Downy Mildew Disease Progress in Resistant and Susceptible Cucumbers Tested in the Field at Different Growth Stages

Downy mildew, caused by the oomycete pathogen Pseudoperonospora cubensis (Berkeley & Curtis) Rostov, is a major foliar disease of cucumber. Ten years after the reemergence of P. cubensis, downy mildew continues to be a major threat to cucumber production in the United States. Cucumber accessions with high levels of resistance have been identified. Development of cultivars with high levels of resistance remains an important objective of cucumber breeding programs. We tested a set of cucumber cultigens, including highly resistant PI accessions and susceptible control lines, to observe the effect of plant age on resistance. Cultigens responded differently to disease across plant developmental stages. In general, older plants had more disease symptoms, even those classified as resistant, such as PI 197088. However, PI 330628 and PI 605996 held their resistance even at late developmental stages. It is possible that these lines were resistant at late stages due to other factors, such as their rapid, indeterminate growth, that allows them to outgrow the disease. However, although PI 197088 appears to have a rapid, indeterminate growth habit, it did not have more resistance at later stages of plant maturity. Regardless of the mechanism involved, plant breeders should use the genetic resistance in PI 330628 and PI 605996 over PI 197088. Cucumber (Cucumis sativus L.) is a major vegetable crop. The crop is cultivated in temperate and tropical countries and is the fourth most widely grown vegetable crop behind tomato (Lycopersicon esculentum Mill.), cabbage (Brassica oleracea var. capitata L.), and onion (Allium cepa L.) (Tatlioglu, 1993). In 2013, the U.S. farm value of cucumber was $363 million, with 3.59 · 10 kg produced for the fresh and processing market (USDA, 2013). In North Carolina, in 2012, the fresh and processing cucumber market was valued at $29 million (USDA, 2012). Downy mildew, caused by the oomycete pathogen P. cubensisRostov, is a major foliar disease of cucumber (Palti and Cohen, 1980). The disease symptoms on cucumber are characterized by the appearance of small, water-soaked lesions on the underside of leaves. Lesions are often angular and bound by leaf veins, eventually turning chlorotic and necrotic. Heavy sporulation can be observed within the lesions on the abaxial leaf surface. The disease has been reported in over 80 countries on 20 genera, including 50 species of the Cucurbitaceae, causing significant economic losses (Lebeda, 1992; Lebeda and Widrlechner, 2003; Olczak-Woltman et al., 2011; Palti and Cohen, 1980). Downy mildew can cause significant reductions in yield due to a short latent period, high secondary infection frequency, rapid spread, and a year-round presence in some cucurbit production areas (Cohen, 1981; Colucci and Holmes, 2010). Before 2004, it was possible to control epidemics of cucumber downy mildew by growing resistant cultivars. However, a resurgence of the disease in 2004 and subsequent breakdown of the resistance resulted in yield losses of up to 40% (Colucci et al., 2006). Presently, 12 years after the appearance of a new race of cucumber downy mildew, the disease continues to threaten production in warm humid regions around the world, including most production areas of the United States. Development of new cultivars with high levels of resistance remains an important objective of cucumber breeding programs. Without high levels of resistance, expensive fungicide programs have been necessary to maintain fruit yield and quality. P. cubensis reproduces both sexually and asexually and is wind dispersed over long distances. Because of the potential for evolution in downy mildew, and the widespread use of fungicides to control the disease, it is likely that resistance to the fungicides will develop. Several studies published since 2004 report the development of resistance to systemic fungicides (Colucci and Holmes, 2007; Hausbeck and Cortright, 2009; Lebeda and Urban, 2007; Mitani et al., 2001). As fungicide resistances continue to develop, cultivars with genetic resistance are needed. New sources of genetic resistance (reduced leaf damage) and tolerance (high yield in the presence of disease) to the new race(s) of downy mildew in the United States were identified in a germplasm screening study and a multiyear re-evaluation of the most resistant and susceptible cultigens conducted at North Carolina State University (Call et al., 2012). Several cultigens were identified with high levels of resistance to the new downy mildew. Among these cultigens, considerable attention has been given to PI 197088. This accession is being used in breeding efforts as the source of high levels of resistance and the inheritance appears to be quantitative (Call, 2012; VandenLangenberg, 2014). Interestingly, the most resistant cultigens identified by Call et al. (2012), were only moderately resistant in the downy mildew screening of Wehner and Shetty (1997). It is likely that the change in the pathogen population in 2004 altered the rankings of these cultigens. Adult plant resistance has been observed in several crop–disease interactions (Chen, 2005; Coelho and Monteiro, 2003; Shaik, 1985). However, to our knowledge, it has not been reported in cucumber. It is possible that the new resistance present in several PI accessions is a form of adult plant resistance. A better understanding of the pattern of resistance to the new downy mildew will help inform breeders and growers on best practices. The objective of this study was to better understand the interaction between the new race(s) of downy mildew and the newly identified, highly resistant