First Report of Brown Leaf Spot Caused by <i>Neopestalotiopsis formicidarum</i> on <i>Rosa roxburghii</i> in China

Rosa roxburghii, medicinal and edible plant endemic to China, exhibits significant nutritional and health-promoting properties. In August 2023, leaf spots were observed on R. roxburghii in Qiannan Prefecture, Guizhou Province, China (107.29°E, 27.05°N) with an incidence of about 80% in a 6.67 ha field. Initially, lesions appeared light to yellowish-brown, subsequently progressing to dark-brown necrotic spots. To identify the pathogen, five symptomatic leaves from five plants were collected. Leaf tissues (3 × 3 mm) were excised from lesion margins, surface disinfected in 75% ethanol for 30 s, rinsed three times in sterile water, plated on potato dextrose agar (PDA), and incubated at 25°C in the dark. After 6 days, isolates with similar morphology were recovered from all five samples. Cultures on PDA had cottony aerial mycelia and concentric rings, with the underside of the culture ranging from white to light yellow. Conidia measured 15.14-30.83 × 4.54-10.06 μm (n = 50) and were fusiform with mostly four septa. Median cells were dark brown. Morphology was consistent with the description of Neopestalotiopsis sp. (Pornsuriya et al. 2020). To confirm the identity of the fungus, internal transcribed spacer (ITS) region, β-tubulin (TUB2), and translation elongation factor 1-alpha (TEF1-α) genes were amplified and sequenced with primers ITS1/ITS4 (White et al. 1990), Bt2a/Bt2b (Glass and Donaldson 1995), and EF1-728F/EF1-986R (Carbone and Kohn 1999), respectively. Sequences from randomly selected strains L11, L22, and L33 were deposited in GenBank (ITS: PV919548-50; TUB2: PV929481-83; TEF: PV929484-86). BLASTn showed the sequences exhibited 99 to 100% similarity with N. formicidarum CBS 115.83, CBS 362.72 and MFLUCC:24-0622 (ITS: KM199344, KM199358, and PV478282; TUB2: KM199444, KM199455, and PV548150; TEF1-α: KM199519, KM199517, and PV548148), respectively. A phylogenetic tree was constructed based on concatenated sequences using neighbour-joining method by MEGA 6 software, confirming that the isolates were most closely related to N. formicidarum. Based on morphology and molecular characterization, isolates were identified as N. formicidarum. Isolate L22 was selected for pathogenicity tests. Branches of three-year-old ‘Guinong No. 5’ plants of R. roxburghii were surface-sterilized with 75% ethanol. Wounded leaves were sprayed with conidial suspension at 1 × 10⁶ conidia/mL. The control group was treated with sterile water. Each treatment comprised three replicates using individual branches as the experimental unit. Inoculated R. roxburghii were grown in the experimental base under natural field conditions. After three days, inoculated leaves exhibited symptoms consistent with those observed under natural field conditions, whereas control plants remained asymptomatic. The fungus re-isolated from symptomatic plants was identical to the original isolate based on morphological characterization and molecular analyses, thus fulfilling Koch's postulates. To our knowledge, this is the first report of brown leaf spot on R. roxburghii caused by N. formicidarum in China and worldwide. Guizhou, hosting >90 % of China’s R. roxburghii (annual output > ¥ 15-billion), faces yield threats from an emergent leaf-spot pathogen; precise etiological diagnosis is imperative for early-warning and targeted control of this economically vital crop.