First Report of <i>Berkeleyomyces rouxiae</i> Causing Black Root Rot of Orange Jasmine ( <i>Murraya paniculata</i> ) Found in Florida, U.S.A.

Murraya paniculata (L.) Jack (Rutaceae), commonly known as orange jasmine, is a widely cultivated ornamental plant and is used in studies related to citrus greening (Skelley and Hoy 2004). In January 2024, 4-year-old orange jasmine plants (3 individuals) collected in Gainesville, Florida (FDACS-DPI LIST 01182024-359) exhibited stunting, chlorosis, leaf drop, and poor health due to inadequate root development with extensive necrosis (plant disease incidence <10%). Signs included the presence of dark-olive fungal aleuriconoidia on necrotic root tissues. Pathogen isolation was performed using the carrot sandwich method (Williamson 2014) on enriched carrot-V8 agar medium (200 ml carrot-V8 juice, 4 g cornmeal, 19 g potato dextrose agar, 2 g calcium carbonate per 1 liter of water), supplemented with 100 mg of ampicillin and 250 mg of chloramphenicol. We deposited a dried sample in the Plant Industry Herbarium Gainesville (PIHG 18281), and an active fungal culture in the USDA Agricultural Research Service Culture Collection (NRRL 64848). Molecular identification was performed using the internal transcribed spacer and large subunit regions of the rRNA genes (White et al. 1990), as well as the beta-tubulin gene (Einax and Voigt 2003) (GenBank accession numbers PQ456796, PQ456797 and PQ462365). Based on a Megablast search (Chen et al. 2015), which showed a high sequence similarity (>99.8%) to the ex-paratype culture CMW5472, and phylogenetic analysis, we identified the pathogen as Berkeleyomyces rouxiae W.J. Nel, Z.W. de Beer, T.A. Duong, & M.J. Wingf. (Ceratocystidaceae) (Nel et al. 2018). In concordance, the fungus produces light-green endoconidiophores, light-green endoconidia of 9 to 27 × 4 to 7 µm, and dark-green aleuriconidia of 35 to 70 × 11 to 14 µm (n = 30), with 4 to 7 septa; in culture. Pathogenicity tests were conducted to evaluate the impact of B. rouxiae on the vegetative growth of 25 treated seedlings and 5 controls (each in duplicate). Seeds were disinfected by soaking for 20 minutes each in 10% bleach, 70% alcohol, and sterile water, followed by overnight soak in sterile water. Seed germination was set up in individual 50-ml sealed conical tubes with sterile perlite in darkness at 28 °C for 14 days. Superficial inoculations were made at the base of the hypocotyl by adding 500 µl of a suspension at 9.1 × 10⁴ spores/ml of endoconidia, while controls received a 0.07% sterile saline solution. Vegetative growth was set up by placing the tubes in a lighted shelf (12/12 light/dark cycle, ~22 °C) with lids sealed using parafilm to enhance airflow. Fertilization and watering were performed every 2-weeks with 2 ml of a 0.1% solution of 20-20-20 NPK fertilizer. At week 12 and after disinfection, roots fragments were placed on V8 for re-isolation and identification of B. rouxiae. In treatments, we observed 100% pathogen infection, as evidenced by the presence of aleuriconidia on necrotic root tissue. This was accompanied by a seedling mortality rate of 24%, while an additional 30% of the surviving seedlings exhibited stunted growth and chlorosis. These symptoms were consistent with those observed in initial samples and closely resembled the disease manifestations caused by B. rouxiae in cotton and tobacco (Korbecka-Glinka et al. 2024, Le et al. 2024). All controls remained asymptomatic throughout the experiment. Based on our literature review, this is the first report of B. rouxiae causing black root rot in M. paniculata, with a notable impact on vegetative growth.