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First Report of Pink Seed of Pea Caused by Erwinia rhapontici in the United States.

In March 2001, the USDA Federal Grain Inspection Service sent for analysis to USDA-ARS, Pullman, WA, 12 discolored seeds of field pea (Pisum sativum L.) from northeastern Montana. Symptoms consisted of pale pinkish brown-to-bright pink discoloration throughout the seed coat. Unlike the pink coloration resulting from application of pink pesticidal seed treatments, the coloration was permanent and could not be removed by extended washing. Ten discolored seeds were disinfested in 0.5% NaOCl for 1 to 2 min and rinsed in sterile distilled water. Five seeds were placed on malt extract agar amended with streptomycin sulfate and tetracycline hydrochloride at 50 mg per liter each, and five seeds were placed on nutrient broth yeast extract agar (NBY) (3) and incubated under ambient lab conditions. No organisms were isolated from peas on the antibiotic agar, but pale pink bacterial colonies were recovered from each of the five peas on NBY. The pink colonies were streaked for selection of single colonies, stored at 4°C, and revived for growth in shaken (120 rpm) NBY broth to optical density (OD) = 0.1 at 640 nm (≈108 CFU/ml). Three pea plants and one control plant were grown in the greenhouse until pods were sufficiently developed to be syringe-inoculated at the suture with 100 μl of bacterial culture. The experiment was repeated using a second bacterial isolate. In each experiment, pods on inoculated plants, but not the control plant, exhibited pale pinkish areas and later produced seeds discolored in shades of pink matching those from the original sample. Some symptomatic seeds were shriveled or aborted as well as discolored. Discolored seeds from the two experiments were plated to NBY and pale pinkish colonies were recovered as before, but no such colonies were recovered from the controls. Two isolates from the original sample and two recovered from the experimental inoculations were tested for anaerobic and aerobic growth using API 50 CHE and API 20 NE tests (bio Mérieux Vitek, Inc., Hazlewood, MO). Known strains of Erwinia rhapontici and E. rubrifaciens from the collection of Dennis Gross (Texas A&M University) were identically analyzed for comparison. All four isolates from pea were gram-negative, facultatively anaerobic, motile rod-shaped bacteria that produced a diffusible pink pigment on NBY. Tests indicated that these strains were E. rhapontici, because the results agreed with previously published data (1,2). Results with the known strain of E. rhapontici were congruent with those from the pea strains. The strain of E. rubrifaciens also produced a pink pigment on NBY, but unlike the other strains, it grew well at 37°C and was negative for acetoin production, nitrate reduction, esculin hydrolysis, and maltose, rhamnose, inositol, and melibiose fermentation and did not assimilate citrate aerobically. Pink seed of pea has been previously reported from Alberta, Canada (2). References: (1) J. G. Holt et al. Bergey's Manual of Determinative Bacteriology, Williams & Wilkins, Baltimore, MD, 1994. (2) H. C. Huang et al. Can. J. Plant Pathol. 12:445, 1990. (3) A. K. Vidaver. Appl. Microbiol. 15:1523, 1967.