First Report of Botrytis cinerea Causing Leaf Spot on Strawberry in Florida.

During the 2020-2021 Florida strawberry season (October to April), strawberry (Fragaria × ananassa) plants showing leaf spots were observed on samples submitted to the Diagnostic Clinic at the University of Florida's Gulf Coast Research and Education Center. Disease incidence was up to 5% and observed on four different farms in Plant City, FL on cultivars SensationTM Florida127 and Florida Brilliance. All the samples were submitted early in the season (November) and shared the same nursery source in California. Symptoms consisted of circular or irregular lesions with purple or brown halos, eventually developing leaf blight with sporulation at the center on advanced lesions. Diseased tissues (0.5 mm2) were surface disinfested with 10% bleach solution for 90 s, rinsed twice in sterile deionized water, and plated on general isolation medium (Amiri et al. 2018). Plates were incubated at 25°C and a 12-h photoperiod. A fungus producing white mycelia with sparse sporulation of Botrytis-like spores was consistently isolated. Isolates were single-spored and grown on HA medium to induce sporulation (Leroch et al. 2013). Three isolates (20-291, 20-293, and 20-295) were selected for identification and pathogenicity assays. Resulting cultures on HA had profuse sporulation resembling gray mold. Conidia (n=50) were round to ellipsoid ranging from 9 to 14.6 μm long (Avg=10.8, SD=1.3) and 6.3 to 9.5 μm wide (Avg=7.7, Sd=0.7). No sclerotia formation was observed on GI and HA medium. Based on morphology, the pathogen was tentatively identified as Botrytis cinerea (Hong et al. 2001; Jarvis 1977). DNA was extracted from the same three isolates using the FastDNA kit (MP Biomedicals, Solon, OH), and the heat shock protein (HSP60), RNA polymerase II-binding (RPB2), and glyceraldehyde 3-phosphate dehydrogenase (G3PDH) genes were amplified (Staats et al. 2004). Sequences were deposited in GenBank (accession nos. MZ288746 - MZ288754). BLASTn searches revealed that isolates were 100% identical to B. cinerea reported causing leaf spot on strawberry in California; accession numbers MK919494 (HSP60, 996/996 bp), MK919495 (RPB2, 1131/1131 bp), and MK919496 (G3PDH, 877/877 bp). To test for pathogenicity, four one-month-old plants of 'Florida Brilliance' were used per isolate and control treatment. Spores were harvested from two-week-old cultures grown on HA medium, and the suspension adjusted to 106 spores/mL in a solution of 0.1% of Tween 20. Plants were spray inoculated until run-off and kept inside clear plastic boxes for 48 h. Control plants were sprayed with sterile deionized water. Afterward, plants were kept in a misting table in the greenhouse with a water regime of 3 s every 10 min during the day. Disease incidence was evaluated weekly, and the experiment repeated once. Two weeks after inoculation, leaf spots were observed in all inoculated plants, while controls remained healthy. Fungi morphologically identical to the original isolates were re-isolated from the diseased tissues. To our knowledge, this is the first report of B. cinerea causing leaf spot on strawberry in Florida. This disease was recently reported in California (Mansouripour and Holmes 2020), which is where the transplants originated from. Considering the disease was observed early in the Florida season, it is likely that it was introduced with transplants from the nursery. This pathogen is also the causal agent of Botrytis fruit rot, which is considered a major disease of strawberry, and a previous study has shown that populations resistant to multiple fungicides are introduced with transplants (Mertely et al. 2018, Oliveira et al. 2018). While Botrytis leaf spot is currently considered rare and of minor significance (Mansouripour and Holmes 2020), it could contribute to the spread of fungicide resistance to from nursery to strawberry fruit production fields. Efforts should be implemented to monitor its occurrence and spread considering the high variability and fungicide resistance profile of this pathogen.