Pan Y-B, Grisham M P, Burner D M, Damann K E, Wei Q
USDA-ARS, Southern Regional Research Center, Sugarcane Research Unit, P.O. Box 470, Houma, LA 70361.
Department of Plant Pathology and Crop Physiology, Agricultural Center, Louisiana State University, Baton Rouge 70803.
Plant Dis. 1998 Mar;82(3):285-290. doi: 10.1094/PDIS.1998.82.3.285.
A polymerase chain reaction (PCR) protocol was developed that specifically detected Clavibacter xyli subsp. xyli, the causal agent of sugarcane ratoon stunting disease. Generic PCR products from the intergenic transcribed spacer (ITS) region of 16S-23S ribosomal DNA of C. xyli subsp. xyli and C. xyli subsp. cynodontis were cloned and sequenced. Based on a multiple sequence alignment among these two sequences and other nonredundant highly homologous sequences from the database, two C. xyli subsp. xyli-specific PCR primers were designed, Cxx1 (5' CCGAAGTGAGCAGATTGACC) and Cxx2 (5' ACCCTGTGTTGTTTTCAACG). These two 20-mer oligonucleotides primed the specific amplification of a 438-bp DNA product from genomic DNA samples of 21 C. xyli subsp. xyli strains. Amplification was not observed with genomic DNA of one C. xyli subsp. cynodontis strain, five strains of four other Clavibacter species, and two strains of two Rathayibacter species. The 438-bp PCR product also was amplified directly from cultured C. xyli subsp. xyli cells and from C. xyli subsp. xyli-infected sugarcane vascular sap with a unique reaction buffer containing polyvinylpyrrolidone and ficoll. Extraction of genomic DNA was not necessary prior to PCR assay.
开发了一种聚合酶链反应（PCR）方案，可特异性检测甘蔗宿根矮化病的病原体——木糖短小杆菌木糖亚种。对木糖短小杆菌木糖亚种和犬齿木糖短小杆菌16S - 23S核糖体DNA基因间隔转录区（ITS）的通用PCR产物进行了克隆和测序。基于这两个序列与数据库中其他非冗余高度同源序列的多序列比对，设计了两条木糖短小杆菌木糖亚种特异性PCR引物，即Cxx1（5' CCGAAGTGAGCAGATTGACC）和Cxx2（5' ACCCTGTGTTGTTTTCAACG）。这两条20聚体寡核苷酸引物能从21株木糖短小杆菌木糖亚种的基因组DNA样本中特异性扩增出一条438 bp的DNA产物。一株犬齿木糖短小杆菌、其他四种短小杆菌属的五株菌株以及两种 Rathayibacter 属的两株菌株的基因组DNA均未观察到扩增现象。438 bp的PCR产物也可直接从培养的木糖短小杆菌木糖亚种细胞以及感染了木糖短小杆菌木糖亚种的甘蔗维管束汁液中，使用含有聚乙烯吡咯烷酮和聚蔗糖的独特反应缓冲液进行扩增。在进行PCR检测之前无需提取基因组DNA。
