Pasche J S, Mallik I, Anderson N R, Gudmestad N C
Department of Plant Pathology, North Dakota State University, Fargo 58108.
Department of Botany and Plant Pathology, Purdue University, West Lafayette, IN 47907.
Plant Dis. 2013 May;97(5):608-618. doi: 10.1094/PDIS-06-12-0554-RE.
An increase in the stringency for higher quality potato tubers and restrictions on the use of soil fumigants, among other factors, has garnered renewed interest in Verticillium wilt, particularly in russet-skinned cultivars grown for processing. In response to the needs of producers, breeders have increased efforts in the development of potato cultivars with resistance to Verticillium dahliae Kleb., the primary cause of Verticillium wilt. These efforts have resulted in the release of numerous russet-skinned cultivars with purported resistance to the pathogen. However, because efficient and effective methods to screen germplasm for true resistance do not exist, breeders typically have reported resistance based on the development of wilt symptoms alone. The studies reported here demonstrate the efficiency and practicality of a QPCR method for quantification of V. dahliae in potato stem tissue. This method, developed to detect the target trypsin protease gene of the pathogen, was compared with traditional methods for V. dahliae quantification which involve plating stem tissue or sap onto semi-selective media, as well as to a recently developed QPCR assay which amplifies a region of the β-tubulin gene of V. dahliae. The QPCR assay developed in the studies reported here was demonstrated to be sensitive to 0.25 pg of DNA. Use of the duplex real-time PCR assay, utilizing the potato actin gene to normalize quantification, resulted in clearer differentiation of levels of resistance among eight russet-skinned potato cultivars inoculated in greenhouse trials when compared with traditional plating assays. However, relative levels of resistance among cultivars were similar between traditional plating and QPCR methods, resulting in correlation coefficients greater than 0.93. The assay described here also detected the pathogen in inoculated stem tissue at higher frequencies than both traditional plating assays and a previously developed QPCR assay. The QPCR assay developed here demonstrates rapid, efficient, and accurate quantification of V. dahliae, providing a tool amenable for use by breeding programs on large numbers of clones and selections, and will aid researchers evaluating other control strategies for Verticillium wilt.
对更高品质马铃薯块茎的要求日益严格，以及土壤熏蒸剂使用受限等因素，重新引发了人们对黄萎病的关注，尤其是对用于加工的褐皮品种。为满足生产者的需求，育种者加大了对培育抗大丽轮枝菌（Verticillium dahliae Kleb.，黄萎病的主要致病菌）马铃薯品种的投入。这些努力已培育出众多据称对该病原菌具有抗性的褐皮品种。然而，由于不存在高效且有效的方法来筛选真正具有抗性的种质，育种者通常仅依据枯萎症状的发展来报告抗性。本文报道的研究证明了一种用于定量马铃薯茎组织中番茄黄萎病菌的QPCR方法的有效性和实用性。该方法是为检测病原菌的目标胰蛋白酶基因而开发的，与传统的将茎组织或汁液接种到半选择性培养基上进行大丽轮枝菌定量的方法，以及最近开发的一种扩增大丽轮枝菌β - 微管蛋白基因区域的QPCR检测方法进行了比较。本文报道的研究中开发的QPCR检测方法被证明对0.25 pg的DNA敏感。使用双链实时PCR检测方法，利用马铃薯肌动蛋白基因进行定量标准化，与传统接种试验相比，在温室试验中接种的8个褐皮马铃薯品种中，能更清晰地区分抗性水平。然而，传统接种和QPCR方法之间品种的相对抗性水平相似，相关系数大于0.93。本文描述的检测方法在接种的茎组织中检测病原菌的频率也高于传统接种试验和先前开发的QPCR检测方法。本文开发的QPCR检测方法证明了对大丽轮枝菌的快速、高效和准确的定量，为育种项目在大量克隆和选择中使用提供了一种适用工具，并将有助于研究人员评估黄萎病的其他防治策略。
