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.
Isolation of pathogenic bacteria from the skin ulcerous symptomatic gourami (Colisa lalia) through 16S rDNA analysis
