Zhou L-H, Han Y, Ji G-H, Wang Z-S, Liu F
College of Plant Protection, Yunnan Agricultural University, Kunming 650201, China.
Plant Dis. 2013 May;97(5):685. doi: 10.1094/PDIS-11-12-1047-PDN.
Panax notoginseng is a species that produces a rare type of Chinese herbal medicine and is cultivated primarily in Yunnan Province. P. notoginseng has a 3-year-long crop cycle before harvest. A new bacterial disease was observed on P. notoginseng plants in the Wenshan Mountain area of Yunnan in 2012. The disease affected primarily leaves. Symptoms were circular or irregular brown leaf spots, surrounded by a yellow halo, located on the edges of the leaves. Eight creamy white pigmented, rounded strains were isolated consistently from leaf spots on Luria-Bertani agar (LB) medium, incubated at 28°C. Three strains (SQYB-1, SQYB-2, SQYB-3) of eight isolates were prepared for further study. Three isolates were purified and characterized: all were gram-negative, rod-shaped, motile, aerobic, non-spore forming, and negative for oxidase, potato soft rot, arginine dehydrolase, presence of tyrosinase and urease, nitrate, and fluorescent pigment production. Moreover, they were positive for levan production, presence of catalase, and for tobacco hypersensitivity. All three strains isolated were identified as Pseudomonas syringae pv. syringae (Pss) based on morphology, metabolic profile (Biolog Microbial ID System), and 16S rDNA sequence analysis (1). The metabolic similarity index between experiment strain SQYB-1 and a type of strain Pss was 0.619. The primers of 16S rDNA sequence amplification were 27F/1492R. Before sequencing, we cloned the PCR products. There was 99% homology in 16S rDNA sequences between one isolate, SQYB-1 (NCBI Accession No. JX876901) and Pss (HQ840766), supporting the identification of the P. notoginseng strains as Pss (3). For Koch's postulates, 10 surface-disinfected young leaves on three plants were inoculated with SQYB-1 isolates by spraying a phosphate-buffered saline cell suspension (3.0 × 10 CFU/ml) onto the leaves (4). Controls were inoculated similarly with sterile phosphate-buffered saline. Plants were covered with polyethylene bags for 24 h at 25°C and then transferred to a greenhouse. Circular or irregular brown spots were observed on all bacteria-inoculated leaves within 9 to 14 days after inoculation. No symptoms were observed on controls. Bacteria reisolated from inoculated leaves were identical to the original isolates as determined by the methods described above. The present study indicated that isolate SQYB-1 could independently infect P. notoginseng leaves, which was different from the finding of Luo et al. concerning involvement of Pss in root rot (2). To our knowledge, this is the first report of Pseudomonas syringae pv. syringae causing bacterial leaf spot on P. notoginseng in China. References: (1) M. R. Gillings et al. PNAS 12:102, 2005. (2) W. F. Luo et al. J. Yunnan Agric. Univ. 14:123, 1999 (in Chinese). (3) C. L. Oliver et al. Plant Dis. 4:96, 2012. (4) H. Ornek et al. New Dis. Rep. 13:40, 2006.
三七是一种能产出珍稀中药材的植物，主要种植于云南省。三七在收获前有一个为期3年的生长周期。2012年，在云南文山地区的三七植株上观察到一种新的细菌性病害。这种病害主要影响叶片。症状表现为叶片边缘出现圆形或不规则的褐色病斑，病斑周围有黄色晕圈。在28°C下于Luria-Bertani琼脂（LB）培养基上从病斑中持续分离出8个乳白色、圆形的菌株。从这8个分离菌株中选取3个菌株（SQYB-1、SQYB-2、SQYB-3）进行进一步研究。对这3个分离菌株进行了纯化和特性鉴定：所有菌株均为革兰氏阴性、杆状、具运动性、需氧、不形成芽孢，氧化酶、马铃薯软腐病、精氨酸脱氨酶、酪氨酸酶和脲酶的存在、硝酸盐及荧光色素产生试验均为阴性。此外，它们在果聚糖产生、过氧化氢酶存在及烟草过敏试验中呈阳性。根据形态学、代谢谱（Biolog微生物鉴定系统）和16S rDNA序列分析（1），所有3个分离菌株均被鉴定为丁香假单胞菌丁香致病变种（Pss）。实验菌株SQYB-1与一种Pss菌株的代谢相似性指数为0.619。16S rDNA序列扩增引物为27F/1492R。在测序前，我们对PCR产物进行了克隆。一个分离菌株SQYB-1（NCBI登录号JX876901）与Pss（HQ840766）的16S rDNA序列同源性为99%，支持将三七菌株鉴定为Pss（3）。为进行柯赫氏法则验证，通过向3株植物上的10片表面消毒的幼叶喷洒磷酸盐缓冲盐水细胞悬液（3.0×10 CFU/ml）接种SQYB-1分离菌株（4）。对照组同样接种无菌磷酸盐缓冲盐水。将植物在25°C下用聚乙烯袋覆盖24小时，然后转移到温室中。接种后9至14天内，在所有接种细菌的叶片上均观察到圆形或不规则的褐色斑点。对照组未观察到症状。通过上述方法确定，从接种叶片上重新分离出的细菌与原始分离菌株相同。本研究表明，分离菌株SQYB-1能够独立感染三七叶片，这与罗等人关于Pss参与根腐病的研究结果不同（2）。据我们所知，这是中国关于丁香假单胞菌丁香致病变种引起三七细菌性叶斑病的首次报道。参考文献：（1）M. R. Gillings等人，《美国国家科学院院刊》12:102，2005年。（2）W. F. 罗等人，《云南农业大学学报》14:123，1999年（中文）。（3）C. L. Oliver等人，《植物病害》4:96，2012年。（4）H. Ornek等人，《新病害报告》13:40，2006年。
