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.
The RB gene from wild potato Solanum bulbocastanum imparts broad-spectrum late blight resistance to cultivated potato. To explore marker associations and haplotype frequencies near RB, we developed, optimized, validated, and employed a set of markers specific to the haplotype associated with the RB resistance allele. Our markers, developed using a mismatch amplification mutation assay (MAMA)-PCR approach, have single nucleotide polymorphism-level sensitivity. MAMA markers were validated via linkage mapping and applied to a collection of 60 S. bulbocastanum genotypes representing the geographic distribution of the species. Considerable marker disassociation within a 100 kb region encompassing RB was revealed. Physical distance between markers was not an effective predictor of marker disassociation. Applied to a collection of four S. bulbocastanum genotypes of known RB allelic status, a set of six MAMA markers accurately predicted the presence of the RB allele. Implications for marker aided genotype selection and association mapping in wild potato species are explored.Resumen El gen RB de la especie Silvestre Solanum bulbocastanum confiere resistencia de amplio espectro al tizón tardío en la papa cultivada. Con el fin de explorar asociaciones de marcadores y frecuencias de haplotipos cercanos a RB, desarrollamos, optimizamos, validamos y empleamos un juego de marcadores específicos al haplotipo asociado con el alelo de resistencia RB. Nuestros marcadores, desarrollados usando una estrategia de un ensayo de amplificación de mutación desajustada (MAMA)-PCR, tienen sensibilidad a nivel de polimorfismo de un nucleó-tido sencillo. Los marcadores MAMA se validaron vía un mapa de ligas y se aplicó a una colección de 60 genotipos de S. bulbocastanum representativos de la distribución geográfica de la especie. Se reveló una disociación considerable de marcadores dentro de una región de 100 kb abarcando RB. La distancia física entre marcadores no fue un factor predictivo efectivo de la disociación de marcadores. Aplicado a una colección de cuatro genotipos de S. bulbocastanum de un estatus conocido de RB alélico, un juego de seis marcadores MAMA predijeron con precisión la presencia del alelo RB. Se exploran las implicaciones para selección de genotipos asistida con marcadores y mapeo de asociaciones en especies silvestres de papa.
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