Abstract
Phytophthora fruit rot, caused by Phytophthora capsici, is prevalent in most watermelon-producing regions of southeastern United States and is known to cause pre- and post-harvest yield losses. A non-wound inoculation technique was developed to evaluate detached mature fruit belonging to U.S. watermelon PIs for resistance to fruit rot caused by P. capsici. Mature fruit were harvested and placed on wire shelves in a walk-in humid chamber [greater than 95% relative humidity (RH), temperature 26 ± 2 °C] and inoculated with a 7-mm agar plug from an actively growing colony of P. capsici. Twenty-four PIs that exhibited resistance in a preliminary evaluation of 205 PIs belonging to the watermelon core collection in 2009 were grown in the field and greenhouse in 2010 and 2011 and evaluated in the walk-in humid chamber. Fruit rot development was rapid on fruit of susceptible controls ‘Black Diamond’, ‘Sugar Baby’, and PI 536464. Several accessions including PI 560020, PI 306782, PI 186489, and PI 595203 (all Citrullus lanatus var. lanatus) were highly resistant to fruit rot. One C. colocynthis (PI 388770) and a C. lanatus var. citroides PI (PI 189225) also showed fruit rot resistance. Fruit from PIs that were resistant also had significantly lower amounts of P. capsici DNA/gram of fruit tissue compared with the susceptible commercial cultivars Sugar Baby and Black Diamond. The sources of resistance to Phytophthora fruit rot identified in this study may prove useful in watermelon breeding programs aimed at enhancing disease resistance.
Phytophthora capsici has been documented as a pathogen on a wide variety of vegetable crops in the families Solanaceae (tomato, pepper, eggplant), Cucurbitaceae (cucumber, watermelon, squash, pumpkin, melon), Fabaceae (lima bean) (Babadoost and Zitter, 2009; Erwin and Riberio, 1996; Gubler and Davis, 1996; Hausbeck and Lamour, 2004), frasier fir (Quesada-Ocampo et al., 2009), and plants belonging to 23 other families (Granke et al., 2012b). The pathogen has also been reported to have caused severe losses in cucurbit crop production since the 1940s resulting in cessation of production in severely infested fields (Babadoost, 2004; Babadoost and Zitter, 2009; Hausbeck and Lamour, 2004; Kreutzer et al., 1940).
Phytophthora fruit rot of watermelons caused by P. capsici was first reported in 1940 (Wiant and Tucker, 1940) and is prevalent in many watermelon-growing regions of the United States (Gevens et al., 2008; Hausbeck et al., 2012; Kousik et al., 2011a; McGrath, 1996). The disease is particularly severe in the southeastern United States where ≈50% of the watermelons are grown (Florida, Georgia, Alabama, South Carolina, North Carolina, and Virginia) and optimal conditions for development of Phytophthora fruit rot are prevalent. Between 2003 and 2008 many watermelon growers in Georgia, South Carolina, and North Carolina did not harvest their crop as a result of severe fruit rot. In some instances fruits rotted after shipping, resulting in rejection of entire loads and loss of revenue (Jester and Holmes, 2003; Kousik et al., 2011a). Such instances led the watermelon growers’ consortium, National Watermelon Association, to list Phytophthora fruit rot as one of their most important research priorities (Morrissey, 2006).
The current recommended strategy for managing P. capsici includes a combination of several control methods, cultural practices that ensure well-drained soils, crop rotation, soil solarization, reducing splash dispersed soil, and application of fungicides (Babadoost, 2004; Granke et al., 2012b; Hausbeck and Lamour, 2004; Kousik et al., 2011a; McGrath, 1996). Several commercial fungicides that are effective in managing Phytophthora fruit rot of watermelon have been identified (Kousik et al., 2011a, 2011b). However, the prevalence of P. capsici isolates insensitive to fungicides such as cyazofamid and mefenoxam also has been well documented (Granke et al., 2012a, 2012b; Hausbeck and Lamour, 2004; Kousik and Keinath, 2008; Jackson et al., 2012). Furthermore, the application of fungicides is not very effective when disease pressure is high (Granke et al., 2012b; Kousik et al., 2011a; McGrath, 1994). Therefore, alternative strategies such as host resistance for managing Phytophthora fruit rot are needed.
Host plant resistance can be considered the cornerstone of an integrated disease management program, and watermelon cultivars with resistance to P. capsici would be extremely useful in management of Phytophthora fruit rot. Host resistance to P. capsici in peppers was first reported by Kimble and Grogan (1960) and additional sources of resistance were subsequently reported by Barksdale et al. (1984) and Peter et al. (1984). Recently, commercial bell pepper hybrids with resistance to P. capsici have become available (Candole et al., 2010; Foster and Hausbeck, 2010). However, there are few reports on resistance of cucurbit crops to P. capsici. Resistance to crown rot caused by P. capsici was reported in a Korean pumpkin cultivar (Lee et al., 2001) and squash (Cucurbita pepo) accessions (Padley et al., 2008). Gevens et al. (2006) identified cucumber varieties that limit development of P. capsici; however, none of the varieties tested had complete fruit rot resistance. Resistance in cucumber was related to the developmental stage of the fruit, increasing with increasing age and size of the fruit (Ando et al., 2009; Gevens et al., 2006). Ando et al. (2009) also noted that there have been no studies to identify sources of resistance to P. capsici in watermelon or melon.
