A method for detecting Xanthomonas cucurbitae in pumpkin seed
A. RAVANLOU (1). (1) University of Illinois, Urbana, IL, U.S.A.
A method was developed to detect <i>Xanthomonas cucurbitae</i>, the causal agent of bacterial spot of cucurbit, in pumpkin seed. Pumpkin seeds were collected from symptomatic fruit from 20 pumpkin fields in Illinois in 2010 and 2011. From each field, 3,000 seeds were tested for presence of <i>X. cucurbitae</i> on seeds. For each test, 600 seeds were added to a flask containing 500 ml of 0.75% NaCl and 0.02% Tween 20, and incubated at 5°C on a shaker (120 rpm) for 12 hr. The seed wash was centrifuged (16,000 x g) for 5 min. The pellet was re-suspended in 1 ml sterilized distilled water, diluted 10<sup>-2</sup> to 10<sup>-4</sup>, and 100 µl of each dilution was streaked onto Kasugamycin-cephalexin agar medium in each Petri plate. Developing bacterial colonies were examined and single-cell cultures were prepared by streaking colonies onto Lauria-Bertani agar medium in Petri plates. In another experiment, 3,000 seeds were surface disinfested by soaking in 0.05% sodium hypochlorite for 1 min. Seeds were dried and their shells were removed in a flow hood. The kernels were tested for the presence of <i>X. cucurbitae</i> using the method described above. Collected bacterial isolates were identified as <i>X. cucurbitae</i> based on the morphological characteristics on yeast extract-dextrose agar and PCR, followed by a pathogenicity test on pumpkin cultivar Howden. <i>X. cucurbitae</i> was detected in 12 of 20 seed lots tested. Also, <i>X. cucurbitae</i> was detected in the kernels of all three seed lots tested.
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