First Report of Pseudomonas syringae pv. coriandricola Causing Bacterial Leaf Spot on Carrot, Parsley, and Parsnip in Serbia.
First Report of Pseudomonas syringae pv. coriandricola Causing Bacterial Leaf Spot on Carrot, Parsley, and Parsnip in Serbia.
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DOI：
10.1094/pdis-10-14-1041-pdn
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发表时间：
2015-02
期刊：
影响因子：
4.5
通讯作者：
T. Popović;Ž. Ivanović;M. Ignjatov;D. Milošević
中科院分区：
文献类型：
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作者：
T. Popović;Ž. Ivanović;M. Ignjatov;D. Milošević
2014年春季，在塞尔维亚伏伊伏丁那省一个0.5 ha的蔬菜农场，发现胡萝卜（Daucus carota）、欧芹（Petroselinum crispum）和防风草（Pastinaca sativa）发生了严重的叶斑病。病害发生在潮湿和凉爽的条件下，三种作物各有5%至25%的植株受到感染。症状表现为棕色角状叶斑，直径约2mm，常受叶脉限制。收集有症状的叶片，在室温下冲洗和干燥，从坏死组织边缘取叶切片，在无菌磷酸盐缓冲液中浸泡，用5% （w/v）蔗糖（NAS）涂抹在营养琼脂上。分离后，白色，圆形，圆顶状，雷氏阳性菌落一致形成。每个寄主（胡萝卜、欧芹和防风草）各5株进行进一步研究。菌株革兰氏阴性，好氧，过氧化氢酶和烟草过敏反应阳性，氧化酶、马铃薯片腐病和精氨酸二水解酶阴性。这些反应对应于LOPAT Ia组，其中包括丁香假单胞菌病原体(3)。使用Rep、ERIC和BOX引物(4)的重复基因外回文序列(Rep)-PCR指纹图谱对所有菌株都是相同的。对三个代表性菌株（每个宿主各一个）的持家基因gyrB和rpoD(1)进行序列分型。gyrB基因序列分别为KM979434 ~ KM979436（分别来自胡萝卜、欧防风草和欧芹）和KM979437 ~ KM979439（分别来自欧防风草、欧芹和胡萝卜），rpoD基因序列分别存入NCBI GenBank数据库。序列与致病菌株丁香假单胞菌pv进行了比较。存放在植物相关和环境微生物数据库（http://genome.ppws.vt.edu/cgi-bin/MLST/home.pl）中的coriandricola ICMP12471。BLAST分析显示gyrB同源性100%，rpoD同源性99%。用来自每个寄主的5个有代表性的菌株对4周龄胡萝卜(cv。南特)，欧芹(cv。NS Molski)和防风草(cv。Dugi beli glatki)采用两种方法：将细菌悬浮液（108 CFU ml-1）喷洒在叶片上直至径流(5)，并用皮下注射器将细菌悬浮液注射到叶片中(2)。每个品系和方法使用4株。采用无菌蒸馏水作为各植物种的阴性对照处理。所有植物在100%湿度的雾室中保存4小时，然后转移到25°C和80%相对湿度的温室中，在三周内检查症状的发展。所有菌株接种后5 ~ 7 d，叶片首先出现水浸病损；14个DAI病变变成深棕色，常被晕包围。对照植株接种无菌蒸馏水后未见症状。为了实现科赫的假设，对NAS进行了重新隔离。从每个接种的宿主中获得重新分离的细菌，并通过LOPAT测试和Rep-PCR指纹图谱确认与原始分离的细菌相同。根据完成科赫假设的致病性试验、序列分析和细菌学试验，确定菌株为p.s. pv。coriandricola。据我们所知，这是塞尔维亚首次报道胡萝卜、欧芹和防风草的细菌性叶斑病。由于新鲜市场的质量要求，它可能对生产构成威胁。参考文献：(1)P. Ferrente和M. Scortichini。植物病理学杂志，2009,29(5):544 - 544。(2) M. Gupta等。植物学报，2013,37(4):418 - 418。(3) R. A. Lelliott等。j:。细菌。29:470,1966。(4) F. J. Louws等。达成。环绕。[j] .中国生物医学工程学报，2002。(5)徐旭，米勒。植物学报，2013,37(2):988 - 988。
During the spring of 2014, a severe leaf spot disease was observed on carrot (Daucus carota), parsley (Petroselinum crispum), and parsnip (Pastinaca sativa) on a 0.5-ha vegetable farm in Vojvodina Province, Serbia. The disease appeared under wet and cool conditions with 5 to 25% of plants infected for each of the three crops. Symptoms were characterized as brown angular leaf spots, ~2 mm in diameter, often limited by veins. Collected symptomatic leaves were rinsed and dried at room temperature, and leaf sections taken from the margin of necrotic tissue were macerated in sterile phosphate buffer and streaked onto nutrient agar with 5% (w/v) sucrose (NAS). After isolation, whitish, circular, dome-shaped, Levan-positive colonies consistently formed. Five strains from each host (carrot, parsley, and parsnip) were used for further study. Strains were gram-negative, aerobic, and positive for catalase and