A majority of the evaluations to identify sources of resistance to P. capsici in pepper, tomato, and some cucurbits has been performed on seedlings. Over four months are usually needed to grow watermelon fruit for evaluations and because of larger fruit, more resources are required to complete the evaluations. Furthermore, resistance at the seedling stage is generally not correlated with resistance in the fruit (Gevens et al., 2006). In pepper, it was reported that resistance is controlled by different genes in the leaves and the roots (Sy et al., 2005; Walker and Bosland, 1999). In cucumbers, the plants are generally resistant to P. capsici; however, the fruit are very susceptible and it is possible that more than one mechanism of resistance may be involved (Gevens et al., 2006). We evaluated seedlings of over 1800 U.S. watermelon PIs for resistance to crown rot (Kousik et al., unpublished data) caused by P. capsici and 205 PIs for resistance to fruit rot (Kousik, 2011) and there appears to be no clear correlation between resistance in fruit and resistance in seedling to the same pathogen.
Because fruit rot is a major problem in watermelon, identifying sources of resistance that can be useful for developing resistant cultivars is important. In 2009, we conducted a preliminary unreplicated evaluation of 205 PIs in the core collection of U.S. watermelon PI available with the Plant Genetic Resources and Conservation unit (PGRCU), Griffin, GA (<http://www.ars-grin.gov>) and reported the results as an abstract (Kousik, 2011). The present studies were conducted to identify and confirm resistance in watermelon PI to Phytophthora fruit rot. In addition, we quantified the development of P. capsici in inoculated fruit tissue using real-time quantitative polymerase chain reaction to further confirm resistance.
Materials and Methods
Seeds of U.S. watermelon PIs.
Seeds of U.S. watermelon PIs belonging to the core collection were obtained from PGRCU, Griffin, GA. The details of the various PIs evaluated in this study are available on the germplasm resources information network (GRIN) web site (<http://www.ars-grin.gov/cgi-bin/npgs/html/desc.pl?151021>). Seeds of commercial watermelon cultivars Black Diamond, Sugar Baby, Mickey Lee, and Charleston Gray used as controls were purchased from Willhite seeds (Willhite Seeds, Pooleville, TX).
Phytophthora capsici isolate.
Phytophthora capsici isolate RCZ-11, which was highly aggressive on watermelon fruit and caused severe rot, was used as the inoculum. The isolate was collected in 2003 from zucchini (Cucurbita pepo) plants in South Carolina and belongs to mating type A2. The isolate was kindly provided by Dr. A.P. Keinath, Clemson University. The isolate was routinely maintained on V8 juice agar amended with antibiotics (PARP) as described by others (Keinath, 2007; Quesada-Ocampo et al., 2009). The isolate also was routinely inoculated and then re-isolated from watermelon fruit to maintain its virulence.
Evaluation of watermelon core collection for resistance.
The first experiment was conducted in the Summer of 2009 to identify potential sources of resistance among the watermelon PIs. Five plants each of 205 PIs from the watermelon core collection were seeded in 50-cell Jiffy trays (Jiffy Products of America, Norwalk, OH) filled with Metro Mix (Sun Gro Horticulture, Bellevue, WA) and allowed to germinate and grow in a greenhouse for 4 weeks. Four-week-old plants were transplanted onto 96-cm wide raised beds covered with white plastic mulch. Beds were spaced 6.4 m apart. Each PI plot was a single row of five plants spaced 46 cm apart with 2.7-m spacing between plots and was not replicated. Plants of susceptible commercial cultivars Mickey Lee and Black Diamond were used as controls. Irrigation was provided by drip tape placed ≈2.5 cm below the soil surface along the center of the raised beds. After bedding but before planting, row middles were sprayed with glyphosate at 1.1 kg·ha−1 and Strategy [ethalfluralin (0.45 kg·ha−1) + clomazone (0.14 kg·ha−1)] for weed management. Weeds between beds were controlled during the season with spot application of glyphosate. Vines of the watermelon plants were turned every week so as to keep the plants from growing into neighboring plots. Standard watermelon production practices with respect to irrigation and weed management were followed (Kemble, 2010; Sanders, 2006). Fruit were harvested when mature as determined by the drying of tendril on the node bearing the fruit. Because the fruit from different PIs matured at different times in this trial, we included fruit from the susceptible cultivars in every evaluation. An average of five fruit were marked and harvested from each plot for inoculations.
Inoculation and fruit rot assessment.
To handle the large number of fruit of varying sizes to be evaluated, a walk-in humidity chamber (4 × 3 × 3.7 m height) with wire shelves was used. Fruit harvested from each plot were numbered and then randomly placed on the wire shelves. Fruit were surface-disinfested with 10% sodium hypochlorite (Gevens et al., 2006; Granke et al., 2012a) before inoculation. Each fruit was inoculated by placing a 7-mm agar plug from a 4-d-old actively growing isolate of P. capsici in the middle (Fig. 1). The agar plug was placed on top of the fruit such that the mycelium and sporangia touched the fruit surface without injuring the fruit. After inoculation, high RH (greater than 95%) was maintained in the room using a humidifier (Charley’s portable fogger, Model M6253; Charley’s Greenhouse, Mt. Vernon, WA). The temperature in the room was maintained at 26 ± 2 °C. The room was continuously illuminated with fluorescent light. Five days after inoculation, the lesion diameter (Fig. 1) on each fruit was measured in two directions perpendicular to each other as described before by others (Gevens et al., 2006; Granke et al., 2012a). The agar plug was considered the center of the lesion. Similarly, the diameter of the area with visible pathogen growth and sporulation was measured (sporulation diameter). The intensity of sporulation within the lesion was recorded on a 0 to 5 scale, where 0 = no visible sporulation; 1 = very sparse sporulation, few seen next to the agar plug; 2 = some sporulation and covering less than ½ the lesion area and not very dense; 3 = medium sporulation covering ½ the lesion area; 4 = heavy sporulation covering ¾ of the lesion area; and 5 = abundant sporangia, very dense and covering most (greater than 85%) of the lesion area. The length and width of each fruit was also recorded to determine the area of each fruit covered by lesion (%) because fruit size varied greatly among the PI.
