Goss's Bacterial Blight and Wilt of Corn Caused by Clavibacter michiganensis subsp. nebraskensis Occurs in Minnesota

Goss's bacterial wilt and blight caused by Clavibacter michiganensis subsp. nebraskensis (Vidaver and Mandel) Davis et al. can be an economically significant disease of corn (Zea mays L.) (1). Corn hybrids with typical leaf and wilt symptoms of Goss's bacterial blight were observed in two western Minnesota fields in Chippewa and Stephens counties in August 2009. Disease incidence was estimated at 40% in one field and 90% in the other. Symptoms consisted of large, tan-to-gray, linear lesions with irregular margins parallel to the veins, with up to 50% of the leaf area symptomatic. Irregular, dark green-to-black, water-soaked spots occurred in the lesions and dried bacterial exudate was present on the lesions. Bacterial streaming from the cut edge of lesions was visible with light microscopy. Fungal structures were not observed in the lesions. Bacteria were isolated from infected leaves collected in both fields. Sections were cut from the margins of the lesions and placed in 0.02 mM phosphate buffer (PB). Bacterial suspensions were spread onto yeast glucose medium (YGM) (3) and incubated for 5 days at 22°C. All colonies were orange and similar in appearance to C. michiganensis subsp. nebraskensis reference strain CIC016 (= CN313.0). Single colonies were subcultured onto YGM and CNS media. Two gram-positive strains, CIC251 and CIC252, were orange, circular, and convex on CNS medium and used to demonstrate Koch's postulates on corn (2). Bacterial suspensions containing 2 × 10⁸ CFU/ml were prepared in PB from 5-day-old cultures grown on YGM. For each of strains CIC251 and CIC252, six plants of the hybrid DKC51-45 were inoculated at the V3 growth stage by swabbing inoculum over the second and third youngest leaves with Carborundum. Three control plants were treated similarly with sterile PB. Plants were incubated in a greenhouse at 20 to 24°C. Linear, water-soaked lesions typical of Goss's wilt began to develop on all inoculated leaves 7 days after inoculation. No symptoms developed on control plants. Two leaf samples with lesions were collected per plant and bacteria isolated as described above. Colonies with characteristics of C. michiganensis subsp. nebraskensis were isolated from all lesions. Presumptive identification of strains CIC251 and CIC252 as C. michiganensis subsp. nebraskensis, as well as colonies isolated from inoculated plants, was validated by rDNA sequencing. Genomic DNA was extracted from 3-day-old colonies on YGM and the 16S region was amplified (~1,480 bp) by PCR assay using primers F27 and r1492 (4). Forward and reverse sequences were aligned and base calls confirmed using Sequencher 4.9. Consensus sequences for each strain were compared with the nucleotide database with BLAST to confirm a 99% match to C. michiganensis subsp. nebraskensis (NCBI GenBank AM410697.1 and U09763.1). This confirms, for the first time (to our knowledge), that Goss's bacterial leaf blight and wilt of corn occurs in Minnesota and could be a production and phytosanitary concern in that state. References: (1) M. Davis et al. Int. J. Syst. Bacteriol. 34:107, 1984. (2) M. Davis and A. Vidaver. Page 221 in: Laboratory Guide for Identification of Plant Pathogenic Bacteria. 3rd ed. N. Schaad et al., eds. The American Phytopathological Society, St. Paul, MN, 2001. (3) S. DeBoer and R. Copeman, Am. Potato J. 57:457, 1980. (4) S. Giovannoni. Page 177 in: Nucleic Acid Techniques in Bacterial Systematics. E. Stackebrandt and M. Goodfellow, eds. John Wiley & Sons, Chichester, New York, 1991.