First report of Robbsia andropogonis causing bacterial leaf spot of bougainvilleas in Taiwan.

Abstract

Bougainvilleas (Bougainvillea spp.) are popular ornamentals commonly grown as bushes, vines, or trees worldwide (Kobayashi et al. 2007). Leaf spot symptoms were observed on a bougainvillea hedge located in North District, Taichung, Taiwan during August of 2022. The lesions were brown, necrotic and had yellow halos (Fig. S1). All the plants at the location showed similar symptoms. Leaf samples were collected from five plants and symptomatic tissues were minced in 10 mM MgCl2. The samples were streaked onto nutrient agar (NA) and after culturing at 28°C for 2 days, small, round, creamy white colonies were consistently isolated from all the samples. A total of five strains (BA1 to BA5) were obtained; each of them was isolated from a different plant. All five strains induced hypersensitive response in tobacco leaves. Amplification and sequencing of the isolated strains' 16S rDNA using primers 27F and 1492R (Lane 1991) revealed that all five strains shared identical sequences (GenBank accession no. OQ053015) with Robbsia andropogonis LMG 2129T (formerly Burkholderia andropogonis and Pseudomonas andropogonis; GenBank accession no. NR104960; 1,393/1,393 bp). Further testing of BA1 to BA5's DNA samples using the pathogen's species-specific primers Pf (5'-AAGTCGAACGGTAACAGGGA-3') and Pr (5'-AAAGGATATTAGCCCTCGCC-3'; Bagsic et al. 1995) successfully amplified the expected 410-bp amplicon in all five samples; the sequences of the PCR products completely matched to those of BA1 to BA5's 16S rDNA. Strains BA1 to BA5 also tested negative for arginine dihydrolase and oxidase activity, and failed to grow at 40°C, all of which are consistent with descriptions of R. andropogonis (Schaad et al. 2001). Pathogenicity of the isolated bacteria was confirmed by spray inoculation. Three representative strains, BA1 to BA3, were used for the assay. Bacterial colonies were scraped from NA plates and suspended in 10 mM MgCl2 added with 0.02% Silwet L-77. The concentrations of the suspensions were adjusted to 4.4-5.8 x 108 cfu/ml. The suspensions were sprayed onto three-month-old, cutting-propagated bougainvillea plants (to runoff). Controls were treated with bacteria-free solutions. Three plants were used for each treatment group (and the controls). The plants were placed in a growth chamber (27/25°C, day/night; 14-hour photoperiod) and bagged for three days. Within 20 days post inoculation, brown, necrotic lesions resembling those observed in the sampling site were observed on all inoculated plants, but not on the controls. One strain was re-isolated for each treatment group and the re-isolated strains all shared the same colony morphology and 16S rDNA sequence with BA1 to BA5. Additional PCR testing of these re-isolated strains using Pf and Pr also produced the expected amplicon. This is the first formal report of R. andropogonis affecting bougainvilleas in Taiwan. The pathogen has been reported causing diseases of betel palm (Areca catechu), corn and sorghum in Taiwan (Hseu et al. 2007; Hsu et al. 1991), some of which are economically important (Lisowicz 2000; Navi et al. 2002). As such, infected bougainvilleas could potentially serve as an inoculum source for these diseases.