Phytophthora fruit rot development on watermelon fruit of a susceptible PI. Agar plug from an actively growing colony of Phytophthora capsici was placed on top of the fruit as the inoculum (center). Large lesion (solid arrow, brown areas) covered with pathogen growth and sporulation (dashed arrow, whitish growth) is observed on the fruit surface. The diameter of the lesion and diameter of the area within the lesion covered with pathogen growth and sporulation (sporulation diameter) and intensity of sporulation were measured.
Citation: HortScience horts 47, 12; 10.21273/HORTSCI.47.12.1682
Phytophthora fruit rot development on watermelon fruit of a susceptible PI. Agar plug from an actively growing colony of Phytophthora capsici was placed on top of the fruit as the inoculum (center). Large lesion (solid arrow, brown areas) covered with pathogen growth and sporulation (dashed arrow, whitish growth) is observed on the fruit surface. The diameter of the lesion and diameter of the area within the lesion covered with pathogen growth and sporulation (sporulation diameter) and intensity of sporulation were measured.
Citation: HortScience horts 47, 12; 10.21273/HORTSCI.47.12.1682
Phytophthora fruit rot development on watermelon fruit of a susceptible PI. Agar plug from an actively growing colony of Phytophthora capsici was placed on top of the fruit as the inoculum (center). Large lesion (solid arrow, brown areas) covered with pathogen growth and sporulation (dashed arrow, whitish growth) is observed on the fruit surface. The diameter of the lesion and diameter of the area within the lesion covered with pathogen growth and sporulation (sporulation diameter) and intensity of sporulation were measured.
Citation: HortScience horts 47, 12; 10.21273/HORTSCI.47.12.1682
Evaluation of selected watermelon PIs grown in the field.
The first evaluation of the most resistant PI was conducted in the summer of 2010. Twenty-four PIs from the experiment conducted in 2009 that had less than 1.0 sporulation intensity (Table 1) were included in this trial. Two susceptible PIs identified in the 2009 trial were also included in this field experiment. The experiment was arranged in a randomized complete block design with two replications for each PI. Only two replications were used because of limited availability of seeds. Four-week-old seedlings of select PI were grown in 50-cell jiffy trays as described previously and transplanted on 4 May onto 96-cm wide raised beds covered with white plastic mulch as described before. Each PI plot was a single row of five plants spaced 46 cm apart with 2.7-m spacing between plots. Plants of susceptible commercial cultivars Mickey Lee, Black Diamond, and Sugar Baby were used as controls. Field plots were managed as described previously. Five fruits were marked (given plot numbers) and harvested from each plot for inoculations as described previously. Five d after inoculation, data on the lesion diameter, sporulation diameter, and sporulation intensity were recorded.
Phytophthora fruit rot development on detached watermelon fruit in a preliminary evaluation of the watermelon core collection of U.S. PIs and controls grown in a field in Summer 2009, Charleston, SC.z
A second trial was conducted in Fall 2010 with 10 of the most resistant and two highly susceptible PIs. The commercial cultivars, Black Diamond, Sugar Baby, and Mickey Lee, were used as susceptible controls. The experimental design was a randomized complete block with four replications for each PI or the controls. Transplants were grown in the greenhouse and transplanted on 6 Aug. 2010. Mature fruit were harvested and inoculated on 22 Sept. 2010 and rated 5 d later as previously described.
In Summer 2011, eight of the most resistant, two highly susceptible PIs, and the controls ‘Sugar Baby’ and ‘Black Diamond’ were seeded in Jiffy trays on 12 May and then seedlings were transplanted to the field on 7 June 2011. The experimental design, similar to the second test in 2010, was a randomized complete block with four replications per plot. Mature fruit were harvested, inoculated, and rated as described previously.
Evaluation of selected PIs grown in the greenhouse.
In Spring 2010, 24 PIs selected on the basis of the 2009 evaluation of the core collection were grown in a greenhouse in Charleston, SC. The greenhouse was maintained at 25 ± 2 °C. Plants were grown in 50-cell Jiffy trays and then transplanted to 11-L (3-gallon) plastic pots. Five plants of each PI were grown. One plant was grown in each pot and was considered as a replication. Each plant was hand-pollinated to produce a self-pollinated fruit. Mature fruit were harvested and placed randomly on shelves in the walk-in humid chamber as described before. Fruit were inoculated and rated for disease development as described previously. After fruit rot ratings were completed, seeds were extracted from the fruit for use in 2011 greenhouse studies. Similar to 2010 evaluations, eight resistant PIs, two susceptible PIs, and the controls ‘Sugar Baby’ and ‘Black Diamond’ were grown and evaluated in the Spring and Fall of 2011.
Assessing P. capsici development in fruit tissue using quantitative polymerase chain reaction.
