Malvick D, Syverson R, Mollov D, Ishimaru C A
Department of Plant Pathology, University of Minnesota, St. Paul, MN 55108.
Plant Dis. 2010 Aug;94(8):1064. doi: 10.1094/PDIS-94-8-1064A.
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 × 10 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.
由密执安棒形杆菌内布拉斯加亚种（Vidaver和Mandel）Davis等人引起的戈斯细菌性枯萎病和疫病可能成为玉米（Zea mays L.）一种在经济上具有重要影响的病害（1）。2009年8月，在明尼苏达州西部奇珀瓦县和斯蒂芬斯县的两块农田中观察到具有典型戈斯细菌性疫病叶片和枯萎症状的玉米杂交种。据估计，一块农田中的发病率为40%，另一块为90%。症状包括大型的、棕褐色至灰色的、边缘不规则且与叶脉平行的线性病斑，多达50%的叶面积出现症状。病斑上出现不规则的、深绿色至黑色的、水渍状斑点，病斑上有干燥的细菌渗出物。在光学显微镜下可见从病斑切口边缘处有细菌溢菌现象。在病斑中未观察到真菌结构。从两块农田采集的感染叶片中分离出细菌。从病斑边缘切取切片，置于0.02 mM磷酸盐缓冲液（PB）中。将细菌悬浮液涂布在酵母葡萄糖培养基（YGM）（3）上，于22°C培养5天。所有菌落均为橙色，外观与密执安棒形杆菌内布拉斯加亚种参考菌株CIC016（= CN313.0）相似。将单菌落转接至YGM和CNS培养基上。两株革兰氏阳性菌株CIC251和CIC252在CNS培养基上呈橙色、圆形且凸起，并用于对玉米进行柯赫氏法则验证（2）。从在YGM上培养5天的培养物中用PB制备含2×10 CFU/ml的细菌悬浮液。对于菌株CIC251和CIC252中的每一株，在V3生长阶段，通过用金刚砂将接种物涂抹在第二和第三片最幼嫩的叶片上，对6株杂交种DKC51 - 45进行接种。用无菌PB对3株对照植株进行类似处理。将植株置于20至24°C的温室中培养。接种7天后，接种的所有叶片上开始出现典型的戈斯枯萎病的线性水渍状病斑。对照植株未出现症状。每株采集两片有病斑的叶片样本，并按上述方法分离细菌。从所有病斑中均分离出具有密执安棒形杆菌内布拉斯加亚种特征的菌落。通过rDNA测序验证了将菌株CIC251和CIC252推定为密执安棒形杆菌内布拉斯加亚种，以及从接种植株中分离的菌落。从YGM上3日龄菌落中提取基因组DNA，并使用引物F27和r1492通过PCR测定法扩增16S区域（约1480 bp）（4）。将正向和反向序列进行比对，并使用Sequencher 4.9确认碱基调用。将每个菌株的一致序列与核苷酸数据库进行BLAST比对，以确认与密执安棒形杆菌内布拉斯加亚种（NCBI GenBank AM410697.1和U09763.1）有99%的匹配。据我们所知，这首次证实了明尼苏达州发生了玉米的戈斯细菌性叶斑病和枯萎病，并且可能成为该州生产和植物检疫方面的一个问题。参考文献：（1）M. Davis等人，《国际系统细菌学杂志》34:107，1984年。（2）M. Davis和A. Vidaver，载于《植物病原细菌鉴定实验室指南》第3版，N. Schaad等人编，美国植物病理学会，明尼苏达州圣保罗，2001年，第221页。（3）S. DeBoer和R. Copeman，《美国马铃薯杂志》57:457，1980年。（4）S. Giovannoni，载于《细菌系统学中的核酸技术》，E. Stackebrandt和M. Goodfellow编，约翰威立国际出版公司，纽约奇切斯特，1991年，第177页。
