First Report of Xanthomonas translucens Causing Etiolation on Creeping Bentgrass Turf in Illinois, Kentucky, and North Carolina.

Symptoms of etiolation, which is an abnormal elongation and yellowing of tillers, have been observed on creeping bentgrass [Agrostis stolonifera L. (CBG)] putting greens for decades; however, symptoms are typically transient and non-problematic. Reports of etiolation have become more frequent recently and research supports the involvement of bacteria (1). During stressful summer periods in 2011 and 2012, 62 CBG putting green samples were submitted to the NCSU Turf Clinic exhibiting symptoms of etiolation, chlorosis, and/or general decline. Microscopic examination of stem and leaf tissue often showed bacterial streaming from the xylem tissue. Symptomatic tissue was surface disinfested in sodium hypochlorite (10% Clorox) for 5 min, blotted dry, and rinsed in sterile dHO. Disinfested tissue was placed in a small drop of sterile dHO on a glass microscope slide and cut to allow bacteria to stream into the water for 2 min. The resulting bacterial suspension was streaked onto three nutrient agar (NA) plates and incubated at 30°C overnight. Bacterial colonies varied in morphology and those present in the greatest number based on morphology were re-streaked to isolate individual colonies. Bacterial isolates were tentatively identified to species using rDNA sequencing of 16S and ITS regions (3). Sequencing results showed isolates obtained from 6 locations (in Illinois, Kentucky, and North Carolina) having a positive match (≥99% 16S and ≥93% ITS) to Xanthomonas translucens (GenBank accessions AY572961, HM181927, JX976312, AY253329, and AB680445). Additional research is needed to confirm pathovar designation as X. translucens isolates were similar to both poae and graminis pathovars. A representative isolate (LW10-12A) was also examined for carbon source utilization using the BIOLOG 3rd Gen Microplate (Biolog Inc., Hayward, CA) resulting in a positive identification of X. translucens. Isolate LW10-12A was used to inoculate 6-week-old seeded creeping bentgrass cv. A1 plants maintained at 1 cm height in 3.5 cm diameter containers. Scissors were dipped in a cell suspension (~10 CFU ml in sterile dHO) and used to cut healthy CBG plants at 1 cm height and the remaining suspension was applied to the foliage until runoff using an atomizer bottle. Non-inoculated plants were cut and misted using sterile dHO. After inoculation, plants were placed in a sealed clear plastic Camwear container (Cambro Co., Huntington Beach, CA) for 48 h and then transferred to the growth chamber bench (30°C) receiving irrigation twice daily with dHO. Etiolation was rated within each of the four replicates by counting the number of etiolated leaves that were easily observed as significantly higher than the rest of the turf canopy. Plants inoculated with X. translucens exhibited etiolation of the youngest leaf within 48 h, whereas the non-inoculated plants did not. Symptoms were similar to observations in the field, as etiolated leaves were chlorotic and easily extracted from the turf surface. Microscopic examination showed bacterial streaming and identification of bacteria, using the previously described methods, was positive for X. translucens. Etiolation symptoms persisted over multiple weeks, but a decline in turf quality was not observed. Etiolation has been previously suggested as a precursor to bacterial wilt, caused by X. translucens pv. poae, on annual bluegrass [Poa annua L. f. reptans (Hausskn) T. Koyama] (2) and Acidovorax avenae has also been shown to produce etiolation on CBG (1). To our knowledge, this is the first confirmation of X. translucens as a cause of etiolation in CBG. References: (1) P. R. Giordano et al. Plant Dis. 96:1736, 2012. (2) N. A. Mitkowski et al. Plant Dis. 89:469, 2005. (3) N. W. Schaad et al. Lab. Guide for Ident. of Plant Path Bac., 2001.