After completing data recording, fruit tissue (10 mm length × 7 mm diameter) from highly resistant and susceptible PIs were collected from either side of the agar plug using a 7-mm cork borer for the fruit collected from the field in Summer 2010. Samples were collected 7 mm away from the agar plug. Similarly, fruit tissue samples were also collected from the field and greenhouse experiments conducted in 2011. The two fruit tissue samples from either side of the agar plug from each fruit were pooled together in one 2-mL microcentrifuge tube. Approximately 0.8 g of fresh tissue of each sample was lyophilized in a 2-mL microcentrifuge tube and ground using glass beads in a FastPrep-24 sample preparation system (M.P. Biomedicals, Irvine, CA). DNA was extracted using techniques described by Lamour and Finley (2006) in 96-well plates. Total DNA extracted from each of the samples was determined using the Qubit fluorometer and Quant-it DNA assay kit (Invitrogen Corporation, Carlsbad, CA), as per the manufacturer’s instructions. Real-time quantitative polymerase chain reaction (qPCR) assay to quantify the amount of pathogen DNA in the fruit tissue was conducted using P. capsici-specific β-tubulin-based primers designed using P. capsici-specific sequences described by Donahoo and Lamour (2008) and primers described by Kousik et al. (2012). The primer sequences were PCBT-F: TAA CTG CCG CGT GTA TGT TC and PCBT-R: GCT CAT CTT CAG ACC CTT GG. Real-time qPCR was conducted using a SYBR green-based assay in a Roche Diagnostics LightCycler 480 (Roche Diagnostics, Indianapolis, IN) following the manufacturer’s protocol (Roche Diagnostics LightCycler 480 Version 1.5.0). The reaction mixture (10 μL) consisted of 2.5 μL of DNA extract and 7.5 μL LightCycler 480 SYBR green master mix and 0.7 μM of each primer. The thermal cycling conditions were as follows: initial denaturation at 95 °C for 5 min followed by 40 cycles of 95 °C for 10 s, 60 °C for 10 s, 72 °C for 30 s, and a final extension step at 72 °C for 5 min. Each sample was run in duplicate to validate the reproducibility of the qPCR method. A standard curve was constructed by plotting known concentrations (0.0001 to 10 ng) of P. capsici DNA against the Ct values obtained from real-time PCR using software provided with the Light Cycler 480 system. The amplification reactions had a high efficiency (R2 = 0.99). After every qPCR run, we also examined the melting curve analysis of all the samples at the end of PCR runs to confirm that only one amplicon was being quantified. The quantity of P. capsici DNA present in unknown samples was calculated using the standard curve and the software provided with the LightCycler480.
Statistical analysis.
All fruit rot data from the field and greenhouse experiments were analyzed using the PROC GLM procedure of SAS (SAS Institute Inc., Cary, NC) and means were separated using Fisher’s protected least significant difference (lsd) (α = 0.05). Correlations between the fruit rot parameters that were measured (lesion diameter, sporulation diameter, and sporulation intensity) were conducted using the PROC CORR procedure of SAS. Data on P. capsici DNA (ng·g−1 fruit tissue) were log-transformed and then analyzed using the PROC GLM procedure of SAS. Transformed means were separated using Fisher’s protected lsd (α = 0.05); however, actual means are presented in the tables.
Results
Phytophthora fruit rot lesion development was observed on the susceptible checks 24 h after inoculation after which the lesion rapidly expanded for the next 3 to 4 d in all the experiments. The preliminary evaluation of 205 PIs helped identify potential sources of resistance in the watermelon core collection available with GRIN. Of the 205 PIs evaluated, the majority were highly susceptible and extensive sporulation was observed on most fruit. Overall we identified and considered 25 PIs (12%) with sporulation intensity less than 1.0 as potential sources of resistance. Twenty-two (12%) of the 159 Citrullus lanatus var. lanatus PIs we evaluated, one C. colocynthis (PI 388770), and two C. lanatus var. citroides PI (PI 189225) showed varying levels of resistance. Details of the reaction of some of the most resistant and susceptible PIs for which four to five fruit were evaluated is presented in Table 1. A complete list of the reactions of the 205 PIs can be obtained from the author and will also be entered into the GRIN database.
Heavy pathogen sporulation on the lesions was observed on the susceptible genotypes in 2010 (Table 2). Significant differences (P ≤ 0.0001) in the response of the various PIs to Phytophthora fruit rot was observed with respect to all the disease parameters measured (lesion diameter, percent fruit area covered by lesion, sporulation diameter, and sporulation intensity) in the first field experiment conducted in Summer 2010. Highly significant correlations (r ≥ 0.91, P < 0.0001) among all the disease parameters measured were observed in this trial. Because of such highly significant correlations, we have presented data on lesion diameter and sporulation intensity in Table 2, leaving out data on sporulation diameter. Significant disease development was observed on the susceptible commercial cultivars, Sugar Baby and Black Diamond, indicating that the fruit rot test had worked effectively (Table 2). PI 435991 was the most susceptible with respect to fruit area covered by lesion and heavy sporulation intensity. Several C. lanatus var. lanatus PIs had significantly smaller lesions and reduced sporulation compared with the susceptible checks. These included PI 457916, PI595203, 560020, and 560002. Similarly, PI 388770 (Citrullus colocynthis) and PI 189225 (C. lanatus var. citroides) were also significantly more resistant to fruit rot compared with the susceptible cultivars.
Phytophthora fruit rot development on detached watermelon fruit of U.S. PIs and controls grown in a field in Summer 2010 and in a greenhouse in 2010, Charleston, SC.
The 24 PIs used in the field study in Summer 2010 also were grown in the greenhouse and evaluated in 2010. Results between the first 2010 field and greenhouse evaluations were similar. Significant differences (P ≤ 0.0001) among the PIs with respect to lesion development were observed. Significant correlations (r > 0.90, P = 0.0001) among the various fruit rot parameters (fruit area covered by lesion, lesion diameter, sporulation diameter, and sporulation intensity) measured were observed. The sporulation intensity on 10 of the PIs was less than 1.0 on the 0 to 5 scale and these PIs were significantly more resistant to fruit rot compared with the susceptible controls (Table 2). PI 306782, PI 494527, PI 186489, PI 560020, and PI 560002 collected in Nigeria and PI 595203 from the United States were observed to be highly resistant in this 2010 greenhouse trial.
Highly significant correlations (r > 0.90, P < 0.0001) were observed among the fruit rot parameters measured in the second field trial conducted in Fall 2010. Fruit of ‘Sugar Baby’, PI 435991, ‘Mickey Lee’, and ‘Black Diamond’ had severe rot (Table 3). Disease ratings for several C. lanatus var. lanatus PIs from Africa, including PI 560002, PI 494527, PI 457916, PI 186489, and PI 560020, were significantly lower than the susceptible controls. PI 189225 (C. lanatus var. citroides) was also resistant as observed in the first 2010 experiment.
