First report of Leaf Blight on Polygonatum cyrtonema Caused by Neofusicoccum parvum in China

Polygonatum cyrtonema Hua (commonly known as Huangjing) is a significant traditional Chinese medicine with centuries of documented use (Zhao et al. 2018). This plant is efficacious in treating various age-related conditions, including diabetes, hyperglycemia, fatigue and physical debility (Cui et al. 2018). In June 2024, symptoms consistent with leaf blight disease were observed in P. cyrtonema cultivation fields in Shanghai, China (31°2′2″N, 121°26′43″E). Approximately 30% of the plants were affected. Infected leaves exhibited progressive drying originating from the tips and advancing inward, forming distinctive concentric rings. To isolate the causal agent, eight symptomatic leaves were randomly collected from different sections of the field. Necrotic tissue margins underwent surface sterilization: triple rinsing with sterile water, 30-second disinfection in 75% ethanol, 2-minute treatment in 2% NaOCl, followed by five sterile water rinses. Samples were plated on potato dextrose agar (PDA) medium supplemented with 50 µg/ml chlortetracycline and incubated at 28°C. Pure cultures were obtained via hyphal-tip transfer. Ten morphologically similar strains were isolated from infected leaves. Colonies appeared white initially, turning gray after five days. Conidia were single-celled, hyaline, ellipsoidal, and aseptate, measuring 13.6 to 20.4 × 4.1 to 5.8 µm (n = 50, counted from the two selected isolates). The morphological characteristics aligned with descriptions of Neofusicoccum sp. (Pavlic et al. 2009). Molecular identification of representative isolates (YK2 and YK6) targeted the internal transcribed spacer (ITS) region of rDNA using primer ITS1/ITS4 (White et al. 1990), transcription elongation factor 1-alpha (EF1-α) using EF1-668F/EF1-1251R (Alves et al., 2008), and β-microtubulin (TUB2) using T1/Bt-2b (Damm et al. 2009). The obtained ITS (GenBank accessions nos: PQ163849 and PX242873), EF1-α (PQ180387 and PX278747), TUB2 (PQ180390 and PX278748) all showed > 99% identity with several GenBank sequences of Neofusicoccum parvum (ITS: ON209688 and KJ193654; EF1-α: OP095382 and PV591898; TUB2: OP095386 and JX462257). A concatenated phylogenetic analysis of these loci confirmed the isolate as N. parvum. For pathogenicity tests, six potted two-year-old P. cyrtonema seedlings were used. Their leaves were surface-cleaned with sterile water. Nine leaf tips (three from each of the six seedlings) were wounded with a sterile needle. A 20 µl droplet of a conidial suspension (1×106 spores/mL) of isolate YK2 was then applied to each wound. Similarly, a 20 µl droplet of sterilized water was applied to another nine leaf tips as controls. Each seedling was separately covered with clear polyethylene bags to maintain high humidity and kept in a climatic chamber at 28°C. Characteristic lesions developed on inoculated leaves within ten days, whereas controls remained asymptomatic. The fungus re-isolated from symptomatic tissues was verified as N. parvum based on morphology and DNA sequence. N. parvum is a destructive fungal pathogen affecting numerous crops globally. This study constitutes the first documented report of N. parvum causing leaf blight disease on P. cyrtonema in China, and it provides an important reference for the biology and epidemiology of N. parvum.