First report of bacterial spot caused by Robbsia andropogonis in Loropetalum chinense

In the Southern United states, loropetalum (Loropetalum chinense Oliv. var. rubrum) is a popular evergreen shrub (Feng et al. 1999). The foliage varies in color from red to burgundy depending on the cultivar and environmental conditions. Plants are utilized as specimen plants or as hedges. In 2017, leaf spots were observed on loropetalum plants in multiple production facilities in Central Florida. The leaf spots were dark and water soaked, and when plants were heavily infested leaf drop occurred. Isolations on nutrient agar from symptomatic tissue consistently yielded smooth, cream-colored colonies of gram-negative bacteria. Two isolates of the bacterium, P845 and P846, were selected for further identification and characterization. Strains were first characterized by using fatty acid methyl ester analyses with the MIDI identification system (Sherlock version 6.2B, Microbial ID, Newark, DE). The two strains had similarity indexes of 0.944 and 0.958, respectively, to Robbsia andropogonis (formerly Burkholderia andropogonis). PCR amplifications of a partial 16S rRNA gene with universal primers 27F/1492R (Chen et al. 2015), gyrB gene with LJ23f/LJ24r, and rpoD gene with LJ25f/LJ26r (Duan et al. 2009) were sequenced. All sequences for both strains were identical with a 100% sequence identity to R. andropogonis. The 16S rDNA, gyrB, and rpoD sequences of P845 were deposited in NCBI GenBank under accession numbers MK063729, MK071276, and MK071277, respectively. To satisfy Koch’s postulates, both strains were inoculated onto loropetalum ‘Plum Delight’ and sorghum ‘WGF’. Loropetalum plants were approximately 15 cm in height with 25 to 50 leaves, whereas sorghum plants consisted of community pots with 20 to 30 seedlings, approximately 10 cm in height. Inoculations were done in a greenhouse with temperatures between 21 and 32°C and light levels between 9,688 and 12,917 lm/m². Suspensions of each strain at a concentration of 1 × 10⁸ CFU/ml were sprayed on plants to runoff. Plants were bagged to raise humidity and incubated for 24 h. A 150 mM NaCl buffer solution and R. andropogonis type strain ATCC 23061 were used as negative and positive controls, respectively. The experiment was repeated three times. Within 2 weeks, leaf spots similar to the original ones observed developed on both hosts with all three bacterial strains, whereas the buffer control inoculated plants were asymptomatic. Identical colonies were isolated from the spots and sequenced as described above. The sequences were identical to R. andropogonis deposited in NCBI GenBank. To our knowledge, this is the first report of R. andropogonis infecting and causing leaf spot on L. chinense.