Phytophthora fruit rot development on detached watermelon fruit of U.S. PIs and controls grown in a second field experiment, Charleston, SC, Fall 2010.
The field experiment in Summer 2011 was conducted on eight PIs that were most resistant in the 2010 trials. The susceptible controls were severely rotted and significant differences (P ≤ 0.0001) were observed among the reaction of the PI to fruit rot. These eight PIs were significantly more resistant than the susceptible controls PI 536464, ‘Black Diamond’, and ‘Sugar Baby’ (Table 4). Minimal to no sporulation was observed on the inoculated fruit of these PIs (PI 560020, PI 457916, PI 494530, PI 595203, PI 186489, PI 306782, PI 560002, and PI 185635).
Phytophthora fruit rot development on detached watermelon fruit of U.S. PIs and controls grown in a field, Charleston, SC, Summer 2011.
Greenhouse evaluations in the spring and fall of 2011 confirmed the resistance in the selected PIs. Like in previous evaluations, highly significant differences between the controls and the select resistant PIs were observed. The controls PI 536464 and ‘Sugar Baby’ had severe fruit rot with large lesions and heavy sporulation (Table 5). The resistant PIs (PI 306782, PI 560020, PI 595203, PI 186489, PI 185635, and PI 560002) had very minimal to no sporulation.
Phytophthora fruit rot development on detached watermelon fruit of U.S. PIs and controls grown in a greenhouse in the Spring and Fall of 2011.
Significantly lower amounts of P. capsici DNA were detected in fruit tissue of PI 388770, PI 560020, PI 457916, PI 595203, and PI 560002 compared with the susceptible controls ‘Sugar Baby’ and ‘Black Diamond’ (Table 6) in the Summer 2010 field trial. The susceptible PI had greater than 2400 ng of pathogen DNA per gram of fruit tissue compared with less than 713 ng·g−1 of fruit tissue in the resistant PIs. Highly significant correlations between the amount of pathogen DNA in the fruit tissue and the fruit rot parameters, lesion diameter (r = 0.82, P ≤ 0.0001), sporulation diameter (r = 0.79, P ≤ 0.0001), and sporulation intensity (r = 0.72, P = 0.0003) were observed. Similar results were observed on the fruit tissue samples collected from the field-grown watermelon in Summer 2011 (Table 6).
Phytophthora capsici DNA detected in field-grown inoculated fruit tissue of select U.S. PIs in Summer 2010 and 2011 using real-time quantitative polymerase chain reaction.
‘Black Diamond’ had significantly more pathogen DNA (12,855 ng·g−1 fruit tissue) followed by the other susceptible checks: PI 435991 (6,882 ng·g−1), ‘Sugar Baby’ (3,601 ng·g−1), and PI 536464 (3,337 ng·g−1) compared with the resistant PIs in the greenhouse experiment in 2011. PI 185635 (296 ng·g−1), PI 560002 (258 ng·g−1), PI 494527 (181 ng·g−1), PI 560020 (41 ng·g−1), and PI 186489 (15 ng·g−1) all had significantly (P = 0.05) lower amounts of pathogen DNA in the fruit tissue confirming their resistance to fruit rot. No P. capsici DNA was detected in PI 306782 and PI 595203 in this greenhouse experiment. Significant correlations (r = 0.73, P ≤ 0.0001) between the levels of pathogen DNA present in fruit tissues and the visual ratings of fruit rot parameters were observed.
Discussion
An effective technique to evaluate detached watermelon fruit for resistance to Phytophthora fruit rot was developed. This technique should be useful for investigating inheritance of resistance to fruit rot, where a large number of fruit will need to be evaluated. In a preliminary study in 2008 we evaluated the watermelon core collection in the field. However, because of environmental conditions, fruit rot development in susceptible controls was not adequate to confidently assess resistance. Hence, we developed the detached fruit assay to evaluate watermelon fruit for resistance. A similar detached fruit assay was developed to evaluate cucumber for resistance to Phytophthora fruit rot (Gevens et al., 2006). Mature watermelon fruit were used in the current study because in commercial fields, fruit rot is usually observed when fruits are nearing harvest. Furthermore, fruit rot is also an important post-harvest problem in watermelon (Jester and Holmes, 2003; Kousik et al., 2011a). Mature watermelon fruit vary greatly in size among the PIs (especially during evaluation of the core collection), from very large to small fruit, and hence we used several different measurements to assess fruit rot severity. Therefore, we also calculated the approximate surface area covered by the lesion to compare the various PIs. Significant correlations were observed among the fruit rot parameters measured, indicating that use of lesion diameter and sporulation intensity may be sufficient to assess resistance. The inoculum plug was placed directly on the fruit without injuring the fruit surface because injuring the surface may break down some of the natural defense mechanisms. We did not cover the inoculum plug with eppendorf tubes and petroleum jelly (Gevens et al., 2006; Granke et al., 2012a) to prevent drying of the agar plugs; instead, we increased the RH (greater than 95%) in the walk-in humid chamber to prevent drying of the agar plugs. This fruit rot evaluation method developed to screen PIs for resistance has also been useful in evaluating post-harvest effectiveness of fungicides against Phytophthora fruit rot of watermelon (Kousik et al., 2011b).
In studies with cucumbers it was observed that infection generally occurred on the blossom end in the field, which was found to be more susceptible (Ando et al., 2009). However, we observed no difference in the level of infection when watermelon fruit were inoculated at the peduncle, middle, or the blossom end with respect to lesion diameter or sporulation intensity (Kousik et al., unpublished data) on resistant or susceptible accessions. In addition, it was easier to measure the disease parameters in the middle of the fruit with the advantage that the agar plug did not slide and fall off despite the high RH in the room.
