Gabriele Gusmini

Inheritance of Resistance to Gummy Stem Blight in Watermelon

Gummy stem blight (GSB), caused by three related species of Stagonosporopsis [Stagonosporopsis cucurbitacearum (syn. Didymella bryoniae), Stagonosporopsis citrulli, and Stagonosporopsis caricae], is a major disease of watermelon [Citrullus lanatus (Thunb.) Matsum. & Nakai] in most production areas of the United States. We studied the inheritance of resistance to GSB using three PI accessions of watermelon. Four families of six progenies (Pr, Ps, F1, F2, BC1Pr, and BC1Ps) were developed from four crosses of resistant PI accessions by susceptible cultivars. Each family was tested in 2002 and 2003 in North Carolina under field and greenhouse conditions for resistance to GSB. Artificial inoculation was used to induce uniform and strong epidemics. The effect of the Mendelian gene for resistance, db, was tested. Partial failure of the data to fit the singlegene inheritance suggested that resistance to GSB of PI 482283 and PI 526233 may be under the control of a more complex genetic system. Gummy stem blight is a major disease of watermelon [C. lanatus (Thunb.) Matsum. & Nakai]. It is caused by three genetically distinct Stagonosporopsis species, S. cucurbitacearum (syn. Didymella bryoniae), S. citrulli, and S. caricae (Stewart et al., 2015). The three species are pathogenic to cucurbits, but S. caricae also causes leaf spot and stem and fruit rot in papaya (Carica papaya) (Stewart et al., 2015). This disease was first observed in 1891 by Fautrey and Roumeguere in France on cucumber (Cucumis sativus L.) and in Delaware in watermelon (Chiu and Walker, 1949; Sherf and MacNab, 1986). In 1917, GSB was reported in the Southern United States, affecting watermelon fruit in Florida (Sherbakoff, 1917). Gummy stem blight remains an important limiting factor for watermelon production in Florida (Keinath, 1995; Power, 1992). Gummy stem blight on watermelon plants is evident as crown blight, stem cankers, and extensive defoliation, with symptoms observed on the cotyledons, hypocotyls, leaves, and fruit (Maynard and Hopkins, 1999). Stagonosporopsis cucurbitacearum is seedborne (Lee et al., 1984), airborne (van Steekelenburg, 1983), and soilborne (Bruton, 1998; Keinath, 1996). Adequate control of GSB through fungicide applications (Keinath, 1995, 2000, 2016) and appropriate cultural practices (dos Santos et al., 2016; Rankin, 1954; Keinath, 1996) is difficult, particularly during rainfall when relative humidity remains high for extended times (Caf!e-Filho et al., 2010). In addition, there is concern among pathologists and breeders for the development of resistance by S. cucurbitacearum to fungicides (Avenot et al., 2012; Kato et al., 1984; Keinath and Zitter, 1998; Li et al., 2016; Malathrakis and Vakalounakis, 1983; Miller et al., 1997; Thomas et al., 2012; van Steekelenburg, 1987). Resistance to GSB has received attention since the 1970s as a possible alternative to chemical control (Lou et al., 2013; Norton et al., 1986, 1993, 1995). Differences in GSB resistance among commercial cultivars of watermelon (C. lanatus) were reported, with ‘Congo’ the least susceptible, ‘Fairfax’ intermediate, and ‘Charleston Gray’ the most susceptible (Schenck, 1962). Resistance assays by controlled inoculation of watermelon plants using spore suspensions of S. cucurbitacearum identified PI 189225 and PI 271778 as the most resistant accessions available in the USDA-ARS watermelon germplasm collection (Sowell, 1975; Sowell and Pointer, 1962). In crosses with susceptible ‘Charleston Gray’, a single recessive gene db was determined to confer resistance in PI 189225 (Norton, 1979). Resistant watermelon cultivars were developed from two crosses (‘Jubilee’ · PI 271778 and ‘Crimson Sweet’ · PI 189225) by selecting diseaseresistant seedlings from backcrossed families that had a high yield of excellent quality fruit (Norton et al., 1986). ‘AU-Jubilant’, ‘AU-Producer’ (Norton et al., 1986), ‘AUGolden Producer’ (Norton et al., 1993), and ‘AU-Sweet Scarlet’ (Norton et al., 1995) were released, with moderate resistance to GSB. However, they were found less resistant to GSB than the resistant parents PI 189225 and PI 271778. To date, no cultivars of watermelon have been released that have a high level of resistance to natural epidemics of GSB. The expanding watermelon industry in the southeastern United States and the increasing losses due to GSB outbreaks in the last decade led to a new set of studies for the use of genetic resistance to control GSB in watermelon (Gusmini et al., 2005; Li and Brewer, 2016). The watermelon breeding program at North Carolina State University developed an efficient screening method for testing watermelon germplasm (Gusmini and Wehner, 2002; Song et al., 2004), including systems for mass production of inoculum of S. cucurbitacearum for large field screening experiments (Gusmini et al., 2003), and a disease assessment scale for rating foliar and stem lesions (Gusmini et al., 2002). Available