Abstract
Phytophthora capsici causes seedling death, crown and root rot, fruit rot, and foliar blight on squash and pumpkins (Cucurbita spp. L.). A total of 119 C. moschata accessions, from 39 geographic locations throughout the world, and a highly susceptible butternut squash cultivar, Butterbush, were inoculated with a suspension of three highly virulent P. capsici isolates from Florida to identify resistance to crown rot. Mean disease rating (DR) of the C. moschata collection ranged from 1.4 to 5 (0 to 5 scale with 0 resistant and 5 susceptible). Potential resistant and tolerant individuals were identified in the C. moschata collection. A set of 18 PIs from the original screen were rescreened for crown rot resistance. This rescreen produced similar results as the original screen (r = 0.55, P = 0.01). The accessions PI 176531, PI 458740, PI 442266, PI 442262, and PI 634693 were identified with lowest rates of crown infection with a mean DR less than 1.0 and/or individuals with DR = 0. Further selections from these accessions could be made to develop Cucurbita breeding lines and cultivars with resistance to crown rot caused by P. capsici.
Cucurbitaceae L. includes ≈120 genera and over 800 species. The family is predominantly of tropical origin with a few members that have been able to adapt to temperate climates. The genus Cucurbita consists of five cultivated species and 10 wild species, with perennials or annuals plants (Teppner, 2004).
The most important cultivated species in the genus Cucurbita are C. pepo L. ‘summer squash’, C. maxima Duchesne ‘winter squash’, and C. moschata ‘winter and crookneck squash’, and of minor importance, C. argyrosperma Huber ‘silver-seed gourd’ and C. ficifolia Bouché ‘fig-leaf cucurbit’.
Cucurbita value is based on the use of immature and mature fruit of summer and winter squashes, respectively (Ferriol and Picó, 2008). The Statistic Division of the Food and Agriculture Organization of the United Nations (FAOSTAT data, 2007) ranked world cucurbit production (including watermelons, cucumbers, squash, pumpkins, and gourds) among the 10 leading vegetable crops worldwide. Pumpkin, squash, and gourd production was ≈20 million tons with China as the major producer followed by India, Russia, and the United States.
Cucurbita moschata is a highly diverse winter squash species adapted to hot and humid weather and low altitudes. Fruit size and color are highly variable. Flesh colors range from light yellow to dark orange. Cucurbita moschata's largest genetic variability occurs in the American Tropics with variation increased by hybridization with wild species (Ferriol and Picó, 2008). The Butternut type of C. moschata is one of the most widely known in Europe and the United States.
Exotic germplasm and wild species from the USDA germplasm collection are used as genetic resources for breeding. Two wild squash species, C. lundelliana Bailey and C. okeechobeensis ssp. okeechobeensis Bailey, have been recently studied as useful sources for disease resistance such as powdery mildew and P. capsici crown rot resistance (Cohen et al., 2003; Contin and Munger, 1977; Metwally et al., 1996; Padley, 2008). Cucurbita lundelliana is native to the Yucatan peninsula and can be hybridized with C. moschata, C. maxima, C. ficifolia, C. pepo, and C. argyrosperma (Ferriol and Picó, 2008; Sitterly, 1972; Whitaker, 1959). Cucurbita okeechobeensis is formed by subspecies okeechobeensis and martinezii Walters & Decker. Subspecies martinezzi is endemic to Mexico, growing in the same region as C. moschata ssp. sororia Merrick & Bates (Ferriol and Picó, 2008). Cucurbita okeechobeensis ssp. okeechobeensis is endemic to Florida. This subspecies is currently categorized as rare or endangered in the National Germplasm Repository (GRIN, 2009), and it can be hybridized with C. ecuadorensis Cutler & Whitaker, C. moschata, C. argyrosperma, and C. pepo. Cucurbita lundelliana and C. okeechobeensis are also cross-compatible (Ferriol and Picó, 2008).
Interest in breeding for resistance to P. capsici is of importance because of the economic impact P. capsici syndromes can have on cucurbit production. This oomycete pathogen affects a wide range of solanaceous and cucurbitaceous plants worldwide (Erwin and Ribeiro, 1996; Tian and Babadoost, 2004). Infection can occur at any plant stage, producing damping-off, root rot, crown rot, foliar blight, and fruit rot symptoms (Hausbeck and Lamour, 2004). Losses resulting from P. capsici in commercial production can reach 100%. Chemical, cultural, and mechanical practices have been reported to reduce P. capsici infection, but multiple cycles of infection and spore production have made its control difficult (Babadoost et al., 2008; Hausbeck and Lamour, 2004). Furthermore, resistance to the fungicides mefenoxam and metalaxyl has been reported in P. capsici (Parra and Ristaino, 2001; Ristaino and Johnston, 1999). Genetic resistance to P. capsici would constitute an important component of P. capsici management.
