Ma X, Lewis Ivey M L, Miller S A
Department of Plant Pathology, The Ohio State University, Ohio Agricultural and Research Development Center, Wooster 44691.
Plant Dis. 2011 Dec;95(12):1584. doi: 10.1094/PDIS-05-11-0448.
In 2009 and 2010, outbreaks of bacterial spot characterized by significant fruit spotting occurred in at least 2,000 ha of commercial processing tomatoes in northwest Ohio and southeast Michigan. Losses were estimated at $7.8 million. Diseased fruit and foliage were collected from 32 Ohio and Michigan fields in 2010. Excised lesions from fruit and leaves were dipped briefly in 70% ethanol, air dried, and chopped into pieces in 10 mM potassium phosphate buffer (KPB), pH 7.4. Ten-fold serial dilutions in KPB were plated on yeast dextrose carbonate agar medium and 83 yellow mucoid colonies were purified. All isolates were gram negative and induced a hypersensitive response in tobacco (Nicotiana tabacum) plants 24 h after inoculation with a 10 CFU/ml bacterial suspension in water. All 83 isolates were identified as Xanthomonas spp. using genus-specific primers RST65/69 (2). Of these, 11 were identified as X. euvesicatoria and 8 as X. perforans using the species-specific primers RST27/28 (1) and JJ19/22 (5'-AACCCAACTAATTTCCCTC-3' and 5'-AACGAGATTTGTTACGAACC-3'; J. B. Jones, personal communication), respectively. DNA fingerprint profiles of 62 of the 64 remaining strains generated using BOX-PCR assays (4) were identical to the profile of X. gardneri type strain XCGA2. The DNA profiles of 2 of the 64 Xanthomonas strains did not resemble those of any reference strains. The 16S rDNA and ITS1 genes from two representative strains (SM174-10 and SM230-10) were PCR amplified, direct sequenced, and aligned using nBLAST with the same gene region from XCGA2 (GenBank Accession No. AF123093). Strains SM174-10 and SM230-10 differed from XCGA2 by 2 bp (99% nucleotide similarity). Pathogenicity tests were performed twice on 6-week-old tomato seedlings (cv. Peto 696). Three tomato seedlings were sprayed until runoff with strain SM174-10 (~10 CFU/ml), three seedlings were sprayed similarly with water (control treatment), and all six plants were grown under high relative humidity (24 s of mist per 12 min) at day/night temperatures of 29/23°C for 15 days. Seedlings inoculated with SM174-10 exhibited water-soaked lesions and chlorosis on the foliage, similar to field symptoms, within 14 days. Seedlings sprayed with water did not develop symptoms. Isolates cultured as described above from all three pathogen-inoculated seedlings were similar in morphology to strain SM174-10; no cultures were recovered from water-inoculated plants. The BOX-PCR fingerprint profile of a representative reisolated colony was identical to that of SM174-10. Although bacterial spot of tomato is a common disease in Ohio and Michigan, to our knowledge this is the first report of X. gardneri infecting tomatoes in these states and provides evidence that there may have been a shift in the primary causal agent of bacterial spot from X. euvesicatoria (3) to X. gardneri. References: (1) H. Bouzar et al. Phytopathology 84:39, 1994. (2) A. Obradovic et al. Eur. J. Plant Pathol. 11:285, 2004. (3) F. Sahin. Ph.D. Diss. The Ohio State University, Columbus, 1997. (4) D. J. Versalovic et al. Methods Mol. Cell. Biol. 5:25, 1994.
2009年和2010年，俄亥俄州西北部和密歇根州东南部至少2000公顷的商业加工番茄爆发了细菌性斑点病，其特征是果实上出现大量斑点。损失估计达780万美元。2010年从俄亥俄州和密歇根州的32个田地收集了患病果实和叶片。将果实和叶片上切除的病斑在70%乙醇中短暂浸泡，风干后，在pH 7.4的10 mM磷酸钾缓冲液（KPB）中切碎。在KPB中进行10倍系列稀释后，接种到酵母葡萄糖碳酸盐琼脂培养基上，纯化得到83个黄色黏液菌落。所有分离株均为革兰氏阴性，在接种10 CFU/ml细菌悬液（溶于水）24小时后，在烟草（Nicotiana tabacum）植株上引发过敏反应。使用属特异性引物RST65/69（2）将所有83个分离株鉴定为黄单胞菌属。其中，分别使用种特异性引物RST27/28（1）和JJ19/22（5'-AACCCAACTAATTTCCCTC-3'和5'-AACGAGATTTGTTACGAACC-3'；J. B. Jones，个人交流）将11个鉴定为丁香假单胞菌（X. euvesicatoria），8个鉴定为穿孔假单胞菌（X. perforans）。使用BOX-PCR分析（4）对其余64个菌株中的62个生成的DNA指纹图谱与加德纳氏黄单胞菌（X. gardneri）模式菌株XCGA2的图谱相同。64个黄单胞菌菌株中的2个DNA图谱与任何参考菌株的图谱均不相似。对两个代表性菌株（SM174-10和SM230-10）的16S rDNA和ITS1基因进行PCR扩增、直接测序，并使用nBLAST与XCGA2（GenBank登录号AF123093）的相同基因区域进行比对。菌株SM174-10和SM230-10与XCGA2有2个碱基对的差异（99%核苷酸相似性）。对6周龄番茄幼苗（品种Peto 696）进行了两次致病性测试。用菌株SM174-10（~10 CFU/ml）对三株番茄幼苗喷雾至径流，同样用水对三株幼苗喷雾（对照处理），所有六株植物在日/夜温度为29/23°C、相对湿度较高（每12分钟喷雾24秒）的条件下生长15天。接种SM174-10的幼苗在14天内叶片上出现水渍状病斑和黄化，与田间症状相似。喷水的幼苗未出现症状。从所有三株接种病原体的幼苗上按上述方法培养的分离株在形态上与菌株SM174-10相似；从接种水株上未分离到培养物。一个代表性的重新分离菌落的BOX-PCR指纹图谱与SM174-10的相同。尽管番茄细菌性斑点病在俄亥俄州和密歇根州是一种常见病害，但据我们所知，这是关于加德纳氏黄单胞菌在这些州感染番茄的首次报道，并提供了细菌性斑点病的主要病原菌可能已从丁香假单胞菌（3）转变为加德纳氏黄单胞菌的证据。参考文献：（1）H. Bouzar等人，《植物病理学》84:39，1994年。（2）A. Obradovic等人，《欧洲植物病理学杂志》11:285，2004年。（3）F. Sahin，博士论文，俄亥俄州立大学，哥伦布，1997年。（4）D. J. Versalovic等人，《分子细胞生物学方法》5:25，1994年。
