 595203, and PI 255137 found to be resistant to ZYMV also had some resistance in this study to PRSV-W. Other accessions reported to have resistance to ZYMV were not tested in this study. One accession, PI 595202, reported to have ZYMV resistance did not show resistance to PRSV-W in this study. PI 244018 and PI 244019 found to be resistant to WMV by Gillaspie and Wright (1993), showed resistance to PRSV-W. Other accessions that were found to be resistant to WMV (PI 189316, PI 189317, and PI 248178) did not have resistance to PRSV-W. Provvidenti and Gonsalves (1982) worked with C. metuliferus and found that the accessions they identified as resistant to WMV were also resistant to PRSV. This was the case for some of the accessions screened in this study.

Gillaspie and Wright (1993) found there were plants that tested negative for virus in ELISA that later tested positive. This is consistent with what we found. Thus, even though there were plants that tested negative for virus with ELISA, all plants eventually showed symptoms of PRSV-W and died. The issue of escapes should not be underestimated. Studies for resistance to WMV and ZYMV found, during retests, that some accessions which were initially rated resistant were actually just escapes. It is likely that some of the accessions reported to be resistant here are escapes.

The susceptible check used for this study was Charleston Gray, a widely available cultivar. However, we identified accessions having more susceptibility to PRSV-W. Those accessions would make excellent susceptible checks because they have high germination rates, and best ratings of 9.0 compared with Charleston Gray, which had a best rating of 8.2 (Table 1). Any of the following accessions could be used as susceptible checks: PI 379253, PI 512383, PI 169270, PI 178877, PI 169244, PI 560016, PI 368495, PI 357727, PI 381696, PI 502318, PI 176485, PI 176921, PI 368497, PI 368515, PI 379245, PI 178873, PI 279461, PI 176490, PI 500309, PI 508442, PI 357690, PI 357735, PI 278027, PI 507859, PI 195928, PI 532817, PI 176499, PI 500332, PI 512358, PI 379246, PI 357675, PI 512391, PI 512359, PI 296337, PI 169283, PI 179661, PI 295850, PI 179662, PI 169252, PI 183399, PI 357728, PI 357692, PI 512407, PI 169258, PI 234603, PI 357698, PI 357736, PI 169245, PI 490382, PI 378613, and PI 296342 (data in GRIN).

The PI accessions with the most resistance along with complete data (missing in no more than one replication) were PI 244017, PI 244018, PI 482342, PI 234287, and PI 482303 (Table 2). The PI accessions that showed resistance to other watermelon viruses in addition to resistance to PRSV-W in this study were PI 244018, PI 244019, PI 482226, PI 595203, PI 255137, and PI 482299.

The final retest was conducted at the completion of the germplasm screening. The final retest results paralleled those of the early retest results, in that there were escapes that were not resistant to PRSV-W (Table 4). Of the 60 resistant PI accessions in the final retest, eight had resistance with a rating of 3.6 or less for the best, average and maximum ratings: PI 244019, PI 244017, PI 482342, PI 482318, PI 485583, PI 482379, PI 595203, and PI 244018. PI 244017 was given the highest resistance rating in both the germplasm screening and the final retest experiments. The 12 susceptible PI accessions had about the same level of susceptibility to PRSV-W as the Charleston Gray check.

Further research is needed to determine whether other isolates of PRSV-W from different geographic regions react the same on the resistant and susceptible cultigens from this experiment. Additional research is needed to determine the inheritance of resistance to PRSV-W in the cultigens identified. The accessions with the highest resistance to PRSV-W should be used to develop inbreds with the highest possible resistance for use in developing resistant cultivars.

	ACKNOWLEDGMENTS

 
The authors gratefully acknowledge the technical assistance of Tammy L. Ellington and Nischit V. Shetty.

Received for publication June 25, 2001.

	REFERENCES

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS AND DISCUSSION REFERENCES

 

• Adlerz, W.C., and J.M. Crall. 1967. Epidemiology of control of watermelon mosaic virus. Florida Agric. Exp. Stn. Annu. Rep. 403.
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• Boyhan, G., J.D. Norton, B.J. Jacobsen, and B.R. Abrahams. 1992. Evaluation of watermelon and related germplasm for resistance to zucchini yellow mosaic virus. Plant Dis. 76:251–252.
• Gillaspie, A.G., and J.M. Wright. 1993. Evaluation of Citrullus sp. germplasm for resistance to watermelon mosaic virus 2. Plant Dis. 77:352–354.
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• Munger, H. M., T.A. More, and S. Awni. 1984. A preliminary report on screening watermelons for resistance to watermelon mosaic viruses 1 and 2. Cucurbit Genet. Coop. Rep. 7:61–62.
• Namba, S., K. Ling, C. Gonsalves, J.L. Slightom, and D. Gonsalves. 1992. Protection of transgenic plants expressing the coat protein gene of watermelon mosaic virus II or zucchini yellow mosaic virus against six potyviruses. Phytopathology 82:940–946.
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