Recently, resistance to the crown rot syndrome in squash has been introgressed into University of Florida breeding lines, Fla. 27-12 and Fla. 27-17, from C. lundelliana and C. okeechobeensis ssp. okeechobeensis through a series of hybridizations and single plant selections (Padley et al., 2008). The genetic composition of each line is 62.5% C. moschata, 25% C. lundelliana, and 12.5% C. okeechobeensis. Preliminary data also indicated that each line was segregating for resistance to foliar blight (Kabelka, personal communication). Unfortunately, transfer of these sources of resistance into a C. moschata background has proven to be challenging as a result of unmarketable fruit quality and hybridization barriers between species. The purpose of this research was to identify sources of resistance to P. capsici within the USDA C. moschata germplasm collection. This study included the evaluation of the crown rot screen's consistency, identification of resistant individuals, and the response of S1 progeny from resistant selections to P. capsici.
Materials and Methods
Plant material.
One hundred nineteen germplasm accessions of C. moschata, representing diverse geographic locations (39 countries), were randomly chosen and used for these studies. These accessions were available in the Plant Genetic Resources Conservation and Utilization S-9 collection in Griffin, GA. C. moschata ‘Butterbush’ (W. Atlee Burpee & Co.), a known highly susceptible cultivar, was used as a control for all these studies (Table 1).
Mean disease ratings of Cucurbita moschata accessions screened with a suspension of three Phytophthora capsici isolates from Florida.z
Disease rating (DR), 0 to 5 scale, for response of Cucurbita moschata germplasm to crown rot caused by Phytophthora capsici. Symptoms rated 21 d after inoculation: (A) score 0 = no symptoms; (B) score 1 = small brown lesion at base of stem; (C) score 2 = lesion has progressed up to the cotyledons causing constriction at the base; (D) score 3 = plant has partially collapsed with apparent wilting of leaves; (E) score 4 = plant has completely collapsed with severe wilting present; and (F) score 5 = plant death.
Citation: HortScience horts 46, 4; 10.21273/HORTSCI.46.4.536
Inoculum preparation.
Three highly virulent Floridian P. capsici mating-type A1 isolates (01-1983A, RJM98-739, and RJM98-805) recovered from squash obtained from Dr. P. Roberts (SWREC, Immokalee, FL) were used. Inoculum was prepared as described by Padley et al. (2008) as follows. One 5-mm mycelial plug from cornmeal agar for each P. capsici isolate was transferred to a 20% clarified V8 agar plate to grow at room temperature. After 7 d, 10 5-mm V8 agar mycelial plugs for each isolate were placed into a 20% clarified V8 broth plate to grow for an additional 7 d in a 28 °C incubator. Broth was then drained and mycelium was washed two times with sterilized distilled water. Sterilized distilled water was added to cover mycelial growth. Plates were placed under incandescent lights at 28 to 30 °C to induce sporangial development. After 24 h, sporangia were chilled at 4 °C for 45 min to induce zoospore release. Mycelium was strained through cheesecloth. A 1-mL encysted zoospore sample was counted using a hemacytometer. Each isolate was adjusted to 2 × 104 zoospores/mL concentration. A combined suspension of motile zoospores containing equal amounts of the three isolates was used in our study for inoculation.
Screening.
Protocols to evaluate the crown response to P. capsici in squash were previously described by Tian and Babadoost (2004). Padley et al. (2008) tested and modified these protocols for screening crown rot resistance in C. pepo germplasm. Padley's et al. (2008) protocol was used to evaluate the C. moschata germplasm collection as follows.
Cucurbita moschata accessions were evaluated using a completely randomized design. Eight seeds per accession and the susceptible control were individually sown in 15.2-cm azalea plastic pots containing Fafard #3S potting mix (Fafard Inc., Agawam, MA). Greenhouse temperatures were maintained between 19 to 34 °C. Seedlings were watered daily and each received 1 g of slow-release fertilizer at the cotyledon stage (Osmocote 14-14-14 NPK; The Scotts Company LLC, Marysville, OH). At the third to fourth true-leaf-stage, each seedling was inoculated at its crown with 5 mL of the 2 × 104 zoospores/mL suspension of the three Floridian P. capsici isolates using a pipette. The infection conditions were optimized by saturating the soil 24 to 36 h before inoculation.
Twenty-one d after inoculation, the plants were visually rated based on a scale ranging from 0 to 5, in which 0 = no symptoms, 1 = small brown lesion at base of stem, 2 = lesion has progressed up to the cotyledons causing constriction at the base, 3 = plant has partially collapsed with apparent wilting of leaves, 4 = plant has completely collapsed with severe wilting present, and 5 = plant death (Fig. 1). A mean DR, calculated as a weighted average, sd, and a percentage of plants with a DR 1 or less, was calculated for each accession and the susceptible control. Plants with a DR less than 2 were considered resistant/tolerant and with a DR of 2 to 5 were considered susceptible to crown rot produced by P. capsici.
