First Report of Pink Seed of Pea Caused by Erwinia rhapontici in North Dakota.

Abstract

In 2006, a seed lot of dry pea cv. DS Admiral obtained from Bowman County, North Dakota contained seed with bright-to-pale pink discoloration on the seed coat. Five discolored seeds and five seeds with normal appearance were surface disinfected in a 0.5% NaOCl solution for 1 min and rinsed with sterilized distilled water for 1 min. Seeds were placed onto potato dextrose agar (PDA) and incubated at 22°C. Three days later, the discolored seeds produced pink bacterial colonies and a pink pigment that diffused throughout the PDA. The pink bacterial colonies were tentatively identified as Erwinia rhapontici on the basis of colony and pigment color (2,3). No fungi or bacteria grew from the seed with normal appearance. A pink bacterial colony growing from one of the discolored seeds was streaked onto PDA and a single colony was obtained. A streaked plate incubated at 37°C showed no growth, which distinguishes E. rhapontici from Brenneria rubrifaciens (formerly E. rubrifaciens) (1-3). To confirm the identity, the isolate was sent to the Bacterial Identification and Fatty Acid Analysis Laboratory at the University of Florida, Gainesville. Fatty acid analysis indicated a similarity index of 0.515 for E. rhapontici. For an additional confirmation of identity, the 16S ribosomal DNA (rDNA) gene was amplified from the E. rhapontici isolate with universal primers fD1 and rP1 (4). The PCR product was cloned into pGEM-T easy vector (Promega, Madison, WI) and sequenced with primers SP6 and T7 at the Keck Biotechnology Center at the University of Illinois, Urbana. The resulting nucleotide sequence was compared with 16S rDNA sequences deposited in the ribosomal database ( http://rdp.cme.msu.edu/seqmatch/seqmatch_intro.jsp ) and showed highest identity to sequences of E. rhapontici or E. persicinus strains. To confirm pathogenicity, the basal ends of five pods on each of six pea plants (cv. Carneval) were syringe injected with 0.1 ml of suspension containing the obtained E. rhapontici isolate in the greenhouse by the methods as previously described (2). As a control, five pods on each of two plants were injected with 0.1 ml of sterile distilled water. Twenty-eight of the 51 seeds obtained from the bacteria-inoculated pods had pink seed symptoms, while seeds from the control pods appeared normal. Isolations from symptomatic and asymptomatic seed were performed as described above, and E. rhapontici was obtained from symptomatic seed. To our knowledge, this is the first report of pink seed of pea caused by E. rhapontici in North Dakota. The first report of this disease on pea in the United States was from Montana (3).