First Report of Anthracnose on caused by in China.

Ilex cornuta (Aquifoliaceae) is a dark green evergreen shrub with glossy leaves that is widely distributed in China and East Asia and used as an ornamental and medicinal plant. In March 2022, typical symptoms of anthracnose were observed on I. cornuta leaves (with approximately 30% of leaves affected) in Jiangxi Academy of Forestry, Nanchang city, Jiangxi Province, China. The early symptoms were light brown spots on the edge or tip of the leaves. The spots gradually expanded to ovoid-shaped lesions and eventually become necrotic, dry, and gray with a dark brown margins. To isolate the pathogen, ten symptomatic leaves were randomly collected, the edges between diseased area and healthy area were cut into small pieces (4×4 mm), surface sterilized by dipping in 70% ethanol for 30 s and 1% sodium hypochlorite (NaClO) for 30 s, and then washed three times with sterile distilled water. Leaf pieces were then placed on potato dextrose agar (PDA) plates at 28℃ in the dark. Subsequently, six isolates were obtained using the single-spore method, five of them were similar in morphological characteristics. Colonies grown on PDA for 7 days were 75-85 mm in diameter, and were cottony, dense, and pale white on the surface and white to grayish-green on the reverse side. Conidia were single-celled, transparent and subcylindrical to clavate. The contents of the conidia were granular and 15.63-20.63 × 5.63-7.50 μm in size (=17.78 ± 1.41× 6.50 ± 0.55 μm, n = 40). The species was also identified by analysis of the internal transcribed spacer (ITS) region, actin (ACT), glyceradehyde-3-phosphate dehydrogenase (GAPDH) and β-tubulin (TUB2), calmodulin (CAL), glutamine gynthetase (GS), DNA Lyase (APN2), intergenic spacer and partial mating type (ApMat) genes using ITS4/ITS5, ACT-512F/ACT-783R, GDF/GDR, T1/Bt2b, CL1C/CL2C, GSF/GSR, ColDL-F3/CgDL-R1 and CgDL-F6/CgMAT1F2 primers, respectively (Weir, et al. 2012; Maharachchikumbura, et al. 2014; Khodadadi, et al. 2020). The sequences were deposited in GenBank (Accession Nos. OQ600619, OQ603370, OQ603373, OQ603379, OQ974177, OQ974176, OQ974178, and OQ974175). BLASTN analysis in GenBank showed that these genes exhibited 100% similarity to the sequences of Colletotrichum aenigma (MT476812, MN525817, MN525878, MN525904, MN525836, KX620296, and MN338281) and 99% similarity to the sequence of Colletotrichum sp. strain (MT071110). Concatenated sequences of these eight genes and Colletotrichum species sequences from GenBank were then used to construct a phylogenetic tree by using the maximum likelihood method in IQtree V1.5.6. Isolate JFRL 03-1005 was grouped into a clade with C. aenigma with a high bootstrap value. Thus, the isolate was identified as C. aenigma based on morphological and molecular data. To verify Koch's postulates, pathogenicity was tested on 2-year-old healthy potted plants of I. cornuta. Ten disinfected leaves were wounded with a sterile scalpel, and then inoculated with 10 μl of conidial suspension (1 × 106 conidia/ml) from isolate JFRL 03-1005. The control leaves were inoculated with 10 μl of sterile water. Then, the potted plants were incubated at 28°C with a 12 h photoperiod and 80% humidity. After 10 days, distinct spots appeared on all inoculated leaves, whereas control leaves remained asymptotic. C. aenigma was reisolated from the spots and identified by sequencing the ITS, ACT, GAPDH, TUB2, CAL, GS, APN2 and ApMat genes. Previous studies reported that C. aenigma can caused anthracnose on the leaves of various cash crops in China, such as apple, tree peonies, mulberry, and walnut (Wang et al. 2020; Zhang et al. 2021; Wang et al. 2022; Zhu et al. 2022). To the best of our knowledge, this is the first report of C. aenigma causing anthracnose on I. cornuta in China, this report further confirmed that C. aenigma has a wide range of hosts in nature. the anthracnose on I. cornuta caused by C. aenigma has seriously affected its ornamental value. Therefore, more attention should be paid to this disease and appropriate control strategies should be formulated.