Rescreen.
Eighteen accessions were chosen from the original screening results for crown rot resistance to P. capsici as follows: 14 accessions with a DR of 0 to 2.8, two accessions with a DR of 3.4, and two accessions with a DR of 5.0 (Table 1). These accessions were selected to represent low, medium, and high DR from the original screen. Selected accessions were rescreened for crown rot resistance to P. capsici using the protocol described previously. The susceptible cultivar ‘Butterbush’ was also included as a control in these studies. A mean DR, sd, and percentage of plants with a DR 1 or less were calculated for each accession and the susceptible control. Consistency between the original and the rescreen DRs were compared using Spearman's rank correlation coefficient.
Screening of S1 progeny from resistant individuals.
Two plants of PI 211996 and PI 176531, identified in the original screening results were grown and self-pollinated to obtain S1 seed (Table 2). Eight S1 seeds per resistant selection and a susceptible cultivar, Butterbush, were planted and grown. Plants were screened for crown rot resistance to P. capsici as described previously. A mean DR, sd, and a percentage of plants with a DR 1 or less were calculated.
Segregation for resistance to Phytophthora crown rot in S1 progeny generated by selfing resistant seedlings (DR = 0) of PI 211996 and PI 176531 from the original screen results.z
Disease ratings were compared by using a Wilcoxon analysis (P < 0.05). Data analysis was performed using the NPAR1WAY procedure of SAS (Statistical Analysis System Version 9.1; SAS Institute, Cary, NC).
Results and Discussion
Potential sources of resistance to crown rot produced by P. capsici were identified in the C. moschata collection. Mean DR for the screened C. moschata germplasm ranged from 1.4 to 5.0 (Table 1). PI 634693 (from India) and PI 483347 (from Korea) with a mean DR of 1.4 and 1.6, respectively, had the lowest mean DR values and were considered possible sources of resistance and/or tolerance to P. capsici. Additional PIs from these results were considered susceptible with higher DR values. The number of accessions with a mean DR of 2.0 to 2.9, 3.0 to 3.9, 4.0 to 4.9, and 5.0 were 15, 21, 40, and 41, respectively. Among these screened PIs, only two plants of PI 211996 (Iran) and two plants of PI 176531 (Turkey) were resistant (DR = 0) to P. capsici. However, ≈39.5% of the 119 screened PIs had at least one individual with a resistant phenotype with a DR = 1.
Fourteen accessions representing a low mean DR of 0 to 2.8, two accessions with a medium mean DR = 3.4, and two accessions with a high mean DR = 5.0 were chosen from the original screen and were rescreened for P. capsici resistance. Mean DR for the rescreened accessions ranged from 0.7 to 5.0 (Table 1). PI 458740 (from Paraguay), PI 442266 (from Mexico), PI 442262 (from Mexico), and PI 634693 (from India) had a mean DR less than 1.0 and were selected as sources of resistance to crown rot. The number of accessions for the remaining PIs with a mean DR of 1.0 to 1.9, 2.0 to 2.9, 3.0 to 3.9, 4.0 to 4.9, and 5.0 were 2, 6, 1, 2, and 3, respectively. The rescreen identified a total of seven seedlings from accessions PI 458740 (two), PI 442266 (one), PI 442262 (one), PI 634693 (one), PI 418965 (one), and PI 442273 (one) resistant to P. capsici (DR = 0). Fourteen accessions had at least one genotype with a DR = 1.
Cucurbita moschata cv. Butterbush was highly susceptible with a mean DR of 5.0 and 4.3 and sd of 0 and 1.5 in the screen and rescreen experiments, respectively. The susceptible control ‘Butterbush’ was highly susceptible in all the experiments when inoculated with P. capsici, confirming the efficiency of the screening method because of the absence of escapes.
The presence of a large number of seedlings with DR = 1 was attributed to the genetic variation within each accession. However, genotypes with DR = 1 were not used for further breeding and selection because of their unknown genetic make-up. One or several genes could be associated with resistance to P. capsici in the C. moschata germplasm. Padley et al. (2009) reported that resistance to crown rot caused by P. capsici was conferred by three dominant genes in a F7 breeding line derived of C. lundelliana, C. okeechobeensis, and C. moschata species in its pedigree.
