In November of 2007, 6-inch rooted cuttings of Mandevilla (= Dipladenia) splendens ‘Red Riding Hood’ were submitted from a greenhouse in Indiana to the Purdue Plant and Pest Diagnostic Lab. Plants exhibited leaf dieback, wilting, and reduced top growth. Microscopic observation revealed no fungal structures within the roots, stems, and leaves; however bacterial streaming was observed from the cut edge of stem and root tissue using ×100 magnification with phase contrast. A Ralstonia solanacearum ImmunoStrip test (Agdia Inc., Elkhart, IN) was used to determine that the samples (roots and stem) were positive for R. solanacearum, the causal agent of southern wilt. A bacterial suspension was prepared from infected tissue and streaked onto King's Medium B (KB). Gram-negative, nonfluorescent, oxidase-positive bacteria were consistently isolated from the diseased tissues and determined as R. solanacearum by BIOLOG (Hayward, CA) carbohydrate utilization. A culture of R. solanacearum and infected plant material were submitted to USDA APHIS PPQ as per select agent protocol. (3) CPHST NPGBL generated pure cultures and together with submitted plant materials they tested positive for R. solanacearum using Agdia ImmunoStrips. Culture and plant material tested positive for R. solanacearum and negative for biovar 2 using the Fegan conventional PCR (1) and the Central Science Lab (CSL, York, UK) real-time PCR (4). Pure cultures were determined to be negative for biovar 2 but positive for biovar 3 using the biovar carbohydrate utilization plate assay (2). On the basis of these results, the bacteria were identified as R. solanacearum biovar 3 and not as the select agent R. solanacearum race 3 biovar 2. Koch's postulates confirmed pathogenicity of the isolated bacteria on tomato, a susceptible host. Three 6-week-old plants were mechanically inoculated with a bacterial suspension of approximately 1 × 108 CFU/ml prepared from cultures grown on KB for 2 days at 28°C. Inoculum (0.1ml of bacterial suspension) was injected into stem axils with a 22-gauge hypodermic needle. Three 6-week-old control plants were inoculated with sterile water. Plants were kept at 24°C with supplemental 400W high-pressure sodium light. Within 5 days, all three inoculated plants exhibited wilt symptoms. No symptoms were observed in control plants. Bacteria were reisolated from symptomatic plants on KB medium as described above, and gram negative, nonfluorescent, oxidase-positive bacteria were obtained. Reisolated strains were identical to R. solanacearum using BIOLOG carbohydrate utilization testing, confirming the causal agent of the disease. Personal correspondence with other diagnosticians also confirms the presence of R. solanacearum biovar 3 in Mandevilla in Ohio, Michigan, and Minnesota. To our knowledge, this is the first documented report in the world of R. solanacearum biovar 3 on Mandevilla.
References: (1) M. Fegan et al. Page 34 in: Bacterial Wilt Disease Molecular and Ecological Aspects. P. Prior et al., eds. INRA Editions-Springer. Verlag, Germany, 1998. (2) E. R. French et al. Fitopatologia 30:126, 1995. (3) USDA/APHIS/PPQ New Pest Response Guidelines. Ralstonia Solanacearum race3 biovar 2, from http://www.aphis.usda.gov/import_export/plants/manuals/emergency/ downloads/nprg-ralstonia.pdf, retrieved May 2008. (4) S. A. Weller et al. Appl Environ. Microbiol. 7:2853, 2000.
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