The preliminary evaluation in 2009 identified 24 PIs as potential sources of resistance (Kousik, 2011). However, because of limited availability of seeds and in some instances poor germination, few fruits of some PIs were evaluated. Hence, in 2010, we evaluated all 24 PIs in the greenhouse and field and determined that some PIs (example PI 189317) that were considered as a potential source of resistance after the initial 2009 screen were susceptible and we stopped evaluating them further. The preliminary screen helped identify several resistant PIs that were confirmed as highly resistant by repeated evaluations.
The potential sources of resistance to Phytophthora fruit rot including PI 560002, PI 560020, PI 186489, PI 306782, PI 494527, and PI 494530 are all considered egusi-type watermelon and were collected in Nigeria (<http://www.ars-gring.gov>). These PIs are also called wild watermelon or egusi melon, because the seeds have a fleshy pericarp and these melons are widely cultivated in Nigeria for the high protein and carbohydrate content in their seeds, which are edible (Gusmini et al., 2004). They can also be easily crossed with commercial cultivated melon for breeding purposes (Gusmini et al., 2004). Many of the PIs with resistance to Phytophthora fruit rot were of the egusi type. However, not all egusi-type PIs we evaluated were resistant.
Gevens et al. (2006) identified cucumber varieties that limit development of P. capsici; however, none of the varieties tested had complete fruit rot resistance. The resistance in cucumber was related to developmental stage of the fruit and increased age and size of the fruit (Ando et al., 2009; Gevens et al., 2006). However, watermelon fruit of a susceptible cultivar, Crimson Sweet, in the study by Ando et al. (2009) were susceptible at all developmental stages, similar to observations in our preliminary studies on ‘Sugar Baby’ and PI 536464 (Kousik and Ikerd, 2012). However, the resistant fruit were resistant at all developmental stages (Kousik and Ikerd, 2012).
Some of the resistance sources identified in this study have also been identified with resistance to other diseases. For example, PI 595203, resistant to Phytophthora fruit rot, is also reported to have resistance to the aphid transmitted Zucchini yellow mosaic virus (ZYMV) and Papaya ringspot virus (Guner, 2004; Strange et al., 2002). In addition, markers linked to ZYMV resistance in PI 595203 have been identified and developed (Harris et al., 2009). Several of the markers are in a resistance gene analog region identified in watermelon (Harris et al., 2009; Ling et al., 2009). It would be interesting to determine if the genes for resistance to Phytophthora fruit rot are linked to the same region in PI 595203 as those for resistance to ZYMV. PI 560020 and PI 560002 that were highly resistant to Phytophthora fruit rot in this study were reported with moderate levels of resistance to powdery mildew (Davis et al., 2007). It would be interesting to explore the possibility of developing multiple disease-resistant germplasm and varieties using such accessions.
Variability among plants within a PI for their response to fruit rot was observed in this trial. Similar variability within a PI has been observed by others during evaluations of watermelon germplasm for resistance to viral and fungal diseases (Davis et al., 2007; Gillaspie and Wright, 1993; Strange et al., 2002). One of the reasons suggested for this variability is that many of the accessions in the collections were increased by open pollination at some time, thus providing the chance for cross-pollination of some of the accessions (Gillaspie and Wright, 1993; Strange et al., 2002). In addition, these accessions were originally collected in the open from various regions of the world and may have been cross-pollinated before collection.
The potential for the existence of races of P. capsici based on their reaction to various pepper and tomato genotypes has been reported (Glosier et al., 2008; Quesada-Ocampo and Hausbeck, 2010; Sy et al., 2005). However, little information is available on resistance of cucurbits to P. capsici or the existence of races based on cucurbit cultivars. The P. capsici isolate RCZ-11 (mating type A2) isolated from zucchini was used to evaluate for fruit rot resistance because it was highly aggressive and produced abundant visible sporangia and pathogen growth on susceptible watermelon cultivars compared with other isolates (Fig. 1). The most resistant lines identified in this study were also resistant to an aggressive isolate (mating type A1) from watermelon (Kousik, unpublished results). The type of plant from which a P. capsici isolate is collected does not define the host specificity of the isolate. Phytophthora capsici isolates collected from bean in Michigan were reported to be more aggressive on zucchini fruit than the cucurbit isolates (Quesada-Ocampo et al., 2010). Similarly, a P. capsici isolate named 12889, isolated from bell pepper in Michigan, was reported to be more aggressive and caused severe root and crown rot on cucurbits compared with some isolates from similar cucurbit crops (Enzenbacher and Hausbeck, 2012). Granke et al. (2012a) compared the virulence of over 100 isolates on fruit of tomato, cucumber, zucchini, and peppers and also observed that isolates from fabaceous hosts were highly virulent on cucumber and zucchini. Granke et al. (2012a) also suggested that a highly aggressive isolate of P. capsici on a host to be evaluated should be used regardless of the host from which it was isolated. It will be interesting to evaluate the resistant PIs against P. capsici isolates from different hosts (such as bean) and states of the United States to determine the potential existence of races with respect to resistance in watermelon and to determine the breadth of resistance.
Results on quantification of P. capsici in susceptible and resistant fruit tissue suggest that the qPCR system can be used to detect pathogen development in the fruit tissue for confirming resistance. However, it will be essential to visually observe symptom development on the fruit in addition to quantification using real-time PCR before using such resistance in breeding programs. Significant correlations between the amounts of pathogen DNA detected and visual fruit rot ratings were noticed confirming the effectiveness of the various methods for detecting resistance. In many of the resistant fruit samples, no rot was observed and no pathogen DNA was detected. In most cases, the fruit surface of the resistant accessions was very firm with very minimal to no fungal growth compared with the susceptible lines, which were severely rotted and were very soft. Overall we detected significantly less amounts of P. capsici DNA in the resistant fruit tissue.