PI accessions (totaling 1274) from the USDA-ARS watermelon germplasm collection, along with 51 adapted cultivars, were tested to identify new genetic sources of resistance to GSB (Gusmini et al., 2005). A total of 59 new accessions were identified that had resistance to GSB as good as or better than PI 189225 and PI 271778 at the field and greenhouse tests. Two of the best were PI 482283 and PI 526233. The objective of this study was to determine the inheritance of resistance to GSB in watermelon accessions PI 482283 and PI 526233, along with the previously identified accession PI 189225. Because of the unsuccessful breeding history for this trait, we hypothesize that resistance to GSB is due to a more complex mode of inheritance, which will be tested by validating the monogenic inheritance of db gene in PI 482283 and PI 526233. Material and Methods Plant material. We used four families developed from the four crosses PI 189225 · ‘NHMidget’, PI 482283 · ‘NHMidget’, PI 482283 · ‘Calhoun Gray’, and PI 526233 · ‘Allsweet’. ‘NH Midget’, ‘Calhoun Gray’, ‘Allsweet’, and PI 526233 were C. lanatus subsp. vulgaris (Chomicki and Renner, 2015). PI 189225 and PI 482283 were Citrullus amarus (Chomicki and Renner, 2015). PI 189225, PI 482283, and PI 526233 were used as resistant parents, and ‘NH Midget’, ‘Calhoun Gray’, and ‘Allsweet’ were used as susceptible parents (Gusmini et al., 2005). The cultivars were obtained from commercial seed stocks, and the PI accessions were obtained from the Southern Regional Plant Introduction Station at Griffin, GA. For each family, we developed six progenies (Pr, Ps, F1, F2, BC1Pr, and BC1Ps) using greenhouses at North Carolina State University in Raleigh, NC. Plating and management. In the greenhouse, temperatures averaged 23 to 43 !C (0800–2000 HR) and 12 to 24 !C (2000–0800 HR) when the assays were performed. We seeded directly in plastic pots (100 · 100 mm size, 600 mL volume) filled with a soilless mix (Canadian sphagnum peatmoss, perlite, Received for publication 22 May 2017. Accepted for publication 12 Sept. 2017. We thank Tammy L. Ellington for her assistance with plant production in the field and greenhouse and Gerald J. Holmes and Anthony P. Keinath for their advice on pathogen handling. Corresponding author. E-mail: lariver2@ncsu. edu. HORTSCIENCE VOL. 52(11) NOVEMBER 2017 1477 vermiculite, processed pine bark). We planted two seeds per pot and thinned to one to ensure a uniform experiment. In the field, seeds were sown on raised, shaped beds on 3.1 m centers in single hills, 1.2 m apart. Border rows of the susceptible ‘Charleston Gray’ and ‘Calhoun Gray’ were planted around each test. We conducted our tests in greenhouses at North Carolina State University in Raleigh, NC, and in the field at the Horticultural Crops Research Station at Clinton, NC. The two families PI 526233 · ‘Allsweet’ and PI 482283 · ‘Calhoun Gray’ were tested in 2002, whereas the other two were tested in 2003. Inoculum preparation.Originally, the isolate of S. cucurbitacearumwas obtained from diseased cucumber tissues harvested from naturally infected plants in Charleston, SC, in 1998. In the fall of 2001, we reisolated S. cucurbitacearum from watermelon plants that were artificially inoculated with the isolate from South Carolina and developed a new stock of inoculum from single spores. Pycnidia were identified with a dissecting microscope (20·) and transferred to petri plates containing potato dextrose agar (PDA) (25 mL/petri plate). Isolates were selected from the first subculture on PDA based on macroscopic observations: colonies dark in color and showing concentric circles of growth were kept and transferred to fresh PDA. Cultures that did not appear contaminated by other fungi or bacteria were transferred to a medium containing 25% PDA to stimulate abundant sporulation. Finally, we observed pycnidia/pseudothecia and spores to verify that their shape and size matched those of S. cucurbitacearum as published (Zitter et al., 1996). For long-term storage (Dhingra and Sinclair, 1995), we transferred the fungus onto sterile filter paper (Whatman #2, 70 mm diameter), subcultured the fungus for 2–4 weeks, dehydrated the filter paper disk and the mycelium for 12–16 h at room temperatures (24 ± 3 !C) under a sterile laminar flow hood, cut the filter paper into squares (5 · 5 mm), and stored them in sterile test tubes in a refrigerator (3 ± 1 !C) in the dark. Cultures of S. cucurbitacearum were grown in Nalgene autoclavable pans (420 · 340 · 120 mm) containing 1000 mL of 50% PDA (Gusmini et al., 2003) before inoculation. We incubated the Nalgene pans for 2–4 weeks at 24 ± 2 !C under alternating periods of 12 h of fluorescent light (40–90mmol·m·s photosynthetic photon flux density) and 12 h of darkness until pycnidia formed. For all inoculations, we prepared a spore suspension by flooding the culture plates with 10 mL of sterile, distilled water, and gently scraping the surface of the agar with an Lshaped sterile glass-rod to remove the spores fro