During a routine nursery inspection in Hernando County, Florida in July 2011, leaf spot symptoms were observed on the popular sugar substitute stevia (Stevia rebaudiana). Spots were roughly circular to irregular in shape, variable in size, and dark brown with a yellow halo. White to cream-colored, circular, convex, gram-negative bacterial colonies were isolated on nutrient agar and identified as Pseudomonas cichorii based on the LOPAT scheme (2). DNA from four individual colonies inoculated in nutrient broth was extracted using DNeasy columns (Qiagen Inc., Valencia, CA). The PCR-amplified product from four different genes, 16S rRNA (1.5 Kbp), gyrB (0.9 Kbp), rpoB (1.2 Kbp), and rpoD (0.7 Kbp), was sequenced (GenBank Accession Nos. JQ994483, JQ994484, JQ994485, and JQ994486). Nucleotide and translated amino acid sequences for each gene were compared to the nucleotide and protein databases, respectively. The best matches were always with P. cichorii with nucleotide identities ranging from 98 to 99% and amino acid identities from 99 to 100%. Four healthy stevia plants were spray inoculated with 20 ml of a 108 CFU ml–1 suspension prepared from a 24-hour-old culture, of which two were sprayed with carborundum (silicon carbide) immediately prior to inoculation. Two additional plants were sprayed with carborundum only or sterile tap water only and served as healthy controls. Symptoms began to develop 4 days after inoculation. Spots originated at the tips or edges of the leaves and enlarged over time. Enlarging lesions progressed to encompass the entire leaf, accompanied by water soaking, curling, and necrosis. Blighting typically spread down to the stem and caused dieback. The pathogen was successfully reisolated from the lesions and produced identical LOPAT scheme results. Based on the information collected, it is believed that this is the first confirmed report of bacterial leaf spot caused P. cichorii on stevia worldwide. This find is significant due to the rising popularity of stevia cultivation for its sweetness and medicinal properties (1). To our knowledge, stevia is not currently being grown commercially in Florida; however, there is commercial acreage being developed elsewhere in the United States.
References: (1) D. Patil et al. Asian J. Pharm. Clin. Res. 5:1, 2011. (2) N. W. Schaad et al. Laboratory Guide for Identification of Plant Pathogenic Bacteria. 3rd Edition. The American Phytopathological Society. St. Paul, MN, 2001.