First Report of <i>Colletotrichum fructicola</i> Causing Leaf Anthracnose on <i>Scrophularia ningpoensis</i> in China

Scrophularia ningpoensis is a perennial herb widely cultivated in southern China for its medicinal properties, including the treatment of fever, constipation, swelling, rheumatism and inflammation (Guo et al. 2023). In June 2022, leaf anthracnose was observed on S. ningpoensis plants that had been cultivated in the experimental field for three months. The relative humidity was 65% in Jinhua, Zhejiang Province, China (29.12°N, 119.64°E). The affected area covered approximately 200 m2 with a disease incidence of 65%. Symptoms initially manifested as circular or irregular dark brown spots, ranging from 0.25 to 3 mm in diameter, which progressively enlarged and coalesced into necrotic lesions with perforations. Six symptomatic leaves tissues (5×5 mm) were surface-sterilized using 75% alcohol for 30 s, followed by disinfection with 0.1% mercuric chloride solution for 30 s. Samples were then rinsed three times with sterile water (1 minute each) and transferred onto potato dextrose agar (PDA) plates for incubation at 25℃ in the dark. Fungal isolates (SNPF01-06) were obtained via hyphal-tip isolation. Colonies on PDA were circular, initially producing white, cottony mycelium that later developed a gray center with a white peripheral margin. Conidia were oval, unicellular, hyaline, smooth-walled, measuring 9.1-17.3×3.4-7.6 µm (mean 13.3×5.8 µm, n=50). The morphological characteristics of isolate SNPF05 were consistent with those of Colletotrichum spp. (Weir et al. 2012). Molecular identification involved amplification of five gene loci: internal transcribed spacer (ITS), actin (ACT), chitin synthase 1 (CHS-1), glyceradehyde-3-phosphate dehydrogenase (GAPDH) and beta-tubulin (TUB2), using primers ITS1/ITS4, ACT-512F/ACT-783R, CHS-79F/CHS-345R, GDF/GDR and T1/Bt2b, respectively (Weir et al. 2012). The obtained sequences were deposited in GenBank under accession numbers PQ687468(ITS), PQ763649 (ACT), PQ763650 (CHS-1), PQ763648 (GAPDH), and PQ763651 (TUB2). BLASTn analysis revealed 99%-100% identity with the C. fructicola type strain ICMP:18581 across all loci: ITS (JX010165; 548/549 bp), ACT (FJ907426; 218/219 bp), CHS-1 (JX009866; 285/287 bp), GAPDH (JX010033; 246/247 bp), and TUB2 (JX010405; 699/699 bp). A maximum-likelihood phylogenetic tree was constructed from the concatenated sequence dataset using MEGA 11; isolate SNPF05 clustered with Colletotrichum fructicola. For pathogenicity testing, six healthy S. ningpoensis plants were selected; three were inoculated via foliar spray with a spore suspension (1×10⁶ conidia/mL) of SNPF05, while three control plants were sprayed with sterile water. All plants were maintained at 25°C under 12-hour light/dark cycles while with 65% humidity. Characteristic leaf blight symptoms appeared on inoculated plants after six days, mimicking field observations. No symptoms developed on control plants. The pathogen was reisolated from symptomatic tissues and identified morphologically and via sequence analysis of ITS, ACT, CHS-1, GAPDH, and TUB2, fulfilling Koch’s postulates. This study represents the first record of C. fructicola causing leaf anthracnose on S. ningpoensis in China. Accurate identification of the pathogen will provide a basis for future prevention and control of leaf anthracnose on S. ningpoensis.