Confirmation of Burkholderia gladioli as the Causal Agent of Bacterial Scab on Gladiolus ( Gladiolus spp.) in Ohio

Ohio is one of the top five floriculture producers in the United States, grossing over $200 million annually (NASS 2019). Within the international floriculture trade, gladiolus cut flowers represent the fifth highest grossing crop (Ahmed et al. 2002). In September 2021, the Ornamental Crops Pathology Lab at the Ohio State University received a gladiolus ( spp.) sample of an unknown cultivar from a home garden in Franklin Co., OH where several plants had failed to grow from planted corms or were stunted and displaying symptoms of disease. Bleached, water-soaked spots with necrotic margins along the flowering stems, stunted flowers with partial necrosis, and necrotic bracts were observed on the submitted sample. Bacterial isolations were performed by surface disinfesting small sections of bract tissue from the border of a lesion by soaking in 10% bleach for 30 sec and rinsing twice in sterile water, macerating the tissue in sterile water, and streaking the suspension on nutrient agar (NA) plates. Plates were incubated at 28°C for 48 hours and the resulting colonies were purified by re-streaking a single colony on NA twice. Bacterial colony morphology on NA presented as cream-colored and shiny with an irregular form and undulate margin. Five tests were performed using one representative isolate to identify the bacterium to the genus level: (1) confirmed levan production, (2) confirmed pectinolytic activity, (3) confirmed ability to grow at 40°C, (4) inability to grow under anaerobic conditions, and (5) a negative oxidase test (Schaad et al. 2000). All test results identified the genus as . To identify to species level, gyrase subunit B ( ) and RNA polymerase subunit D ( ) markers were PCR amplified and sequenced using primers UP1-E/AprU, and 70F2/70R2, respectively (Maeda et al. 2006). NCBI GenBank BLASTn comparison showed that the sequence shared 99.33% identity to the type strain of (CP009323.1), while the sequence showed 99.53% identity (CP009322.1). Sequences were deposited in GenBank under accession numbers ON597852 (gyrB) and ON597853 (rpoD). To confirm pathogenicity, each of two Gladiolus communis 'Mini Elvira' potted plants were inoculated with two bacterial and two control treatments (3 leaves/treatment/plant) as follows: leaf infiltration with 1 mL of either (i) a distilled water-Tween 20 (0.03% v/v) bacterial suspension (10 cfu/mL) or (ii) a sterile water-Tween 20 suspension using a needle-less syringe; foliar spray with either (iii) the bacterial