First Report of Achromobacter xylosoxidans Causing Bacterial Stem and Leaf Blight on Cyrilla arida in Tennessee and the United States.

Scrub titi (Cyrilla arida), broadleaf semi-evergreen shrub, is endemic to central Florida. However, its smaller stature, lustrous, dark-green leaves and abundance of white racemes in late spring make it a potential candidate for future use in Southeastern U.S. landscapes. Three-years-old container grown C. arida plants maintained in a shade house at the Nursery Research Center, McMinnville, TN exhibited black leaf lesions and brown stem lesions (Fig. 1a) in April 2023. The disease severity was 25% of the shoot area and the disease incidence was 10% out of 60 plants. Symptomatic stem and leaf tissues were surface sterilized with 0.525% NaOCl for 1 min. Bacterial colonies were white-colored, opaque, round with smooth edges on lysogen broth agar medium after 2 days of incubation at 28°C. Bacteria were gram-negative and non-fluorescent on King's B. Esculin, catalase, and oxidase tests were positive but arginine dihydrolase and gelatine hydrolysis were negative. Bacterial identity was confirmed by sequencing of DNA from pure cultures (strains FBG5290 and FBG5294). The 16S ribosomal RNA, RNA polymerase sigma factor (rpoD), enolase (eno), and NADH-quinone oxidoreductase subunit L (nuoL) genes were amplified and sequenced using the primers 8F/1492R (Galkiewicz et al. 2008), rpoDpF/R (Sarkar and Guttman 2004), enoP1/P2 and nuoLP1/P2 (Spilker et al. 2012), respectively. The sequences were deposited in GenBank with acc. nos.: OR689356, OR689357 (16S); OR751366, OR751367 (rpoD); OR792456, OR792457 (eno); and OR792458, OR792459 (nuoL). The closest identified species to our two identical strains was Achromobacter xylosoxidans (CP054571), showing 99.6%, 95.2%, 96.2%, and 95.0% identity with >99% coverage to the above mentioned gene sequences, respectively. Phylogenetic analysis, using concatenated sequences along with the genome sequences of other closely related taxa (Fig. 2), suggest that A. xylosoxidans is presently the identified species, but given the results of the MLST, it may be that this organism will be classified as new species in the future. The pathogenicity of the strains was confirmed on 1-year-old C. arida by inoculating five plants per strain. Stems were inoculated by depositing 15 µl of bacterial suspension (1x108 CFU/mL) into the stem wounded using a scalpel. The inoculation sites were covered with moist cotton and wrapped with Parafilm. Inoculation was also performed on three leaves per plant by using a needleless syringe to infiltrate bacteria into the intercellular spaces (1x108 CFU/mL). Sterile water was used for five control plants. Plants were kept in a greenhouse at 21-23°C, 70% RH, and 16-h photoperiod. All inoculated plants showed brown lesions in stems (Fig. 1b and 1c) and leaves (Fig. 1d) 7-10 days after inoculation, while control plants remained asymptomatic (Fig. 1e and 1f). The bacteria were re-isolated from inoculated plants and confirmed as A. xylosoxidans using morphological and molecular methods. Achromobacter spp. are commonly known as human pathogens, and cross-kingdom pathogenic bacterium in animal (mice) and fungi (Coprinus comatus) (Ye et al. 2018). However, A. xylosoxidans was recently reported as the causal agent of stem rot of Amorphophallus konjac in China (Wei et al. 2023). To our knowledge, this is the first report of A. xylosoxidans causing bacterial stem and leaf blight of C. arida in Tennessee and the U.S. Identification of this novel disease lays the foundation development of effective management strategies.