Quantification and determination of inoculum threshold levels of Fusarium commune in Douglas-fir nurseries

Damping-off of Douglas-fir seedlings by Fusarium/italic> commune is a major problem in conifer nurseries in the Pacific Northwest. A more thorough understanding of the quantity and identity of soil-borne pathogens is needed in order to reduce use of these increasingly restricted fumigants. A quantitative real-time PCR (qPCR) assay was developed to quantify F. commune and distinguish it from F. oxysporum, a less virulent and morphologically indistinguishable species. Standard curves designed with pure culture isolates had efficiencies of r2 = 0.9910 and r2 = 0.9801 for F. commune and F. oxysporum, respectively. Correlations between the average colony-forming-units per gram (CFU g-1), the current method of quantification, and the cycle threshold (Ct) value of the F. commune qPCR assay ranged from r2 = 0.5153 to 0.9871. The quantity of DNA (ng/microl) calculated from the F. commune qPCR assay was correlated with the average seedling mortality with r2 = 0.3715 and r2 = 0.4398, respectively (p < 0.001). Results of this study suggest that a qPCR assay for F. commune has the potential to determine the quantity of the pathogen in nursery soils. Disease thresholds for Fusarium/italic> spp. in Douglas-fir nurseries have been speculated upon but not published. Greenhouse inoculum threshold trials were established to determine the mortality threshold for Douglas-fir seedlings. Soil should be managed below a level of approximately 500 CFU g-1 F. commune and 1.00 ng/microl F. commune DNA to keep Douglas-fir damping off below 5%.;Trials were established to determine the disease threshold level in field nursery beds and to validate the qPCR assay using infested field nursery soil. While most isolates had significant, but weak correlations (p < 0.10) between the observed average CFU g-1 and the logarithmically transformed quantity of DNA (ng/microl) (r2 = 0.2749 - 0.5467), the DNA quantity was not significantly correlated with mortality. Furthermore, seedling shoot height and caliper were generally highest in the non-treated control plots, but there were very few differences between added inoculum concentrations. This study was subject to several external factors and should be repeated to determine if the qPCR assay can be used as a field diagnostic tool.