Abstract

Quantification of soilborne pathogen inoculum loads is important in both agricultural and wildland settings. Quantitative Polymerase Chain Reaction (qPCR) methods using SYBR Green chemistry have been shown to be useful for quantifying fungal inoculum loads in environmental samples. The purpose of this study was to develop a method to quantify fungal pathogen inoculum loads in soil seed banks using a qPCR method with SYBR Green chemistry. The invasive annual grass Bromus tectorum was chosen for this seed bank study. There were three objectives: 1) to design target-specific primers for three fungal pathogens known to be important in Bromus tectorum seed banks, 2) to develop a procedure for measuring inoculum loads in field samples, including optimization of qPCR standard curves and protocols, for these pathogens, and 3) to perform qPCR using this methodology on a representative set of field samples to quantify pathogen DNA in seed bank soil and surface litter. The three pathogens were chosen for quantification based on their hypothesized roles in Bromus tectorum stand failure: the seed pathogen Pyrenophora semeniperda, an undescribed species of Fusarium seed rot pathogen belonging to the F. tricinctum species group (FTSG), and the newly-described causal agent of bleach blonde syndrome (Clarireedia capillus-albis). Primers designed for each pathogen were shown to be target-specific in tests against each other and 12 other fungal species cultured from B. tectorum seed banks. Subsequently developed standard curves for each pathogen had R2 values > 0.98, efficiencies between 90 and 110 percent, and generally optimal dissociation curves. Inoculum loads were expressed for each pathogen as picograms of DNA per microliter of extracted soil or surface litter. Significant differences in measured inoculum loads were found between the targeted pathogens and between soil and litter samples for each pathogen. The data provided reinforces that the SYBR Green qPCR method provides a potentially useful tool for the study of field seed and seedling diseases across a wide spectrum of both wildland and agronomic applications.

Degree

College and Department

Life Sciences; Plant and Wildlife Sciences

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