First Report of Bacterial Wilt on Chrysanthemum Caused by Dickeya chrysanthemi (syn. Erwinia chrysanthemi) in Hungary.
First Report of Bacterial Wilt on Chrysanthemum Caused by Dickeya chrysanthemi (syn. Erwinia chrysanthemi) in Hungary.
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匈牙利首次报道由Dickeya chrysanthemi（异名Erwinia chrysanthemi）引起的菊花青枯病。
DOI：
10.1094/pdis-09-13-0948-pdn
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发表时间：
2014
期刊：
影响因子：
4.5
通讯作者：
L. Palkovics
中科院分区：
文献类型：
--
作者：
A. Végh;Zs. Némethy;P. Salamon;Z. Mándoki;L. Palkovics
菊花（Chrysanthemum spp.）是匈牙利流行的盆栽和切花观赏植物。 2012年9月，从布达佩斯附近的布达卡拉斯市和皮利斯市的不同温室采集了表现出枯萎症状的菊花植物（Chrysanthemum morifolium Ramat. cv. Palisade）。受影响的植物的叶子和茎上有深棕色至黑色的病变。叶子上的斑点先被水浸湿，然后坏死，植株枯萎。据种植者称，病害症状发展迅速，导致损失接近100%。该疾病给匈牙利布达卡拉斯和皮利斯市的种植者造成了约 2,000 欧元的损失。种植者和消费者的损失可能达到五十万欧元。采用10份样品进行疾病诊断，并按照Schaad等人的方法进行细菌分离。 （3）。简言之，将患病的叶和茎组织浸软并在 King's 培养基 B (KB) 上划线。 KB 上的菌落为白色且无荧光。所有 10 个菌株均在 26°C 下生长，呈革兰氏阴性，并可诱导烟草（Nicotiana tabacum L. 'White Burley'）叶子产生过敏反应 (1)。还采用生化测试进行鉴定，API 20E（Biomérieux，Marcy l'Etoile，法国）结果表明该细菌属于肠杆菌科。该菌株的β-半乳糖苷酶和柠檬酸盐利用、乙偶姻和吲哚生产、明胶酶以及葡萄糖、甘露醇、蔗糖、蜜二糖和阿拉伯糖的利用呈阳性。为了对病原体进行分子鉴定，使用通用引物对 (63f/1389r) 从菌株 DCBK-1H 中扩增 16S rDNA 基因 (2)。将 PCR 产物克隆至 pGEM T-Easy 质粒载体（Promega，麦迪逊，威斯康星州）并转化至大肠杆菌 DH5α 细胞中。使用M13正向和反向引物对重组质粒(2A2.5)进行测序。该序列存入NCBI GenBank（登录号HF913430），与数据库中发现的多种Dickeya chrysanthemi菌株具有99%至100%的序列同一性，其中模式菌株HM590189、GQ293897、GQ293898与序列FM946179具有99%的相似性和100%的同一性。综合症状、菌落形态、生化检测及16S rDNA序列同源性，鉴定病原菌为菊花菊。通过将回收的菌株接种到三个 1 个月大的健康盆栽菊花插条（C. morifolium cv. Palisade）上来测试致病性。通过将约 10 μl 含有 107 CFU/ml 的细菌悬浮液注射到每个叶子和茎中，对三个“Palisade”品种的每个叶子和茎进行接种。作为阴性对照，一株植物的四片叶子和茎各用水接种。将植物封装在塑料袋中，并在 80% 遮荫、白天 26°C、夜间 17°C 的温度下在温室中培育。 24小时内，接种后的叶片出现水渍，植株枯萎。水控制看起来正常。 D. chrysanthemi 如上所述被重新分离和鉴定；这样，科赫的假设就得到了满足。据了解，这是匈牙利首次报道由菊花枯萎病引起的菊花青枯病。参考文献：(1) Z.克莱门特。 《自然》199:299, 1963。 (2) A. M. Osborn 等人。环境。微生物。 2:39, 2000。 (3) N. W. Schaad 等人。欧文氏软腐菌群。第 56 页：植物病原菌鉴定实验室指南。第三版。 N.W.Schaad 等人编辑。美国植物病理学会，明尼苏达州圣保罗，2001 年。
Chrysanthemum (Chrysanthemum spp.) is a popular potted and cut plant ornamental in Hungary. In September 2012, chrysanthemum plants (Chrysanthemum morifolium Ramat. cv. Palisade) showing wilt symptoms were collected from different greenhouses in the cities of Budakalász and Pilis near Budapest. Affected plants had dark brown to black lesions on the leaves and stems. Spots on the leaves were first water soaked and then became necrotic, and the plants wilted. According to the growers, disease symptoms developed rapidly, resulting in losses of nearly 100%. The disease caused a loss of ~€2,000 for the growers in cities of Budakalász and Pilis in Hungary. Losses for growers and consumers could have reached half a million euros. Ten samples were used for disease diagnosis and bacteria were isolated according to the method of Schaad et al. (3). Briefly, diseased leaf and stem tissues were macerated and streaked onto King's medium B (KB). Colonies on KB were white and non-fluorescent. All 10 strains grew at 26°C, were gram negative, and induced a hypersensitive response on tobacco (Nicotiana tabacum L. 'White Burley') leaves (1). Biochemical tests were also used for identification, and the results of API 20E (Biomérieux, Marcy l'Etoile, France), demonstrated that the bacterium belonged to the Enterobacteriaceae. The strain was positive for β-galactosidase and citrate utilization, acetoin and indole production, gelatinase, and utilization of glucose, mannitol, saccharose, melibiose, and arabinose. For molecular identification of the pathogen, the 16S rDNA gene was amplified from strain DCBK-1H with a general primer pair (63f/1389r) (2). The PCR products were cloned into a pGEM T-Easy plasmid vector (Promega, Madison, WI) and transformed into Escherichia coli DH5α cells. A recombinant plasmid (2A2.5) was sequenced using the M13 forward and reverse primers. The sequence was deposited in NCBI GenBank (Accession No. HF913430) and showed 99 to 100% sequence identity with a number of Dickeya chrysanthemi strains found in the database, including type strain HM590189, GQ293897, GQ293898 with 99% similarity and 100% identity with sequence FM946179. On the basis of the symptoms, colony morphology, biochemical tests, and 16S rDNA sequence homology, the pathogen was identified as D. chrysanthemi. Pathogenicity was tested by inoculating the recovered strains onto three 1-month-old, healthy potted chrysanthemum cuttings (C. morifolium cv. Palisade). Four leaves and stem each of three 'Palisade' cultivars were inoculated by injecting ~10 μl of a bacteria suspension containing 107 CFU/ml into each leaf and stem. As a negative control, one plant was inoculated with water in each of four leaves and stem. Plants were enclosed in plastic bags and incubated in a greenhouse under 80% shade at 26°C day and 17°C night temperatures. Within 24 h, water-soaked spots appeared on inoculated leaves and the plants were wilted. The water control appeared normal. D. chrysanthemi was re-isolated and identified as described above; thus, Koch's postulates were fulfilled. To our knowledge, this is the first report of bacterial wilt caused by D. chrysanthemi on chrysanthemum in Hungary. References: (1) Z. Klement. Nature 199:299, 1963. (2) A. M. Osborn et al. Environ. Microbiol. 2:39, 2000. (3) N. W. Schaad et al. Erwinia soft rot group. Page 56 in: Laboratory Guide for Identification of Plant Pathogenic Bacteria. 3rd ed. N. W. Schaad et al., eds. American Phytopathological Society, St. Paul, MN, 2001.