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.

Dollar spot (DS) (Sclerotinia homoeocarpa F.T. Bennett) is an important disease on golf course turfgrasses in the United States. The objective of this study was to elucidate disease reduction mechanisms related to lightweight rolling. Creeping bentgrass (Agrostis stolonifera L.) plots that were subjected to daily (5 d wk−1) rolling treatments (1x a.m., 1x p.m., 2x a.m.) showed significant DS reductions when compared with a nonrolled control. Treatments rolled in the afternoon (after dew and guttation fluid dissipated) exhibited similar area under disease progress curve (AUDPC) values as treatments rolled in the morning in 2008, 2009, and 2010. Both 1x rolling treatments resulted in significant DS reductions compared with the control in 2009 and 2010. The 2x d−1 treatment exhibited significantly less DS than the control in all years as well as significantly lower AUDPC values than both 1x rolling treatments in 2009 and 2010. Rolling 2x d−1 improved turfgrass quality compared with all other treatments in each year of the study. All rolling treatments exhibited higher root zone volumetric water content (%VWC) compared with the control in 2010, and significant effects on %VWC were observed on individual dates among treatments. Results indicate previously unidentified effects of rolling on putting greens, including added physical and biological effects, which may be contributing to DS suppression.

Giordano, P. R., Chaves, A. M., Mitkowski, N. A., and Vargas, J. M., Jr. 2012. Identification, characterization, and distribution of Acidovorax avenae subsp. avenae associated with creeping bentgrass etiolation and decline. Plant Dis. 96:1736-1742.

In June of 2009, a golf course putting green sample of creeping bentgrass (Agrostis stolonifera L.) cv. Penn G-2 from a golf club in North Carolina was submitted to the Michigan State University Turfgrass Disease Diagnostic Laboratory for diagnosis. The sample exhibited symptoms of general wilt, decline, and characteristic necrosis from the leaf tips down. Fungal pathogens were ruled out when no phytopathogenic fungal structures were observed with microscopic examination of infected tissue. Symptoms appeared similar to those of annual bluegrass affected by bacterial wilt caused by Xanthomonas translucens pv. poae. Bacterial streaming was present in all of the cut infected tissue of the Penn G-2 bentgrass sample when observed with a microscope. To isolate the causal agent, cut leaf tissue (1- to 3-mm tips) exhibiting bacterial streaming was surface disinfected for 1 min in 10% sodium hypochlorite solution and rinsed for 1 min with sterile distilled water. Leaf blades were placed into Eppendorf microtubes with 20 μl of sterile phosphate-buffered saline (PBS) solution (pH 7) and macerated with a sterile scalpel. Serial dilutions up to 1 × 10–4 were performed in sterile PBS; 10 μl of each suspension was plated onto nutrient agar (NA) (Becton Dickinson, Sparks, MD) and incubated at room temperature for 5 days. Pure cultures of three commonly observed single bacterial colonies growing on plates from serial dilutions were made on NA medium. These pure cultures were grown for 5 days and used to inoculate three replicates of 5-week-old Penn G-2 plants that had uniformly filled in 8.5-cm-diameter pots grown under greenhouse conditions. Uninfected Penn G-2 creeping bentgrass plants were inoculated with 1 ml of 1.3 × 109 CFU/ml of bacterial suspension by adding drops of the suspension to blades of sterile scissors used to cut the healthy plants. Of the three different bacterial cultures selected to inoculate healthy plants, only one resulted in slight browning of leaf tips just 2 days after inoculation. The symptoms progressed, and by 5 days after inoculation, browning, twisting and leaf dieback to the sheath were observed. When leaf tips of the inoculated plants were cut, bacterial streaming was observed. Isolation of the bacterium from inoculated Penn G-2 plants was performed to fulfill Koch's postulates. Once isolated, a single bacterial colony was identified by 16S rDNA sequencing (Microcheck Inc. Northfield, VT). 16S rDNA sequencing results indicated that the causal agent of bacterial infection was a member of the Acidovorax genus, with a 100% sequence match to Acidovorax avenae subsp. avenae (2). The same nonflorescent, aerobic, gram-negative bacterium has been consistently isolated from inoculated plants exhibiting symptoms thus far. A member of the Acidovorax genus has also been identified as a pathogen of creeping bentgrass in Japan (1). To our knowledge, this is the first report of a bacterial disease affecting creeping bentgrass caused by Acidovorax spp. in the United States. References: (1) N. Furuya et al. J. Fac. Agric. Kyushu Univ. 54:13. 2009. (2) N. Schaad et al. Syst. Appl. Microbiol. 31:434. 2008.

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.