tobacco hypersensitive reaction but negative for oxidase, rot of potato slices, and arginine dihydrolase. These reactions corresponded to LOPAT group Ia, which includes Pseudomonas syringae pathovars (3). Repetitive extragenic palindromic sequence (Rep)-PCR fingerprint profiles using the REP, ERIC, and BOX primers (4) were identical for all strains. Sequence typing of the housekeeping genes gyrB and rpoD (1) was performed for three representative strains (one from each host). Sequences were deposited in the NCBI GenBank database as accessions KM979434 to KM979436 (strains from carrot, parsnip, and parsley, respectively) for the gyrB gene and KM979437 to KM979439 (strains from parsnip, parsley and carrot, respectively) for the rpoD gene. Sequences were compared with pathotype strain Pseudomonas syringae pv. coriandricola ICMP12471 deposited in the Plant Associated and Environmental Microbes Database ( http://genome.ppws.vt.edu/cgi-bin/MLST/home.pl ). BLAST analysis revealed 100% homology for gyrB and 99% homology for rpoD. Pathogenicity was tested with five representative strains from each host on four-week-old plants of carrot (cv. Nantes), parsley (cv. NS Molski), and parsnip (cv. Dugi beli glatki) using two methods: spraying the bacterial suspension (108 CFU ml-1) on the leaves until runoff (5) and injecting the bacterial suspension into leaves with a hypodermic syringe (2). Four plants were used per strain and method. Sterile distilled water was applied as a negative control treatment for each plant species. All plants were kept in a mist room with 100% humidity for 4 h, then transferred to a greenhouse at 25°C and 80% relative humidity and examined for symptom development over a period of three weeks. For all strains, inoculated leaves first developed water-soaked lesions on the leaves 5 to 7 days after inoculation (DAI); 14 DAI lesions became dark brown, often surrounded by haloes. No symptoms were observed on control plants inoculated with sterile distilled water. For fulfillment of Koch's postulates, re-isolations were done onto NAS. Re-isolated bacteria were obtained from each inoculated host and confirmed to be identical to the original isolates using the LOPAT tests and Rep-PCR fingerprinting profiles. Based on the pathogenicity test accompanied by completion of Koch's postulates, sequence analysis, and bacteriological tests, the strains were identified as P. s. pv. coriandricola. To our knowledge, this is the first report of bacterial leaf spot of carrot, parsley, and parsnip in Serbia. It may present a threat to production due to quality requirements for fresh market. References: (1) P. Ferrente and M. Scortichini. Plant Pathol. 59:954, 2010. (2) M. Gupta et al. Plant Dis. 97:418, 2013. (3) R. A. Lelliott et al. J. Appl. Bacteriol. 29:470, 1966. (4) F. J. Louws et al. Appl. Environ. Microb. 60:2286, 1994. (5) X. Xu and S. A. Miller. Plant Dis. 97:988, 2013.