Identification and characterization of a new bacterial disease of creeping bentgrass (Agrostis stolonifera L.) caused by Acidovorax avenea subsp. Avenae

A new bacterial disease was discovered in 2009 afflicting several creeping bentgrass (CBG) putting greens on a golf course in Charlotte, North Carolina. The reoccurring symptoms caused by this previously uncharacterized disease include chlorosis, etiolation, and a general thinning and decline of irregular shaped areas of turfgrass. Affected plants exhibited heavy bacterial streaming from vascular tissues when viewed microscopically. Initial isolation, pathogenicity tests and biochemical and molecular identification assays determined the causal agent to be the Gram-negative, aerobic, non-fluorescent member of the beta-proteobacteria Acidovorax avenae subsp. avenae (Aaa). Hosts range inoculations showed the Aaa to be pathogenic on all cultivars of CBG with little to no pathogenicity on other hosts tested including several species of amenity turfgrass. Solicitation of symptomatic samples from golf courses between 2010-2013 confirmed Aaa-associated outbreaks of etiolation and decline on more than 30 different sites in 13 states. Inoculations of Aaa suspensions on CBG under a range of temperatures showed significant necrosis above 25°C and severe decline between 30°-35°C with high relative humidity. Root inoculation assays confirmed systemic movement of the turfgrass pathogen and the association of creeping bentgrass etiolation with Aaa infection. Controlled environment experiments with oxytetracycline and streptomycin sulfate significantly reduced symptoms of bacterial decline when pretreated onto plants as foliar spray suspensions prior to inoculation with Aaa. Field studies with inoculations of Aaa induced etiolation and showed the growth regulator trinexapac-ethyl (TE) to significantly increase symptoms when compared to an untreated control. Additionally, ammonium sulfate and the combination of TE + ammonium sulfate consistently increased disease incidence in field plots. No treatment consistently suppressed symptoms of etiolation. Genetic investigations into Aaa using a previously designed Multilocus sequence analysis (MLSA) scheme show turfgrass isolates forming two distinct clades among other closely related Acidovorax spp. Whole genome sequencing of 12 representative isolates among the two turfgrass clades and three other Acidovorax isolates from different plant hosts allowed for the alignment-free design of discriminatory polymerase chain reaction (PCR) primer sets from draft genome assemblies. Predicted diagnostic primer sets were validated against a panel of genomic DNA from 21 target Aaa isolates and 16 non-target bacterial species via traditional PCR. Two primer sets, 0017 and 0019, showed the desired specificity to turfgrass isolates with 100% amplification accuracy. Probes were designed for the primer sets for use in real-time quantitative PCR assay development. For both primer sets, TaqMan and Zen probe assays were designed. Sensitive and specific detection of target organisms was accomplished from purified genomic DNA. Confirmation of detection specificity and sensitivity was accomplished from frozen samples of affected and unaffected turfgrass tissue. This assay allows for rapid and reliable diagnosis of Acidovorax avenae from turfgrass samples, and sets the stage for ecological and epidemiological research related to pathogen detection and disease proliferation.