Leaf Spot on Muscadine Grape ( <i>Vitis rotundifolia</i> ) Caused by <i>Neopestalotiopsis clavispora</i> in Georgia

Muscadine (Vitis rotundifolia Michx), native to the southeastern United States, is cultivated for table grapes, juice, and wine production. In October 2024, leaf spots were observed on muscadine vines (cv. ‘Carlos’) at the University of Georgia Ponder Farm, Tifton, GA. Approximately 40% of the vines within this vineyard block were affected. Lesions were irregularly distributed, varied in size, and appeared brown with darker margins. Dark structures were present on the adaxial leaf surface, and black cirrhi were observed emerging from fruiting bodies. For pathogen isolation, symptomatic leaf tissues (3 × 3 mm) were excised, surface sterilized with 0.5% sodium hypochlorite for 3 minutes, rinsed in deionized water for 3 minutes, transferred to Petri dishes containing potato dextrose agar or oatmeal agar, and incubated at 25°C under fluorescent light. After eight days, cultures developed dense white mycelia, with black acervuli forming on the white mycelial mat. Microscopic examination revealed light brown, five-celled conidia with appendages consistent with Neopestalotiopsis species previously reported to cause leaf spot disease in strawberry in Georgia (Madrid et al. 2024). The partial internal transcribed spacer (ITS), β-tubulin (β-tub), and translation elongation factor 1-alpha (tef1) regions were amplified from a single pure culture isolate (‘AD21-GaMus1’) using primers ITS1/ITS4 (White et al., 1990), Bt2a/Bt2b (Glass and Donaldson, 1995), and EF1-526F/EF1-1567R (Rehner, 2001), respectively. Obtained sequences (accession numbers: PV467457, PV492409, and PV492410) exhibited 100%, 99.51%, and 99.62% similarity, respectively, to Neopestalotiopsis clavispora ex-type isolate MFLUCC 12-0281 (accession numbers JX398979, JX399014, and JX399045) based on BLAST analysis. A phylogenetic analysis utilizing concatenated sequences from these three loci was conducted according to the methods described in Maharachchikumbura et al. (2014) using ex-type and other isolates. This analysis confirmed the placement of the isolate from muscadine within the same clade as other N. clavispora isolates. Isolate pathogenicity was confirmed in the greenhouse on intact plants (cv. ‘Carlos’) and detached muscadine leaves. Conidial suspensions (3 × 106 conidia/ml) were sprayed onto six leaves per plant, and a control plant was sprayed with de-ionized water only. Plants were placed into sealed containers to maintain ~ 85% relative humidity and incubated at 25°C. Four plant replicates were assessed, and a similar experiment was conducted using detached leaves. Thirteen days post-inoculation, brown lesions consistent with those initially observed in the field were observed along the leaf margins, with dark masses (acervuli) embedded within the lesions. N. clavispora conidia were observed both outside and within acervuli. By contrast, no lesions were observed on uninoculated leaves. The fungus was successfully reisolated from inoculated leaves and reidentified utilizing the previously described methods. Although N. clavispora has been reported as a pathogen of strawberry (Obregón et al. 2018), blueberry (Borrero et al. 2018), and apple (Shi et al. 2024) in various regions worldwide, this study represents the first report of N. clavispora infecting muscadine grapes in the U.S. This report will aid disease recognition based on field symptoms.