Final Report for GNC15-217
Bacterial spot, caused by Xanthomonas cucurbitae, has become a serious threat to the pumpkin industry in Illinois and other Midwest states of the United States. X. cucurbitae infects leaves and fruits, causing up to 100% yield losses. X. cucurbitae is considered as a seedborne pathogen and planting pathogen-free seed is an important step in management of the disease. This study was conducted to develop a reliable seed treatment to eradicate the pathogen in the seeds. We evaluated hot-water, hydrochloric acid, and sodium hypochlorite for eradication of X. cucurbitae in naturally-infected and artificially-infested pumpkin seeds. Hot-water treatment at 55°C for 15 min and hydrochloric acid treatment at 0.5% concentration for 40 min eradicated X. cucurbitae in pumpkin seeds without any significant adverse effect on either seed germination or seedling vigor.
Bacterial spot, caused by Xanthomonas cucurbitae, has become a serious threat to pumpkin industry in Illinois and other Midwest states of the United States. The pathogen was first reported in 1926 on ‘Hubbard’ squash in New York. Subsequently, the disease was reported in other cucurbit growing areas in Asia, Australia, Europe, and North America on cucumber, pumpkin, squash, and watermelon.
This pathogen can infect both leaves and fruit. Infected leaves develop small, round to angular, and yellow to beige lesions surrounded by a yellow halo. On some pumpkin cultivars, the lesions may appear more angular, similar to the lesions caused by angular leaf spot, caused by Pseudomonas lachrymens. Lesions may coalesce to cover large parts of the leaves. The lesions on fruit begin as small (1-3 mm in diameter), circular, and slightly sunken with a beige center and brown halo. The lesion may reach 10-15 mm in diameter. Infected fruits are colonized by the opportunistic fungi and bacteria, resulting in decay of fruits.
During 2011-2013, field surveys were conducted in Illinois, Indiana, Iowa, Kansas, Michigan, Missouri, Nebraska, Ohio, and Wisconsin to assess incidence and severity of the bacterial spot in pumpkin and squash. During four weeks of the harvest, 111, 132, and 126 fields in 2011, 2012, and 2013, respectively, were visited. Symptomatic fruit were observed in 95 (86%), 117 (89%), and 113 (90%) of the fields visited, with overall 34, 23, and 19% fruit infected, in 2011, 2012 and 2013, respectively. In some of the fields, more than 90% of fruits were affected.
X. cucurbitae is considered as a seedborne pathogen and planting pathogen-free seed is an important step in management of the disease. Seed-borne inoculi plays an important role in spreading seed-borne diseases, especially in long-distances.
The objective of this study was to develop a reliable seed treatment to eradicate Xanthomonas cucurbitae in pumpkin seeds.
Preparation of treated seeds
For hot-water seed treatment, seeds were treated in water at temperatures of 49, 50, 51, 52, 53, 54, 55, and 56°C for 5, 10, 15, 20, 25, and 30 min. An electrical water-bath was used for hot-water treatment of the seeds. For hydrochloric acid, diluted HCl solutions were prepared by adding a concentrated HCl solution into sterile distiller water (SDW) by gently stirring the solution. Solution of 0.5, 1.0, and 2.0% (v/v) HCl were used in this study. Seeds for treatment were wrapped in two-layer of cheesecloth and immersed into the HCl solutions. Seeds were treated for 10, 20, 30, 40, 50, and 60 min at each of HCl concentration. HCl treated seeds were rinsed with SDW three times, each time for 3 min. Treated seeds were then spread onto sterile paper towel in a laminar flow hood and allowed to dry overnight. The same method used for HCl treatment, was also used in sodium hypochlorite treatment. Seeds were soaked in NaClO solutions with concentrations of 0.5, 1.0, 1.5, and 2.0% for 1, 2, 3, and 4 min in each of the prepared NaClO solutions.
Seed germination and seedling vigor
The effects of the seed treatments on seed germination and seedling vigor were evaluated in both the laboratory and a greenhouse. In the laboratory, samples of 200 seeds were tested each time. The germination test was repeated three times. Untreated pumpkin seeds were used as control. Seeds were placed 0.5 cm apart on the moistened germination paper and the paper was rolled and placed on plastic bags each with 50 ml water. Bags were placed in an incubator with 24±1°C for 4 days. All tested seeds were examined, and the percentage of germinated seeds was calculated. Seedling vigor was assessed using 10 seeds from each treatment, with three replications (a total of 30 seeds from each treatment). Untreated seeds were used as control. Ten seeds were placed 2 cm apart on moistened germination paper, the paper was rolled, and incubated at 24±1°C for 4 days. Lengths of shoots and roots of the seedlings were measured at the end of incubation period.
Seed germination and seedling vigor were also tests in greenhouse using 32-cell trays filed with a mixture of soil:peat:perlite (1:2:1). Seed germination was assessed by planting 20 seeds from each treatment, with three replications (a total of 60 seeds for each treatment and control). Trays were placed in a greenhouse at 24-28°C. Three weeks after sowing seeds, germinated seed were counted, and the percent of seed germination was calculated. Seedling vigor was evaluated 3 weeks after sowing seed, using a 0-4 scale, 0 for non-germinated seed and 4 for vigorously growing seedling.
Hot-water treatment at 55°C for 15 min and hydrochloric acid treatment at 0.5% concentration for 40 min eradicated X. cucurbitae in both naturally-infected and artificially-inoculated pumpkin seeds without any significant adverse effect on either seed germination or seedling vigor. Sodium hypochlorite treatment did not eradicate X. cucurbitae in pumpkin seeds. Effects of hot-water and hydrochloric acid treatments should be evaluated in the fields with different soil types and weather conditions.
Educational & Outreach Activities
Publications are in preparation. The findings of the study have been presented to the extension educators and scientists.
The results from this study showed that seed-borne X. cucurbitae in cucurbits can be eradicated effectively without reducing seed germination or seedling vigor.
Illinois produces approximately 25,000 acres of pumpkin every year. Pumpkin is also a major crop in other states of the United States, as well as worldwide. At this time, we do not know the exact economic impact of the seed treatment developments developed in this study. But, considering the fact that X. cucurbitae is a seedborne pathogen, the pathogen affect most of the cucurbit crops, and the disease is causing substantial crops losses, economic value of the findings in this study is expected to be considerable.
Farmers have not adopted the developed seed treatments yet.
Areas needing additional study
The developed methods should be tested in fields with various soil types and under different environmental conditions.