Determining the Host-range and Developing Seed-treatment for Managing Bacterial Spot on Pumpkin

Determining the Host-range and Developing Seed-treatment for Managing Bacterial Spot on Pumpkin

Summary

Pumpkin is a high-value crop grown throughout the United States (US). Approximately 1.5 billion pounds of pumpkins are produced in the US every year and Illinois with about 25,000 acres ranks first in pumpkin production in the US. More than 90% of processing pumpkin are grown and processed in Illinois. Bacterial spot, caused by Xanthomonas cucurbitae has become one of the most economically important diseases on pumpkins and a serious threat to the industry. Leaf spots and fruit lesions and rot are major symptoms of this disease, causing up to more than 75% yield losses. In the past five years, the disease has become more prevalent in Illinois and other Midwestern states and many growers complain about the yield losses and of lack of effective disease management measures. Developing effective management of bacterial spot of pumpkin has not been feasible because of lack of sufficient information on the etiology of the disease, especially on the host-range and seed-borne aspect of the pathogen. This is proposal for a two-year project to generate necessary information on the etiology of bacterial spot of pumpkin and develop effective methods for management of the disease. The specific objectives of this research are to: (1) identify the host-range of X. cucurbitae; (2) determine the importance of seed-borne aspect of the pathogen; and (3) develop management of the disease, emphasizing effective seed-treatments and cropping rotations. Result of this study will be presented to growers, industry personnel, extension educators, students, and scientists at the local, statewide, regional, and national meetings. The results also will be published in the newsletters, websites and refereed-journals.

Objectives/Performance Targets

This is proposal for a two-year project to generate necessary information on the etiology of bacterial spot of pumpkin and develop effective methods for management of the disease. The specific objectives of this research are to: (1) identify the host-range of X. cucurbitae; (2) determine the importance of seed-borne aspect of the pathogen; and (3) develop management of the disease, emphasizing effective seed-treatments and cropping rotations.

Accomplishments/Milestones

This study was conducted to assess transmission of Xanthomonas cucurbitae in pumpkin seed. Twenty nine pumpkin seed lots were tested for presence of X. cucurbitae, which included 17 lots from commercial pumpkin fields in Illinois, three lots from pumpkin research plots at the University of Illinois Vegetable Research Farm in Champaign, Illinois, and nine lots from three seed companies from the United States, but outside Illinois. Each seed lot was from one pumpkin cultivar, and 3,000 seeds from each lot were tested. In each test, 600 seeds were added to a flask containing 500 ml of 0.75% NaCl solution with 0.02% Tween 20, the flask was shaken at 120 rpm on a shaker for 12 hr at 5°C. The resulting seed-wash was centrifuged at16,000 g for 5 minutes. The pellet was suspended in 1 ml sterilized buffer (0.15 M NaCl, 0.01 M EDTA with pH 8.0). Dilution of 10-2, 10-3, and 10-4 of the suspension were prepared, and100 µl from each dilution was streaked onto kasugamycin-cephalexin agar in each Petri plate. Xanthomonad-like yellow colonies were subcultured on Luria-Bertani agar medium in Petri plates and single cell colonies were prepared. Bacterial isolates were identified as X. cucurbitae based on the morphological characteristics on yeast extract-dextrose agar (yellow and mucoid colonies); biochemical and physiological characteristics (Gram negative, oxidase negative, catalase positive, oxidative and non-fermentative); and producing a 1.4 kb amplicon in PCR test using RST2 and RST3 primers. Pathogenicity testing of the bacterial isolates was conducted on pumpkin cultivar Howden in a greenhouse. X. cucurbitae was detected in nine of 17 seed lots from commercial fields, all three lots from research plots, and none of the seed lots provided by seed companies. In another study, seeds from three lots with X. cucurbitae were tested to determine whether the pathogen is carried on or in the seed. From each lot, 4,000 seeds were surface disinfested using NaClO solution. Each time, 500 seeds were added to a 1,000 ml flask containing 500 ml of 0.5% NaClO solution and shaken by hand. Seeds were treated in NaClO solution for1, 3 or 5 min. Non-surface-disinfested seeds (control seeds) were soaked in sterilized distilled water (SDW) for 1, 3, or 5 min. Treated seeds were dried and their shells were separated from kernels under a laminar flow hood. The kernels and shells were tested for presence of X. cucurbitae using the method described for whole seeds. X. cucurbitae was detected in kernels of all seed samples that were treated in NaClO and sterilized distilled water (SDW), while the pathogen was isolated from shells of seeds that were treated in NaClO for 1 min and seeds treated in SDW. Transmission of X. cucurbitae from naturally-infected seeds to seedlings was studied in a greenhouse. Six hundred forty seeds from each of three seed lots, with X. cucurbitae, were planted in plastic trays (12x40x60 cm) (40seeds per tray), and trays were placed in a greenhouse at 24 to 28°C. Seven to ten days after planting seeds, leaf lesions were observed on the cotyledons leaves and X. cucurbitae was isolated from the in affected tissues.

Impacts and Contributions/Outcomes

The results of this study showed that Xanthomonas cucurbitae is carried on and in the seed and infected seed give rise to infected plants. The conclusions are: (i) try to plant pathogen-free seed, and (ii) develop effective seed treatments for managing seed-borne inoculum of the pathogen.