Xu M L, Melchinger A E, Lübberstedt T
Department of Agronomy, Yangzhou University, 225009 Yangzhou, PR China.
Institute of Plant Breeding, Seed Science, and Population Genetics, University of Hohenheim, D-70593 Stuttgart, Germany.
Plant Dis. 1999 Apr;83(4):390-395. doi: 10.1094/PDIS.1999.83.4.390.
Head smut of maize, caused by Sporisorium reiliana, may substantially reduce grain yield. The objective of the present study was to develop a highly specific and sensitive DNA-based assay for detection of S. reiliana and its differentiation from Ustilago maydis, a maize fungus inducing the symptomatically similar common smut disease. Plasmid libraries of S. reiliana and U. maydis were constructed using a shotgun cloning procedure. Clones containing strongly hybridizing species-specific DNA were selected by screening libraries with their own labeled genomic DNA, followed by cross-hybridization with genomic DNA of maize and other maize-pathogenic fungi. The selected clones were used to generate subclones with short insert fragments to facilitate PCR amplification for labeling and primer design for a PCR assay. Using Dig-dUTP labeled inserts, detection of less than 0.16 ng of fungal DNA was possible by dot blot hybridization. Sequences of insert fragments were determined to design primer pairs for a PCR-based assay. Primer pairs SR1 and SR3 are species-specific for S. reiliana, and UM11 is species-specific for U. maydis. The PCR-based assays can detect fungal DNA of less than 1.6 pg using SR1 and SR3, and 8 pg using UM11, irrespective of the presence of maize DNA. Use of SR1 and SR3 allowed detection of S. reiliana in the extracts of pith, node, and shank from S. reiliana-infected plants, but not in leaves. Thus, both the dot blot hybridization and the PCR-based assays provide a highly sensitive and reliable tool for detection and differentiation of corn smut caused either by S. reiliana or by U. maydis.
由玉蜀黍丝轴黑粉菌引起的玉米丝黑穗病会显著降低谷物产量。本研究的目的是开发一种基于DNA的高度特异性和灵敏性的检测方法，用于检测玉蜀黍丝轴黑粉菌，并将其与引起症状相似的玉米普通黑粉病的玉米黑粉菌区分开来。采用鸟枪法克隆程序构建了玉蜀黍丝轴黑粉菌和玉米黑粉菌的质粒文库。通过用自身标记的基因组DNA筛选文库，然后与玉米和其他玉米致病真菌的基因组DNA进行交叉杂交，选择含有强杂交物种特异性DNA的克隆。选择的克隆用于生成具有短插入片段的亚克隆，以促进用于标记的PCR扩增和用于PCR检测的引物设计。使用地高辛-dUTP标记的插入片段，通过斑点杂交可以检测到少于0.16 ng的真菌DNA。确定插入片段的序列以设计基于PCR的检测方法的引物对。引物对SR1和SR3对玉蜀黍丝轴黑粉菌具有物种特异性，UM11对玉米黑粉菌具有物种特异性。无论玉米DNA是否存在，基于PCR的检测方法使用SR1和SR3可以检测到少于1.6 pg的真菌DNA，使用UM11可以检测到8 pg的真菌DNA。使用SR1和SR3可以检测玉蜀黍丝轴黑粉菌感染植株的髓、节和茎提取物中的玉蜀黍丝轴黑粉菌，但不能检测叶片中的。因此，斑点杂交和基于PCR的检测方法都为检测和区分由玉蜀黍丝轴黑粉菌或玉米黑粉菌引起的玉米黑粉病提供了高度灵敏和可靠的工具。
