First report of Achromobacter xylosoxidans causing bacterial stem and leaf blight on Cyrilla arida in Tennessee and the United States

Authors: OKSEL, CANSU - Tennessee State University; AVIN, FARHAT - Tennessee State University; LIYANAPATHIRANAGE, PRABHA - Tennessee State University; Shreckhise, Jacob - Jake; BAYSAL-GUREL, FULYA - Tennessee State University

Submitted to: Plant Disease
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 2/7/2024
Publication Date: 3/25/2024
Citation: Oksel, C., Avin, F.A., Liyanapathiranage, P., Shreckhise, J.H., Baysal-Gurel, F. 2024. First report of Achromobacter xylosoxidans causing bacterial stem and leaf blight on Cyrilla arida in Tennessee and the United States. Plant Disease. https://doi.org/10.1094/PDIS-01-24-0005-PDN.
DOI: https://doi.org/10.1094/PDIS-01-24-0005-PDN

Interpretive Summary: Scrub titi is a broadleaf semi-evergreen shrub native to Southeastern U.S that currently is rarely used in ornamental horticulture. While its small stature, glossy dark-green leaves, and numerous white flowers in late spring make it an attractive accent plant in the landscape, little is known of its susceptibility to diseases. A USDA-ARS researcher in McMinnville, TN worked with Tennessee State University scientists to identify a new disease of scrub titi that causes stem and leaf blight. The researchers found that the pathogen responsible for these symptoms was a species of Achromobacter, a bacterial group that is more commonly known to be plant-growth promoting or pathogenic to humans. To our knowledge, this is the first report of a disease-causing Achromobacter in scrub titi and only the second report of this bacteria causing disease in plants. This discovery will be useful to growers, plant disease diagnostic clinics, and extension agents in identifying and managing a fatal disease of scrub titi.

Technical Abstract: Scrub titi (Cyrilla arida) is a rare broadleaf semi-evergreen shrub endemic to sandy scrublands in central Florida. While lesser known than its more vigorous wetland relative (Cyrilla. racemiflora), C. arida’s smaller stature paired with its lustrous, dark-green leaves and abundance of white racemes in late spring make it a potentially superior candidate for future use in Southeastern U.S. landscapes. Three-year-old container grown C. arida plants maintained in a shade house at Otis L. Floyd Nursery Research Center, McMinnville, TN exhibited black lesions at the edge of leaves and dark brown lesions in the stem (Fig 1a) in April 2023. The disease severity was 25% of the shoot area and disease incidence was approximately 15-20% of 39 plants. Symptomatic stem and leaf tissues were surface sterilized with 0.525% sodium hypochlorite for 1 min, followed by twice consecutive rinses in distilled water. Bacterial colonies were white-colored, opaque, round with smooth edges on lysogen broth agar media after 2 days of incubation at 28°C. Bacteria were gram-negative and non-fluorescent on King’s B medium. Esculin, catalase, and oxidase tests were positive but arginine dihydrolase and gelatine hydrolysis were negative. The bacterial identity was further confirmed by extracting the total genomic DNA directly from pure cultures (strains FBG5290 and FBG5294). The small subunit ribosomal RNA (16S rRNA), 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 of two strains were deposited in GenBank with accession numbers: OR689356, OR689357 (16S); OR751366, OR751367 (rpoD); OR792456, OR792457 (eno); and OR792458, OR792459 (nuoL). The closest identified species was Achromobacter xylosoxidans (CP014060), showing 99.76%, 95.59%, 96.19%, and 94.99% identity to our sequences, respectively. Phylogenetic analysis, using concatenated sequences along with other closely related taxa from GenBank whole genomes, suggests these isolates could represent a new species, referred to as Achromobacter sp. in this report (Fig 2). The pathogenicity of strains FBG5290 and FBG5294 was confirmed on 1-year-old C. arida by inoculating five plants per strain. Stems were inoculated by depositing 15 µl of bacterial suspension (108 CFU/mL) into the stem wounded using a sterile scalpel. The inoculation sites were covered with moist cotton and wrapped with Parafilm. Inoculation was also performed on three leaves per plant by using needleless syringe infiltration (108 CFU/mL). Sterile water was used for the control plants. Plants were kept in a greenhouse at 21-23°C, 70% RH, 16-h photoperiod and irrigated twice a day using an overhead irrigation system. 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 Achromobacter sp. using morphological and molecular methods. Achromobacter spp. are commonly known as human pathogens, plant growth-promoting bacterium, and cross-kingdom pathogenic bacterium between animal (mice) and fungi (Coprinus comatus). More importantly, however, A. xylosoxidans was recently reported as the causal agent of stem soft rot of Amorphophallus konjac in China (Wei et al. 2023, Ye et al. 2018). To our knowledge, this is the first report of Achromobacter sp. causing bacterial stem and leaf blight of C. arida in Tennessee and the United States. Identification of this pathogen is important for expanding our knowledge of stem and leaf blight in C. arida and developing and applying effective management strategies.