First Report of <i>Colletotrichum aenigma</i> Causing Leaf Anthracnose on <i>Aucuba japonica</i> in Henan Province, China

Aucuba japonica is widely planted in China for landscape use, particularly in gardens and parks. In September–October 2024, leaf blight symptoms were observed on A. japonica in Meicheng Park, Nanyang City, Henan Province (32°59′21″ N, 112°32′54″ E). A subsequent survey across different sections of the park recorded a disease incidence of 39% (n = 100 plants). Initial symptoms consisted of small dark-brown spots on leaves that enlarged into irregular leaf-blight lesions; in severe cases, lesions coalesced and caused defoliation and noticeable aesthetic decline. Diseased leaves (n = 20) were collected and surface sterilized. From each leaf, two lesion margins (3–3 mm²) at the interface of healthy and diseased tissue were excised and placed on potato dextrose agar (PDA). In total, 40 fungal isolates were obtained from symptomatic tissues. Thirty-six isolates showed typical Colletotrichum morphology. Isolates shared similar morphology, and three strains (SJSH09, SJSH12, SJSH23) obtained from symptomatic plants located in different sectors of Meicheng Park were selected for detailed analyses. Colonies on PDA were white to pale gray with a cottony texture, typically exhibiting subtle concentric zoning on the colony surface. Conidia were hyaline, aseptate, smooth-walled, and cylindrical to fusiform, measuring 7.3–22.8 × 2.6–5.3 μm (n = 100), consistent with Colletotrichum morphology (Talhinhas et al. 2023). For molecular identification, five loci—the rDNA internal transcribed spacer (ITS), glyceraldehyde-3-phosphate dehydrogenase ( gapdh ), chitin synthase (chs-1), actin (act), and β-tubulin (tub2)—were amplified from the three strains (Zhang et al. 2023). Sequences have been deposited in GenBank under accession numbers PV636156–PV636158 (ITS), PV641785–PV641787 (gapdh), PV641782–PV641784 (chs-1), PV641788–PV641790 (act), and PV641794–PV641796 (tub2). BLASTn analyses showed that all loci had 99–100% identity to Colletotrichum aenigma reference strains, with no conflicting top hits to other species. A maximum-likelihood phylogeny based on the concatenated dataset (ITS, gapdh, chs-1, act, tub2) and ex-type reference sequences was inferred in MEGA. The three strains formed a clade with the C. aenigma ex-type ICMP 18608, TJ325, and XZC02, clearly distinguishing them from other Colletotrichum species. Morphological and molecular evidence together support identification of all three strains as C. aenigma. Pathogenicity was assessed by spraying conidial suspensions (10⁶ conidia mL⁻¹) onto unwounded leaves of five A. japonica plants; five additional plants were sprayed with sterile water as mock-inoculated controls. Plants were incubated at 28 °C and ~90% relative humidity. 14 days post-inoculation, lesions resembling those in the field developed on all inoculated plants, whereas no symptoms appeared on controls. The pathogen was re-isolated. ITS sequences of these re-isolated were 100% identical to those of the inoculated strains, confirming that the same pathogen were recovered and thereby fulfilling Koch’s postulates. Anthracnose of A. japonica caused by Colletotrichum boninense has been reported previously from Guizhou Province, China (Liu et al. 2022). This is the first report of C. aenigma causing leaf anthracnose on A. japonica. Given the widespread use of A. japonica as an ornamental shrub in urban landscapes, this newly recognized anthracnose disease poses a potential threat to plant health and aesthetic value. Accurate identification provides a basis for reliable diagnosis and for the development of targeted fungicide applications and other management strategies to protect ornamental plantings.