Previous research investigating resistance to P. capsici reported that environmental variation can affect the screening results as a result of uncontrolled conditions. Walker and Bosland (1999) reported that efforts in breeding and selection for resistant cultivars to P. capsici could be ineffective in pepper if screening methods do not allow for the separation of resistant and susceptible genotypes for root rot and foliar blight disease syndromes. Similarly, Reifschneider et al. (1986) described the importance of controlling several factors affecting the expression of resistance to blight caused by P. capsici in pepper. Plant age, isolate virulence, zoospore concentration, and inoculation method affected their screening results. They suggested the use of standardized screening methods to identify resistant plants.
Padley et al. (2008, 2009) developed and reported an effective method for screening genotypes with crown rot resistance to P. capsici in squash, which consistently differentiated resistant and susceptible genotypes. Controlled environmental conditions were maintained that excluded sources of variation between the original screen and the rescreen results for all our experiments.
Comparisons between the original screen and the rescreen gave a Spearman's rank correlation coefficient of r = 0.55 (P = 0.01). Variation in these experiments as noted by the Spearman's rank correlation coefficient and their DR was believed to be largely attributed to the presence of heterogeneity for resistance to crown rot in the C. moschata germplasm accessions. Small sample size, unknown number of resistance genes for P. capsici, and bias toward low mean DR accessions during rescreening could be partially responsible for the reduced correlation coefficient between experiments. Furthermore, if the resistant phenotype is conditioned by dominant alleles at three loci as reported by Padley et al. (2009), then the resistant phenotype could represent as few as ≈43% in S1 progeny from heterozygous resistant genotypes. If the trait being selected is polygenic, then a large number of segregating plants will be required to obtain the desired genotype. These factors did not appear to affect the results and selection for resistant genotypes. It was noticed that the screened accessions shifted positions between experiments (ranks) but always had a lower mean DR when compared with susceptible accessions (Table 1).
Heterogeneity for resistance to crown rot in Cucurbita was previously reported by Padley et al. (2008). They found that resistance to crown rot caused by Florida P. capsici isolates was segregating within some of the C. pepo PIs and that additional breeding and selection were necessary to fix the resistance into a breeding line. Donahoo et al. (2009) described the existence of variable levels of host resistance in Cucumis melo L. PIs. Likewise, Kousik and Thies (2010) reported heterogeneity for resistance to P. capsici in bottle gourd, Lagenaria siceraria Standl.
The existence of heterogeneity in our selected germplasm for resistance to P. capsici was analyzed in the S1 progeny of four resistant plants with DR = 0, PI 211996 (two plants) and PI 176531 (two plants). One of the resistant plants (from PI 211996) did not produce seed when self-pollinated. Padley (2008) reported similar fertility problems in advanced breeding lines of C. pepo. The screening of eight seedlings each from the three remaining P. capsici crown rot-resistant selections revealed that the mean DR of selected resistant PIs and their S1 progeny were not significantly different (Table 2). However, an increase of at least one to three plants with a DR 1 or less was observed between PIs and S1 progeny. These results indicated that the PIs were heterozygous for the resistance genes and that the frequency of resistant genotypes could be increased in advanced generations.
Similar findings were reported in tomato, pepper, and pumpkin in which different levels of resistance to P. capsici were attributed to a partial (quantitative) resistance (Hwang and Hwang, 1993; Kim and Hwang, 1992; Lee et al., 2001). Inheritance of P. capsici resistance in pepper and squash was reported to be governed by three genes, inherited in a Mendelian fashion, which formed part of the whole root rot, crown rot, and foliar blight resistance (Padley et al., 2009; Walker and Bosland, 1999).
Resistant and tolerant genotypes to foliar blight and fruit rot caused by P. capsici have been found in breeding lines derived from C. okeechobeensis and C. lundelliana at the University of Florida. These resistant genotypes varied among experiments depending on the syndrome being evaluated. A tissue-specific genetic control for resistance to P. capsici in squash is believed to be present (Chavez and Kabelka, unpublished results). In pepper, tissue specificity for the genes controlling resistance to P. capsici has been reported (Barksdale et al., 1984; Sy et al., 2005; Walker and Bosland, 1999). Further studies to differentiate the effect of each syndrome produced by P. capsici in resistant lines are necessary.
In conclusion, resistance to P. capsici was found in representatives of the USDA C. moschata germplasm collection. Accessions PI 176531, PI 458740, PI 442266, PI 442262, and PI 634693 were the most resistant with a mean DR less than 1.0 and/or individuals with DR = 0. These differences among screen and rescreen results in the C. moschata germplasm were attributed to segregation for resistance to crown rot caused by P. capsici. Similar results were obtained when different accessions progeny showed some differences in mean DR when compared with their parents. Selection of resistant and/or tolerant genotypes for crown rot caused by P. capsici can be done through screening and selection. Future projects will require studying resistance of selected individuals for crown rot, foliar blight, fruit rot, and their interaction.
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