Several other researchers also have demonstrated the use of qPCR in differentiating between resistant and susceptible genotypes of plants (Kousik et al., 2012; Silvar et al., 2005). Silvar et al. (2005) detected less Phytophthora DNA in resistant pepper genotypes compared with the susceptible genotypes. In this study we used β-tubulin sequence-based primers as opposed to ITS sequence-based primers to quantify P. capsici DNA. This choice was based on our previous work with these two primer sets where β-tubulin primers performed better than the ITS primers (Kousik et al., 2012). Quantifying the pathogen DNA helped confirm resistance to Phytophthora fruit rot in resistant watermelon PIs.
An integrated approach has been recommended by most researchers to manage diseases caused by P. capsici on vegetable crops (Granke et al., 2012b; Hausbeck and Lamour, 2004). Therefore, even when watermelon cultivars with resistance to Phytophthora fruit rot become available, a combination of appropriate cultural practices, fungicides, and host resistance should be used to prevent the development of races and breakdown of resistance.
Literature Cited
Ando, K., Hammar, S. & Grumet, R. 2009 Age-related resistance of diverse cucurbit fruit to infection by Phytophthora capsici J. Amer. Soc. Hort. Sci. 134 176 182
Babadoost, M. 2004 Phytophthora blight: A serious threat to cucurbit industries. APSnet April–May 2004. Online. doi: 10.1094/APSnetFeature-2004-0404
Babadoost, M. & Zitter, T.A. 2009 Fruit rots of pumpkin: A serious threat to the pumpkin industry Plant Dis. 93 772 782
Barksdale, T.H., Papavizas, G.S. & Johnston, S.A. 1984 Resistance to foliar blight and crown rot of pepper caused by Phytophthora capsici Plant Dis. 68 506 509
Candole, B.L., Conner, P.J. & Ji, P.J. 2010 Screening Capsicum annuum accessions for resistance to six isolates of Phytophthora capsici HortScience 45 254 259
Davis, A.R., Levi, A., Tetteh, A., Wehner, T.C. & Pitrat, M. 2007 Evaluation of watermelon and related species for resistance to race 1W powdery mildew J. Amer. Soc. Hort. Sci. 132 790 795
Donahoo, R.S. & Lamour, K.H. 2008 Interspecific hybridization and apomixis between Phytophthora capsici and Phytophthora tropicalis Mycologia 100 911 920
Enzenbacher, T.B. & Hausbeck, M.K. 2012 An evaluation of cucurbits for susceptibility to cucurbitaceous and solanaceous Phytophthora capsici isolates. Plant Dis. 96:1404–1414
Erwin, D.C. & Riberio, O.K. 1996 Phytophthora diseases worldwide. American Phytopathological Society, St. Paul, MN
Foster, J.M. & Hausbeck, M.K. 2010 Resistance of pepper to Phytophthora crown, root, and fruit rot is affected by isolate virulence Plant Dis. 94 24 30
Gevens, A.J., Ando, K., Lamour, K.H., Grumet, R. & Hausbeck, M.K. 2006 A detached cucumber fruit method to screen for resistance to Phytophthora capsici and effect of fruit age on susceptibility to infection Plant Dis. 90 1276 1282
Gevens, A.J., Roberts, P.D., McGovern, R.J. & Kucharek, T.A. 2008 Vegetable diseases caused by Phytophthora capsici in Florida. University of Florida, Extension digital information Source SP159. 1 Mar. 2012. <http://edis.ifas.ufl.edu/vh045>
Gillaspie, A.G. & Wright, J.M. 1993 Evaluation of Citrullus sp. germplasm for resistance to watermelon mosaic virus 2 Plant Dis. 77 352 354
Glosier, B.R., Ogundiwin, E.A., Sidhu, G.S., Sischo, D.R. & Prince, J.R. 2008 A differential series of pepper (Capsicum annuum) lines delineates fourteen physiological races of Phytophthora capsici Euphytica 162 23 30
Granke, L.L., Quesada-Ocampo, L.M. & Hausbeck, M.K. 2012a Differences in virulence of Phytophthora capsici isolates from a worldwide collection on host fruits Eur. J. Plant Pathol. 132 281 296
Granke, L.L., Quesada-Ocampo, L.M., Lamour, K. & Hausbeck, M.K. 2012b Advances in research on Phytophthora capsici on vegetable crops in the United States. Plant Dis. 96:1588–1600.
