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

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.

Recombination of Virulence Genes in Divergent Acidovorax avenae Strains That Infect a Common Host.

Bacterial etiolation and decline (BED), caused by Acidovorax avenae, is an emerging disease of creeping bentgrass on golf courses in the United States. We performed the first comprehensive analysis of A. avenae on a nationwide collection of turfgrass- and maize-pathogenic A. avenae. Surprisingly, our results reveal that the turfgrass-pathogenic A. avenae in North America are not only highly divergent but also belong to two distinct phylogroups. Both phylogroups specifically infect turfgrass but are more closely related to maize pathogens than to each other. This suggests that, although the disease is only recently reported, it has likely been infecting turfgrass for a long time. To identify a genetic basis for the host specificity, we searched for genes closely related among turfgrass strains but distantly related to their homologs from maize strains. We found a cluster of 11 such genes generated by three ancient recombination events within the type III secretion system (T3SS) pathogenicity island. Ever since the recombination, the cluster has been conserved by strong purifying selection, hinting at its selective importance. Together our analyses suggest that BED is an ancient disease that may owe its host specificity to a highly conserved cluster of 11 T3SS genes.

Identification, Characterization, and Distribution of Acidovorax avenae subsp. avenae Associated with Creeping Bentgrass Etiolation and Decline.

Bacterial etiolation and decline caused by Acidovorax avenae subsp. avenae is an emerging disease of creeping bentgrass (Agrostis stolonifera) in and around the transition zone, a unique area of turfgrass culture between cool and warm regions of the United States. It is suspected that the disease has been present for many years, although diagnosis of the first occurrence was not reported until 2010. Solicitation of samples from golf courses in 2010 and 2011 was undertaken to investigate the prevalence and dissemination of Acidovorax avenae subsp. avenae on creeping bentgrass. At least 21 isolates from 13 states associated with these outbreaks on golf courses were confirmed as A. avenae subsp. avenae by pathogenicity assays and 16S rDNA sequence analysis at two independent locations. Pathogenicity testing of bacterial isolates from creeping bentgrass samples exhibiting heavy bacterial streaming confirmed A. avenae subsp. avenae as the only bacterium to cause significant disease symptoms and turfgrass decline. Host range inoculations revealed isolates of A. avenae subsp. avenae to be pathogenic on all Agrostis stolonifera cultivars tested, with slight but significant differences in disease severity on particular cultivars. Other turfgrass hosts tested were only mildly susceptible to Acidovorax avenae subsp. avenae infection. This study initiated research on A. avenae subsp. avenae pathogenicity causing a previously uncharacterized disease of creeping bentgrass putting greens in the United States.