Using a Genome-Based PCR Primer Prediction Pipeline to Develop Molecular Diagnostics for the Turfgrass Pathogen Acidovorax avenae

Abstract

Acidovorax avenae is the causal agent of bacterial etiolation and decline (BED) of creeping bentgrass, a poorly understood and often misdiagnosed disease that can result in considerable aesthetic and functional damage to golf course putting greens. Current diagnostics of BED are based on laborious culture-based methods. In this work, we employed a novel alignment-free primer prediction pipeline to design diagnostic primers for turfgrass-pathogenic A. avenae using 15 draft genomes of closely related target and nontarget Acidovorax spp. as input. Twenty candidate primer sets specific to turfgrass-pathogenic A. avenae were designed. The specificity and sensitivity of these primer sets were validated via a traditional polymerase chain reaction (PCR) and a real-time PCR assay. Primer sets 0017 and 0019 coupled with an internal oligo probe showed optimal sensitivity and specificity when evaluated with the target pathogen, closely related bacterial species, and microorganisms that inhabit the same host and soil environment. Finally, the accuracy of the newly developed real-time PCR assay was evaluated to detect BED pathogens from BED-symptomatic and asymptomatic turfgrass samples. The diagnostic results produced by the real-time PCR assay were consistent with results of a cultural-based method. This assay will allow quicker and more effective detection of the BED pathogen, thus potentially reducing misdiagnoses and unnecessary usage of fungicides.

A one‐step multiplex PCR assay for the detection and differentiation of four species of Clarireedia causing dollar spot on turfgrass

“…Comparative genomics also has enabled specific detection and identification of multiple plant-pathogenic bacteria. 20,[28][29][30] However, genomics-informed methods are rarely used for specific detection and differentiation of plant-pathogenic fungi. Our work illustrates how comparative genome-based strategies can be S3.…”

“…Given the situation that new unidentified species of Clarireedia potentially exist in the field, 4,8 we just need to design species‐specific primers to detect new species by sequencing the genomes of a few isolates representing the new species, and the primers detecting the existing species may not require development of new primers. Comparative genomics also has enabled specific detection and identification of multiple plant‐pathogenic bacteria 20,28–30 . However, genomics‐informed methods are rarely used for specific detection and differentiation of plant‐pathogenic fungi.…”

“…Preliminary work indicates that the different species of Clarireedia harbor hundreds of species‐specific genes (Unpublished data). Genomics‐informed PCR methods have been successfully developed to detect a few plant pathogens, including the turfgrass pathogen Acidovorax avenae , 20 which encourages the development of novel PCR‐based methods to detect and differentiate between the known species of Clarieedia causing DS in China using comparative genomics analyses.…”