Development and Validation of a Real-Time PCR Assay for the Quantification of Verticillium dahliae in Potato

Pasche, J. S., Mallik, I., Anderson, N. R., and Gudmestad, N. C. 2013. Development and validation of a real-time PCR assay for the quantification of Verticillium dahliae in potato. Plant Dis. 97:608-618. 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 russetskinned 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. Verticillium wilt in U.S. potato (Solanum tuberosum L.) production is caused largely by Verticillium dahliae Kleb., although V. albo-atrum Reinke & Berthold may be associated with the disease in cooler climates (15,60). V. dahliae, either alone or in conjunction with other fungal pathogens, including Fusarium spp. and Colletotrichum coccodes (Wallr.) S. Hughes, and the root-lesion nematode Pratylenchus penetrans (Cobb) Sher & Allen, may be the primary cause of economic losses in the potato industry in the United States (12,15,49,56,59). This is due not only to yield reductions but also to losses in tuber size and quality from vascular discoloration, as well as losses from the cost of control, primarily soil fumigation using metam sodium (12,27). Symptoms of Verticillium wilt begin to appear at the base of the plant and move acropetally as the season progresses (59,62). The symptoms of general plant wilting can be impossible to differentiate from natural senescence. However, unilateral wilting, the result of differential colonization of individual vascular bundles, and the erect posture of dead plants infected with V. dahliae are diagnostic of Verticillium wilt (14,62). Discoloration often is present in stem as well as tuber vascular tissue. Microsclerotia are the main infective propagules of V. dahliae under field conditions and are reported to survive in the soil for up to 14 years (69). Microsclerotia germinate in the presence of susceptible host plants to infect near the zone of elongation of root tips (6,26). Any movement of soil into a field subsequently may result in the introduction of the pathogen into noninfested soil (58,62). Initial introductions of V. dahliae into fields often are isolated but, when susceptible potato cultivars are grown, infected stem tissue serves as inoculum for subsequent crops, returning microsclerotia to the soil after harvest (62). In potato, 100% stem infection was recorded at soil infestation levels of 6 to 10 microsclerotia/g in Wisconsin (49), whereas, 8 and 18 to 23 microsclerotia/g of soil have been documented as the economic thresholds in Idaho and Colorado, respectively (17,50). Although the relationship between disease incidence and microsclerotial concentration differ among cultivars, environmental conditions, and cropping practices, the reduction of microsclerotia in the soil is paramount in mitigating the effects of this monocyclic pathogen in the absence of genetic resistance (5,70). Several factors have led to the increased importance of Verticillium wilt, including reduced tillage and shorter rotations between potato crops, higher value placed on tuber quality, and an increase in use restrictions of soil fumigants—most recently, metam sodium (12,38,63). Soil fumigation using metam sodium is effective and widely used but also is expensive and detrimental to the environment (12,38,58). Environmental Protection Agency restrictions on the application of metam sodium are mounting and will result in limiting usage by growers (38). Verticillium wilt management also involves reducing soil pathogen populations as well as limiting the effect the pathogen has on the host. Control measures which focus on reducing populations in the soil, in addition to soil fumigation, include crop rotation, tillage, and solarization (12,17,58,63,66). Control measures that focus on minimizing infection and the effects of the pathogen on the host include nutrient and water management, as well as the use of resistant cultivars (12,18,58). Because of the general lack of resistance in commonly grown cultivars, some cultural management practices can aid in decreasCorresponding author: N. C. Gudmestad, E-mail: [email protected] Accepted for publication 4 December 2012. http://dx.doi.org/10.1094 / PDIS-06-12-0554-RE © 2013 The American Phytopathological Society