Hiltunen L H, Kelloniemi J, Valkonen J P T
MTT Agrifood Research Finland, P.O. Box 413, FI-90014 University of Oulu, Finland.
Department of Agricultural Sciences, P.O. Box 27, FI-00014 University of Helsinki, Finland.
Plant Dis. 2014 Sep;98(9):1267. doi: 10.1094/PDIS-03-14-0278-PDN.
Common scab is an important disease in potato (Solanum tuberosum) caused by Streptomyces spp. In Finland, morphological and physiological characterization (5) and comparison of the 16S rRNA gene sequences have suggested that Streptomyces scabies and S. turgidiscabies are the main causal species (2), but occurrence of S. europaeiscabiei has not been previously studied. In September 2011, potato tubers of cvs. Fambo, Melody, Puikula, Rosamunda, Victoria, and Van Gogh showing symptoms of common scab were collected from 10 fields in southern (Lammi), eastern (Liperi), and western Finland (Isokyrö, Kalajoki, Tyrnävä). Symptoms included superficial, raised, and pitted corky lesions ranging from 3 to 10 mm in diameter. Isolations were made from scab lesions on water agar. Colonies phenotypically characteristic of Streptomyces were transferred to glucose yeast malt extract agar (GYM) after 7 days. Pure cultures were obtained through subsequent transfers to fresh GYM medium, on which the strains produced golden brown colonies and white to grey spores. DNA was extracted from bacteria using the E.Z.N.A. SP Plant DNA Mini Kit (Omega Bio-Tek, Inc.). Primers developed for the 16S rRNA gene sequences (4) were used to detect S. scabies and S. turgidiscabies by PCR. Of the 14 strains recovered, nine were assigned to S. scabies, three to S. turgidiscabies, and two remained unidentified. However, S. scabies and S. europaeiscabiei cannot be distinguished by the 16S rRNA gene sequence, but the ITS1 region of the 16S operon sequence amplified by PCR is cleaved by Hpy99I in S. scabies but not in S. europaeiscabiei (1). Altogether, 18 strains were tested, including nine obtained in 2011 and seven Finnish and two Swedish strains isolated and assigned to S. scabies in the mid-1990s (2,4,5). The ITS1 sequence of S. scabies type strain, ATCC49173, was cleaved with Hpy99I, in contrast to all other strains that were consequently assigned to S. europaeiscabiei. To further confirm the identity of the Finnish strains, sequences of atpD, recA, and rpoB genes from three strains (one from 1995, two from 2011) (GenBank Accession Nos. KJ802471 to 79) were found to be 100, 99.8, and 100% identical, respectively, to corresponding S. europaeiscabiei type strain CFBP 4497 sequences (3). Pathogenicity of the S. europaeiscabiei strains isolated in 2011 was confirmed using radish seedling assay (1). All strains prevented or reduced the growth of radish seedlings (cv. French Breakfast) or caused severe necrosis in repeated experiments. No symptoms were observed on the seedlings grown on oat meal agar without bacteria. The pathogenicity of the S. europaeiscabiei strains isolated in the mid-1990s was confirmed using minituber assays (4,5). In addition, two of these strains were tested in a glasshouse experiment and two in a radish seedling assay and shown to be pathogenic. The results suggest that S. europaeiscabiei is an important cause of potato common scab in Finland. In the earlier studies, S. europaeiscabiei may have been mistaken for S. scabies, as the restriction analysis of the ITS1 region was not done. References: (1) R. Flores-Gonzáles et al. Plant Pathol. 57:162, 2008. (2) J. F. Kreuze et al. Phytopathology 89:462, 1999. (3) D. P. Labeda. Int. J. Syst. Evol. Microbiol. 61:2525, 2011. (4) M. J. Lehtonen et al. Plant Pathol. 53:280, 2004. (5) P. Lindholm et al. Plant Dis. 81:1317, 1997.
马铃薯普通疮痂病是由链霉菌属引起的马铃薯(Solanum tuberosum)重要病害。在芬兰,形态学和生理学特征鉴定(5)以及16S rRNA基因序列比较表明,疮痂链霉菌和肿胀链霉菌是主要致病菌种(2),但此前尚未对欧洲链霉菌的发生情况进行研究。2011年9月,从芬兰南部(拉米)、东部(利佩里)和西部(伊索屈勒、卡拉约基、蒂尔纳韦)的10个田地收集了表现出普通疮痂病症状的Fambo、Melody、Puikula、Rosamunda、Victoria和Van Gogh品种的马铃薯块茎。症状包括直径3至10毫米的浅表、凸起和凹陷的木栓质病斑。在水琼脂上从疮痂病斑进行分离。7天后,将表型特征为链霉菌的菌落转移到葡萄糖酵母麦芽提取物琼脂(GYM)上。通过随后转移到新鲜的GYM培养基上获得纯培养物,菌株在该培养基上产生金褐色菌落和白色至灰色孢子。使用E.Z.N.A. SP植物DNA小提试剂盒(Omega Bio-Tek公司)从细菌中提取DNA。用于16S rRNA基因序列的引物(4)用于通过PCR检测疮痂链霉菌和肿胀链霉菌。在回收的14个菌株中,9个被鉴定为疮痂链霉菌,3个为肿胀链霉菌,2个仍未鉴定。然而,疮痂链霉菌和欧洲链霉菌不能通过16S rRNA基因序列区分,但通过PCR扩增的16S操纵子序列的ITS1区域在疮痂链霉菌中可被Hpy99I切割,而在欧洲链霉菌中则不能(1)。总共测试了18个菌株,包括2011年获得的9个菌株以及20世纪90年代中期分离并鉴定为疮痂链霉菌的7个芬兰菌株和2个瑞典菌株(2,4,5)。疮痂链霉菌模式菌株ATCC49173的ITS1序列被Hpy99I切割,而所有其他菌株因此被鉴定为欧洲链霉菌。为进一步确认芬兰菌株的身份,发现来自3个菌株(1个来自1995年,2个来自于2011年)(GenBank登录号KJ802471至79)的atpD、recA和rpoB基因序列分别与相应的欧洲链霉菌模式菌株CFBP 4497序列100%、99.8%和100%相同(3)。使用萝卜幼苗试验(1)证实了2011年分离的欧洲链霉菌菌株的致病性。在重复实验中,所有菌株均抑制或减少了萝卜幼苗(法国早餐品种)的生长或导致严重坏死。在没有细菌的燕麦粉琼脂上生长的幼苗未观察到症状。使用微型块茎试验(4,5)证实了20世纪90年代中期分离的欧洲链霉菌菌株的致病性。此外,其中2个菌株在温室试验中进行了测试,2个在萝卜幼苗试验中进行了测试,结果表明它们具有致病性。结果表明,欧洲链霉菌是芬兰马铃薯普通疮痂病的重要病因。在早期研究中,由于未对ITS1区域进行限制性分析,欧洲链霉菌可能被误认为是疮痂链霉菌。参考文献:(1)R. Flores-Gonzáles等人,《植物病理学》57:162,2008年。(2)J. F. Kreuze等人,《植物病理学》89:462,1999年。(3)D. P. Labeda,《国际系统与进化微生物学杂志》61:2525,2011年。(4)M. J. Lehtonen等人,《植物病理学》53:280,2004年。(5)P. Lindholm等人,《植物病害》81:1317,1997年。