Cheng M, Dong J, Laski P J, Zhang Z, McBeath J H
Department of High Latitude Agriculture, University of Alaska-Fairbanks 99775.
Key Laboratory of Agricultural Biotechnology of Yunnan Province, Institute of Biotechnology and Germplasm Resources, Yunnan Academy of Agricultural Sciences, Kunming, China 650223.
Plant Dis. 2011 Jun;95(6):777. doi: 10.1094/PDIS-12-10-0914.
Potatoes (Solanum tuberosum) are one of the most important crops in China following rice, wheat, and corn. Aster yellows phytoplasma appeared to be widespread in China; it was found to cause diseases on alfalfa, oranges, peaches, periwinkles, bamboo (1), and cactus (4). However, scant information of this pathogen on potatoes is available except for a few short reports published during the 1950s. During the potato disease surveys conducted from 2005 to 2010 in Yunnan and Inner Mongolia, 10 to 35% of potato plants exhibited symptoms of yellowing or purpling of apical leaves, with the top leaves rolling inward and aerial tubers formation. Total DNA was extracted from midveins of leaves and roots of 125 diseased and asymptomatic plants with a DNeasy Plant Mini Kit (Qiagen, Valencia, CA) according to the manufacturer's instructions. A nested PCR was carried out with the first round primer pair P1/P7 followed by the second round primer pair R16F2n/R16R2 (2,3). A PCR product of approximately 1.2 kb was amplified from diseased plants but not from asymptomatic plants. Restriction fragment length polymorphism (RFLP) patterns were analyzed by digesting a 1.2-kb product using restriction enzymes AluI, BfaI, BstUI, HhaI, HpaI, KpnI, MseI, and RsaI. Comparing the RFLP patterns with previously published phytoplasma strains (2), aster yellows phytoplasma found on potato plants in Yunnan and Inner Mongolia belong to group I, subgroup B (16SrI-B). The PCR product from P1/P7, diluted 1:30, was amplified by using primer pair P1A/P7A (3) and P1A/16S-SR (3). The nested-PCR products from P1A/P7A and P1A/16S-SR were cloned into pCR8/GW/TOPO vector (Invitrogen, Carlsbad, CA) and sequenced by the Core Lab of the University of Alaska-Fairbanks and GENEWIZ (South Plainfield, NJ). The nucleotide sequence (GenBank Accession No. HQ599228) was analyzed by iPhyClassifier software and had 99.53% sequence identity to the reference strain (GenBank Accession No. M30790) for 'Candidatus Phytoplasma asteris'. The RFLP similarity is identical (coefficient 1.00) to the reference pattern of 16Sr group I, subgroup B (GenBank Accession No. NC_005303). To our knowledge, this is the first report revealing the molecular characteristics of a phytoplasma associated with aster yellows-diseased potatoes in China. References: (1) H. Cai et al. Plant Prot. 31:38, 2005. (2) I.-M. Lee et al. Int. J. Syst. Bacteriol. 48:1153, 1998. (3) I.-M. Lee et al. Int. J. Syst. Evol. Microbiol. 54:337, 2004. (4) W. Wei et al. Plant Dis. 91:461, 2007.
马铃薯（Solanum tuberosum）是继水稻、小麦和玉米之后中国最重要的作物之一。翠菊黄化植原体在中国似乎广泛存在；已发现它会在苜蓿、橙子、桃子、长春花、竹子（1）和仙人掌（4）上引发病害。然而，除了20世纪50年代发表的几篇简短报告外，关于这种病原菌在马铃薯上的信息很少。在2005年至2010年于云南和内蒙古开展的马铃薯病害调查中，10%至35%的马铃薯植株表现出顶叶黄化或发紫的症状，顶部叶片向内卷曲并形成气生块茎。使用DNeasy Plant Mini试剂盒（Qiagen公司，加利福尼亚州瓦伦西亚）按照制造商的说明从125株患病和无症状植株的叶片中脉及根部提取总DNA。进行巢式PCR，第一轮引物对为P1/P7，随后第二轮引物对为R16F2n/R16R2（2,3）。从患病植株中扩增出约1.2 kb的PCR产物，但从无症状植株中未扩增出。使用限制性内切酶AluI、BfaI、BstUI、HhaI、HpaI、KpnI、MseI和RsaI消化1.2 kb的产物，分析限制性片段长度多态性（RFLP）模式。将RFLP模式与先前发表的植原体菌株（2）进行比较，在云南和内蒙古马铃薯植株上发现的翠菊黄化植原体属于I组，B亚组（16SrI-B）。将P1/P7的PCR产物稀释1:30，使用引物对P1A/P7A（3）和P1A/16S-SR（3）进行扩增。来自P1A/P7A和P1A/16S-SR的巢式PCR产物克隆到pCR8/GW/TOPO载体（Invitrogen公司，加利福尼亚州卡尔斯巴德）中，并由阿拉斯加大学费尔班克斯分校核心实验室和GENEWIZ公司（新泽西州南普莱恩菲尔德）进行测序。通过iPhyClassifier软件分析核苷酸序列（GenBank登录号HQ599228），其与“Ca. Phytoplasma asteris”参考菌株（GenBank登录号M30790）的序列同一性为99.53%。RFLP相似性与16Sr I组B亚组的参考模式（GenBank登录号NC_005303）相同（系数1.00）。据我们所知，这是首次揭示中国与翠菊黄化病害马铃薯相关的植原体分子特征的报告。参考文献：（1）H. Cai等人，《植物保护》31:38，2005年。（2）I.-M. Lee等人，《国际系统细菌学杂志》48:1153，1998年。（3）I.-M. Lee等人，《国际系统与进化微生物学杂志》54:337，2004年。（4）W. Wei等人，《植物病害》91:461，2007年。
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