Gubler, W.D. & Davis, R.M. 1996 Phytophthora root and crown rot, p. 19–20. In: Zitter, T.A., D.L. Hopkins, and C.E. Thomas (eds.). Compendium of cucurbit diseases. APS Press, American Phytopathological Society, St. Paul, MN
Guner, N. 2004 Papaya ring spot virus watermelon strain and zucchini yellow mosaic virus resistance in watermelon. PhD diss., Dept. Hort. Sci., North Carolina State Univ., Raleigh, NC
Gusmini, G., Wehner, T.C. & Jarret, R.L. 2004 Inheritance of egusi seed type in watermelon J. Hered. 95 268 270
Harris, K.R., Ling, K., Wechter, W.P. & Levi, A. 2009 Identification and utility of markers linked to the zucchini yellow mosaic virus resistance gene in watermelon J. Amer. Soc. Hort. Sci. 134 1 6
Hausbeck, M.K., Foster, J.M. & Linderman, S.D. 2012 Managing Phytophthora on cantaloupe, muskmelon and watermelon. 18 June 2012. <http://veggies.msu.edu/Research/FS_MelonPcap.pdf>
Hausbeck, M.K. & Lamour, K.H. 2004 Phytophthora capsici on vegetable crops: Research progress and management challenges Plant Dis. 88 1292 1303
Jackson, K.L., Yin, J. & Ji, P. 2012 Sensitivity of Phytophthora capsici on vegetable crops in Georgia to mandipropamid, dimethomorph and cyazofamid. Plant Disease ‘First Look’ paper. 18 Apr. 2012. <http://dx.doi.org/10.1094/PDIS-12-11-1082-RE>
Jester, W. & Holmes, G.J. 2003 Phytophthora fruit rot –A menace to watermelon production. <http://www.nationalwatermelonassociation.com/scientific_phytophthorafruitrot.php>
Keinath, A.P. 2007 Sensitivity of populations of Phytophthora capsici from South Carolina to mefenoxam, dimethomorph, zoxamide and cymoxanil Plant Dis. 91 743 748
Kemble, J.M. 2010 Southeastern U.S. 2010 vegetable crop handbook. Southeastern Vegetable Extension Workers. p. 99–102
Kimble, K.A. & Grogan, R.G. 1960 Resistance to Phytophthora root rot in peppers Plant Dis. Rep. 44 872 873
Kousik, C.S. 2011 Sources of resistance to Phytophthora fruit rot in watermelon plant introductions Phytopathology 101 S94 (Abstract)
Kousik, C.S., Adams, M.L., Jester, W.R., Hassell, R., Harrison, H.F. & Holmes, G.J. 2011a Effect of cultural practices and fungicides on Phytophthora fruit rot of watermelon in the Carolinas Crop Prot. 30 888 894
Kousik, C.S., Thies, J.A. & Harrison, H.H. 2011b Evaluation of actigard and fungicides for managing Phytophthora fruit rot of watermelon, 2010 PDMR 5 V058
Kousik, C.S., Donahoo, R.S. & Hassell, R. 2012 Resistance in watermelon rootstocks to crown rot caused by Phytophthora capsici Crop Prot. 39 18 25
Kousik, C.S. & Ikerd, J. 2012 Watermelon fruit age and development of Phytophthora fruit rot on resistant and susceptible lines HortScience (Abstract)
Kousik, C.S. & Keinath, A.P. 2008 First report of insensitivity to cyazofamid among isolates of Phytophthora capsici from the southeastern United States Plant Dis. 92 979
Kreutzer, W.A., Bodine, E.W. & Durrell, L.W. 1940 Cucurbit diseases and rot of tomato fruit caused by Phytophthora capsici Phytopathology 30 951 957
Lamour, K.H. & Finley, L. 2006 A strategy for recovering high quality genomic DNA from a large number of Phytophthora isolates Mycologia 98 514 517
Lee, B.K., Kim, B.S., Chang, S.W. & Hwang, B.K. 2001 Aggressiveness to pumpkin cultivars of isolates of Phytophthora capsici from pumpkin and pepper Plant Dis. 85 497 500
Ling, K.S., Harris, K.R., Meyer, J.D.F., Levi, A., Guner, N., Wehner, T.C., Bendahmane, A. & Havey, M.J. 2009 Non-synonymous single nucleotide polymorphisms in the watermelon eIF4E gene are closely associated with resistance to zucchini yellow mosaic virus Theor. Appl. Genet. 120 191 200
McGrath, M.T. 1994 Fungicides provided insufficient suppression of Phytophthora fruit rot of cucurbits when disease pressure was high Phytopathology 84 1373 (Abstract)
McGrath, M.T. 1996 Phytophthora fruit rot, p. 53–54. In: Zitter, T.A., D.L. Hopkins, and C.E. Thomas (eds.). Compendium of cucurbit diseases. APS Press, St. Paul, MN
Morrissey, B. 2006 NWA update. The vineline. April 2006 issue. p. 7–10
Padley, L.D. Jr, Roberts, P.D., French-Monar, R. & Kabelka, E.A. 2008 Evaluation of Cucurbita pepo accessions for crown rot resistance to isolates of Phytophthora capsici HortScience 43 1996 1999
Peter, K.V., Goth, R.W. & Webb, R.E. 1984 Indian hot peppers as new sources of resistance to bacterial wilt, Phytophthora root rot, and root-knot nematodes HortScience 19 277 278
Quesada-Ocampo, L.M., Fulbright, D.W. & Hausbeck, M.K. 2009 Susceptibility of Fraser fir to Phytophthora capsici Plant Dis. 93 135 141
Quesada-Ocampo, L.M., Granke, L.L. & Hausbeck, M.K. 2010 Differences in virulence of Phytophthora capsici isolates from a worldwide collection on zucchini fruits, p. 248–251. In: Thies, J.A., C.S. Kousik, and A. Levi (eds.). Proc. of Cucurbitaceae 2010. American Society of Horticultural Science, Alexandria, VA
Quesada-Ocampo, L.M. & Hausbeck, M.K. 2010 Resistance in tomato and wild relatives to crown and root rot caused by Phytophthora capsici Phytopathology 100 619 627
Sanders, D.C. 2006 Vegetable crop handbook for the southeastern U.S. southeastern vegetable extension workers. p. 86–88
Silvar, C., Diaz, J. & Merino, F. 2005 Real-time polymerase chain reaction quantification of Phytophthora capsici in different pepper genotypes Phytopathology 95 1423 1429
Strange, B.E., Guner, N., Pesic-VanEsbroeck, Z. & Wehner, T.C. 2002 Screening the watermelon germplasm collection for resistance to Papaya ring spot virus type-W Crop Sci. 42 1324 1330
Sy, O., Steiner, R. & Bosland, P.W. 2005 Recombinant inbred line differential identifies race-specific resistance to Phytophthora root rot in Capsicum annuum Phytopathology 98 867 870
Walker, S.J. & Bosland, P.W. 1999 Inheritance of Phytophthora root rot and foliar blight resistance in pepper J. Amer. Soc. Hort. Sci. 124 14 18
Wiant, J.S. & Tucker, C.M. 1940 A rot of winter queen watermelon caused by Phytophthora capsici J. Agr. Res. 60 73 88
