A New TaqMan Real-Time Polymerase Chain Reaction Assay for Quantification of Fusarium virguliforme in Soil

Mbofung, G. C. Y., Fessehaie, A., Bhattacharyya, M. K., and Leandro, L. F. S. 2011. A new TaqMan real-time polymerase chain reaction assay for quantification of Fusarium virguliforme in soil. Plant Dis. 95:1420-1426. The quantification of the soilborne pathogen Fusarium virguliforme inoculum in soil is important for epidemiological studies of soybean sudden death syndrome (SDS). Classical dilution plating methods to determine inoculum density in soil have yielded inconsistent results due to slow growth, variable colony morphology of the pathogen, and the presence of other fungi with similar phenotype. A TaqMan realtime polymerase chain reaction assay was developed based on sequences of the FvTox1 gene of F. virguliforme. The gene differed by four single-nucleotide proteins from the other SDS-causing species. Assay specificity was tested on 48 fungal isolates that varied in taxonomic relatedness. Assay sensitivity was appraised on 10-fold serial dilutions of genomic DNA, conidia suspensions, and soil spiked with conidia. Applicability of the assay was evaluated on field and greenhouse soil samples, and on roots of symptomatic plants. The assay detected only DNA sequences specific to F. virguliforme. The detection limit of the assay was 5 pg/μl, 1,000 conidia/ml, and 1,000 conidia/g soil for genomic DNA, conidial suspensions, and soil with conidia, respectively. The assay was specific to F. virguliforme and was used successfully to quantify inoculum density in soil and soybean roots. The assay can be used as a diagnostic tool for rapid screens of field and greenhouse soil, and for symptomatic and asymptomatic plants. Fusarium virguliforme O’Donnell & T. Aoki is a soilborne fungus that infects soybean roots and causes root rot, crown rot, and vascular discoloration (20). When the fungus colonizes xylem tissues in roots, the toxins it produces are translocated to leaves, where they cause interveinal chlorosis, necrosis (or leaf scorch), premature defoliation, and flower and pod abortion (21,26). Collectively, these symptoms are referred to as sudden death syndrome (SDS) of soybean. This economically important disease is widely distributed throughout the soybean-producing regions of the United States (26), with estimated yield losses ranging from 11 to 75 million bushels per year between 1997 and 2007 (37). In recent years, SDS has caused particular concern in the Midwest, where outbreaks have been more severe (21) and several states have reported the disease for the first time (5,8,17,41). SDS also occurs in South America, where it is caused by F. tucumaniae T. Aoki, O’Donnell, Yosh. Homma & Lattanzi; F. brasiliense T. Aoki & O’Donnell; and an undescribed Fusarium sp. in addition to F. virguliforme, which is the only species known to cause SDS in the United States (1,25). The mechanisms of long-distance spread of F. virguliforme are unclear but dispersal likely involves movement of soil, where the pathogen overwinters in the form of chlamydospores on plant debris and associated with the soybean cyst nematode (Heterodera glycines Ichinohe) (20,26). Chlamydospores, together with conidia and mycelia, may serve as inoculum for infection in subsequent growing seasons. Studies on pathogen distribution and inoculum density in soybean fields have been limited (28,29,32,35) due to the lack of practical methods to specifically detect and quantify F. virguliforme in soil. A tool to accurately and specifically quantify F. virguliforme in soil is needed to clarify the role of inoculum density in disease progress, facilitate SDS risk assessments in soybean fields, and provide insights about mechanisms of pathogen spread. SDS management is challenging and requires the use of multiple approaches, with the selection of resistant varieties being of primary importance (26). However, soybean varieties characterized as resistant to F. virguliforme exhibit partial resistance because resistance is multigenic (16,30), and resistance in some varieties can be overcome by high inoculum concentrations (2,14). Furthermore, screening for resistance to SDS in greenhouse assays does not consistently predict mature-plant responses in field conditions (26,34), possibly due, in part, to differences in inoculum levels in greenhouse versus field studies. Because SDS development in field conditions is highly dependent on environmental factors (26,31), accurate quantification of F. virguliforme in soil could help to improve reliability of resistance screening assays. Soil dilution plating methods have been used to quantify F. virguliforme propagules in soil (28,29,32) but these methods are time consuming, laborious, and often underestimate pathogen density. Isolation is further compounded by fast-growing fungi that can overrun culture plates containing the very slow-growing F. virguliforme (26). Dilution-plate methods are also unsuitable for processing numerous samples that would be needed to assess SDS risk based on inoculum densities in soybean fields. In contrast, realtime polymerase chain reaction (PCR) can be used to quantify density of fungal propagules in soil (7,11,36) and may be applicable for studies of soilborne inoculum levels of F. virguliforme. One of the challenges with quantification of F. virguliforme in field soil is the presence of closely related Fusarium spp. that are ubiquitous in soil (22). Published real-time PCR assays (12,18) for F. virguliforme were based on the mitochondrial small subunit (mtSSU) rDNA region. However, primers developed from this region also amplify DNA from other SDS-causing Fusarium spp., as well as DNA from some F. solani (Mart.) Sacc. strains that do not cause SDS but have been isolated from soybean and corn roots in Iowa (unpublished data). The published primers were developed prior to recent findings that the mtSSU locus is highly conserved and unable to resolve species boundaries within the SDS-bean root rot (BRR) clade of the F. solani species complex (1,25), which groups the four SDS-causing species with a bean root rot F. phaseoli species. Genetic loci that delimit species boundaries within the SDSBRR clade would be useful for the design of species-specific primCorresponding author: L. L. Leandro, E-mail: [email protected] Accepted for publication 16 June 2011. doi:10.1094 / PDIS-02-11-0120 © 2011 The American Phytopathological Society