cucumber cultigens, and to assess the presence of adult plant resistance. Materials and Methods Production methods and ratings. In Summer 2013 and 2014, nine cultigens were direct seeded into beds covered by black plastic mulch on raised beds at the Horticultural Crops Research Station at Clinton, NC. After germination, plots were thinned to five plants each. The soil type in Clinton was a mixture of Norfolk, Orangeburg, and Goldsboro loamy sand. The environment is subtropical during the main growing season, with high humidity and frequent rainfall. Cucumbers were grown using recommended horticultural practices as summarized by Schultheis (1990). Irrigation was applied as needed through plastic drip tape for a total of 25 to 40 mm per week. Plots consisted of five single-plant hills, with individual hills 0.6 m apart within the row. Border rows were planted using the susceptible cultivar Coolgreen about every third row to encourage the even spread of inoculum. No artificial inoculum was used in the field tests in North Carolina. Plots were exposed to natural epidemics encouraged by overhead irrigation during the growing season. The experiment design had multiple planting dates throughout the season. Plantings were made every 2nd week for six planting dates. The first Received for publication 2 Mar. 2016. Accepted for publication 23 June 2016. Corresponding author. E-mail: tcwehner@gmail. com. 984 HORTSCIENCE VOL. 51(8) AUGUST 2016 plantings were on 9 May 2013 and 1 May 2014. Disease severity was evaluated weekly in all planting dates after the onset of observable symptoms in the first planting date for six consecutive weeks. Ratings were based on the percentage of symptomatic leaf area [disease rating scale 0 to 9, where 0 = no damage, 1 = 1% to 10%, 2 = 11% to 20%, 3 = 21% to 30%, 4 = 31% to 40%, 5 = 41% to 50%, 6 = 51% to 60%, 7 = 61% to 70%, 8 = 71% to 80%, and 9 = 81% to 100% (or dead)]; as described by Jenkins and Wehner (1983). The first ratings were taken on 11 July 2013 and 3 July 2014. The first rating day was exactly 62 d from planting in both years. Plant material. Nine cucumber cultivars and PI accessions (hereafter collectively referred to as cultigens) differing in disease resistance were used to evaluate severity of disease at different life-cycle stages. Cultigens were chosen based on previous studies at North Carolina State University (Call, 2012; Call et al., 2012; Shetty et al., 2002; Wehner and Shetty, 1997). ‘Wisconsin SMR 18’ (Wisconsin AES) is highly susceptible, ‘Poinsett 76’ (Clemson and Cornell University) is moderately resistant, and ‘Ashley’ (Clemson University) is moderately susceptible. The PI accessions tested were the most resistant overall environments in a germplasm retest study (Call et al., 2012). They include PI 197085, PI 197086, PI 197088, PI 330628, PI 432875, and PI 605996. Experiment design. We planted nine cultigens (subplots) in each of four replicates through time (planting date) using a split-plot design. In this way, plants in each subsequent planting (main plot) were at an earlier stage of development when inoculum arrived. To observe the true effect of plant age on resistance, we rated the entire study multiple times through the main infection season. A single rating of the entire experiment represents a snapshot of resistance expression on a set of cultigens at different stages of plant development. For example, our fourth rating in 2014 taken on 24 July was 14, 28, 42, 56, 70, and 84 d after planting each sequential set of plots. This range of dates represents a high degree of spread in plant development stage, from first true leaf (14 d), full flower (28 d), early fruit set (42 d), late fruit set (56 d), early seed maturity (70 d), and late seed maturity (84 d). To study the effect of plant stage on resistance, one must look at a set of cultigens at different stages of growth, all rated at the same time. For that, we used the fourth rating, since it had the best array of growth stages in both years. Earlier ratings lacked plants that were at the seed-set stage, whereas later ratings lacked plants at the preflower stage. Statistical analysis of field data. Data were analyzed using analysis of variance (ANOVA) and regression analysis in the statistical packages JMP Pro 10 (SAS Institute Inc., Cary, NC), R (R Development Core Team, 2014) and SAS v9.4 (SAS Institute Inc., 2013). Before analysis, data were checked for normality. Residual plots had a random distribution, indicating that the statistical model was valid and its assumptions were met (Fernandez, 1992). Results and Discussion Severity of downy mildew was higher in 2013 than in 2014 when comparing mean disease severity pooled overall planting dates (Table 1). It is possible that the environment was more conducive to disease development in 2013 than in 2014, resulting in higher overall ratings. In general, 2013 was a wetter year than 2014, an environmental condition that promotes the spread of downy mildew. Because of disease intensity differences between years, we analyzed the data separately between years to highlight any differences that disease intensity may cause. However, in areas of our report where no major differences appeared in our analysis, we pooled over years for simplicity. All model effects were significant in our analysis of the fourth rating date in both 2013 and 2014, including planting date, cultigen, and the planting date by cultigen interaction (Table 2). Mean squares for planting date were 55 and 29 times higher than the planting date by cultigen interaction in 2013 and 2014, respectively. However, a significant planting Table 1. Mean disease severity for resistance of cucumber cultivars by year (2013, 2014) at Clinton, NC, for each of six ratings.