First Report of Neofusicoccum parvum Causing Leaf Spot Disease on Piper sarmentosum Roxb in China

Piper sarmentosum Roxb (Piperaceae) is a perennial herb, widespread in southern China and Southeast Asia, which has medicinal, edible, and ornamental value. In March 2025, brown circular spots on leaves were observed in a 0.1-hectare nursery at Mazhang District, Zhanjiang, Guangdong Province, China (22.6912° N, 110.2793° E). Initially round with yellow halos and dark centers, lesions expanded into irregular brown patches. About 70% of plants were infected, and their leaves were covered with brown necrotic lesions of varying sizes. The disease persists year-round, severely impairing plant growth and diminishing ornamental value. Tissue sections (5 × 5 mm) excised from lesion margins were treated with 3.5% H₂O₂ (3 min), rinsed thrice with sterile water, plated on potato dextrose agar (PDA) containing 50 mg/L ampicillin, and incubated at 25-28°C in darkness for 3 days. Mycelial tips from colony edges were transferred to fresh PDA, and single-spore isolates were obtained with the plate dilution method after sporulation. Colonies with dense aerial mycelia were flat and gray on PDA. Conidia were aseptate, hyaline, smooth, ellipsoidal to fusiform, and 9.3 to 11 × 4.7 to 6.2 µm (average 5.2 × 10.2 µm; n > 50). The morphological characteristics of the fungus were similar to those of Neofusicoccum sp. (Pavlic et al. 2007). For precise identification, the internal transcribed spacer (ITS) regions, translation elongation factor (TEF1), RNA polymerase II subunit (RPB2) and beta tubulin (TUB2) genes from isolates AJ5-3 and AJ5-4, were amplified and sequenced using the primer pairs ITS4/ITS1 (White et al. 1990), EF1-728F/EF-2 (O’Donnell et al. 1998), RPB2-5F2/RPB2-7cR (Reeb et al. 2004; Liu et al. 1999), and TUB2Fd/TUB4Rd (Aveskamp et al. 2009), respectively. The obtained sequences of ITS, TUB2, TEF1 and RPB2 showed more than 99% sequence identity with those of Neofusicoccum microconidium ex-type CERC: 3497 (ITS: PV926511 and PV926537, 559/561 [99%] and 559/562 [99%]; TUB2: PV935313 and PV935314, 398/401 [99%] and 398/401 [99%]; TEF1: PV935311 and PV935312, 270/272 [99%] and 271/272 [99%]; RPB2: PV932132 and PV932133, 615/616 [99%] and 615/616 [99%]). Phylogenetic analysis of the concatenated data from the ITS, TEF1, TUB2, and RPB2 sequences using the maximum-likelihood method placed AJ5-3 and AJ5-4 within the Neofusicoccum microconidium clade. Thus, the isolate was identified as N. microconidium. Pathogenicity testing was performed on five one-year-old potted plants. From each plant, two healthy new leaves were selected and inoculated with spore suspensions (106 spores/ml) and 6-mm fungal plugs, respectively, and sterile water and agar were used as controls. Inoculated leaves were wrapped in plastic bags to maintain high humidity and maintained at 25-28°C. The tests were replicated three times. Within 5-10 days, the sites where the fungal plugs were inoculated showed the same lesions as the original field symptoms, while controls remained asymptomatic. N. microconidium was consistently re-isolated and confirmed through morphological and molecular analysis (ITS, TUB2, TEF1, RPB2). This is the first report of N. microconidium causing brown blotch on Piper sarmentosum in China. This fungus is widely present all over the world and can cause diseases in many plants. This report will help farmers or agricultural production units to prevent, control, and diagnose diseases more precisely, reducing production costs while improving the effectiveness of prevention and control.