Title: Development and evaluation of a TaqMan Real-Time PCR assay for Fusarium oxysporum f. sp. spinaciae Authors
Submitted to: Plant Disease
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: May 20, 2013
Publication Date: July 20, 2013
Citation: Okubara, P.A., Harrison, L.A., Gatch, E.W., Schroeder, K.L., Vandemark, G.J., Du Toit, L.J. 2013. Development and evaluation of a TaqMan Real-Time PCR assay for Fusarium oxysporum f. sp. spinaciae. Plant Disease. 97(7):927-937. Interpretive Summary: The Pacific Northwest of the United States (PNW) is highly suitable for spinach seed production, and Washington and Oregon produce up to 50% of the US and 25% of the annual world spinach seed supply. However, the regional acidic soils are highly conducive to Fusarium wilt of spinach caused by Fusarium oxysporum f. sp. spinaciae, a soilborne pathogen that is becoming the primary limiting factor for spinach seed production in the PNW. We have developed a rapid and sensitive molecular diagnostic assay for the detection of Fusarium oxysporum f. sp. spinaciae in soil and seed samples. This will provide a critical tool for evaluation of management strategies for spinach wilt and planting risks for grower.
Technical Abstract: Fusarium oxysporum f. sp. spinaciae, causal agent of spinach Fusarium wilt, is an important soilborne pathogen in many areas of the world where spinach is grown. The pathogen is persistent in acid soils of maritime western Oregon and Washington, the only region of the USA suitable for commercial spinach seed production. A TaqMan real-time PCR assay was developed for rapid identification and quantification of the pathogen, based on sequencing the intergenic spacer (IGS) region of rDNA of isolates of the pathogen. A guanine single nucleotide polymorphism (G SNP) was detected in the IGS sequences of 36 geographically diverse isolates of F. oxysporum f. sp. spinaciae, but not in the sequences of 64 isolates representing other formae speciales and 33 isolates representing other fungal species or genera. The SNP was used to develop a probe for a real-time PCR assay. The real-time PCR assay detected a range of 3 to 14,056 CFU F. oxysporum f. sp. spinaciae/g soil in 82 soil samples collected over three years from naturally infested spinach seed production sites in western Washington, although a reliable detection limit of the assay was determined to be 11 CFU F. oxysporum f. sp. spinaciae/g soil. A significant (P <0.05), positive correlation between enumeration of F. oxysporum on Komada’s agar and quantification of the pathogen using the TaqMan assay was observed in a comparison of 82 soil samples. Correlations between pathogen DNA levels, Fusarium wilt severity ratings, and spinach biomass were significantly positive for one set of naturally-infested soils, but not between pathogen DNA levels, wilt incidence ratings, and spinach biomass for other soil samples, suggesting that soilborne pathogen population is not the sole determinant of spinach Fusarium wilt incidence or severity. The low incidence of the G SNP detected in one isolate of each of F. oxysporum ff. spp. lageneriae, lilii, melongenae, and raphani, and reaction of the real-time PCR assay with 16 of 22 non-pathogenic isolates of F. oxysporum associated with spinach plants or soil in which spinach had been grown potentially limits the application of this assay. The high level of detection by the real-time PCR assay of non-pathogenic F. oxysporum isolates associated with spinach suggests that isolates of F. oxysporum f. sp. spinaciae may be more closely related to non-pathogenic isolates of this species than to isolates of other formae speciales, and that some isolates of the spinach pathogen may have evolved from non-pathogenic isolates or vice versa. Nonetheless, since all isolates of F. oxysporum f. sp. spinaciae tested positive with the real-time PCR assay, the assay may provide a valuable means of screening for resistance to Fusarium wilt by quantifying development of the pathogen in spinach plants inoculated with the pathogen.