First Report of Colletotrichum gloeosporioides Causing Leaf Blight on Cercis chinensis Bunge in China

Cercis chinensis Bunge, commonly used as an ornamental plant, is native to southeastern China and extensively cultivated in gardens across major cities in the country. In August 2023, a new high-incidence disease was discovered at Huangshan University in Huangshan, Anhui Province, China. The symptoms initially began as small brown spots, which gradually expanded into large irregular brown spots with black-brown edges. The disease was investigated at both Jilingshan Park and Huangshan University, where C. chinensis Bunge was planted, revealing an average incidence rate of was 85 % at these sites. Seventy two leaf tissue samples (3 to 4 mm²) were collected from the margins of the lesion and subjected to surface sterilization with 75% ethanol for 30 seconds followed by 1% sodium hypochlorite for 90 seconds. Subsequently, the tissues were rinsed with sterile H2O, placed on potato dextrose agar (PDA) medium, and incubated at 25℃ for 5 days. The same fungus was isolated from 90% of the tissues, and pure cultures were obtained by monosporic isolation. Representative isolates ZJ 2-1, ZJ 2-2 and ZJ 2-3 were selected for morphological and molecular characterization. The colonies displayed a color range from white to gray, with white margins and aerial hyphae, while the reverse side of the colonies appeared gray to brown. Conidia were cylindrical, aseptate, with obtuse to slightly rounded ends, measuring 15.8±1.8×4.7±0.56 μm (n = 50). The morphological characteristics were generally consistent with those of Colletotrichum gloeosporioides species complex (Weir et al. 2012). Five conserved regions of isolates (ZJ 2-1, ZJ 2-2 and ZJ 2-3), including the internal transcribed spacer (ITS), glutamine synthase (GS), calmodulin (CAL), actin (ACT), and chitin synthase 1(CHS1) gene regions, were amplified using specific primers ITS1/ITS4 (Gardes et al. 1993), GSR1/GSF1 (Guerber et al. 2003), CL1C/CL2C (Li et al. 2018), ACT-512F/ACT-783R, and CHS-79F/CHS-345R (Zhu et al. 2019), respectively. Using the BLAST, ITS, GS, CAL, ACT and CHS1 gene sequences (GenBank accession nos. PP514751, PP448025, PP448026, PP448027 and PP448028, respectively) were 100% (594 out of 594 bp), 100% (864 out of 864 bp), 100% (299 out of 299 bp), 100% (732 out of 732 bp) and 100% (282 out of 282 bp) identical to C. gloeosporioides (GenBank accession nos. JX010152, JX010085, JX009818, JX009731 and JX009531, respectively). A Maximum Likelihood phylogenetic tree, constructed by combining all sequenced loci in MEGA7, showed that the isolates ZJ 2-1, ZJ 2-2 and ZJ 2-3 clustered within the C. gloeosporioides clade with 99% bootstrap support (Fig. S1). To fulfill Koch's postulates, five C. chinensis Bunge plants were tested for pathogenicity in the field with isolates ZJ 2-1, ZJ 2-2 and ZJ 2-3 at Huangshan University. Twelve leaves from each tree were wounded and inoculated with mycelial plugs (approximately 4 mm in diameter) and 10 μl of a spore suspension (1.0 × 106 conidia/ml) of C. gloeosporioides. Inoculation with sterile PDA plugs and pure water on leaves of each tree served as negative controls. Plastic bags were used to wrap the leaves, and sterile H2O was sprayed into the bags to maintain moisture conditions (Zhang et al.2020). The experiment was repeated two times, and within 5 days, all inoculated points displayed lesions similar to those observed in the field, whereas controls remained asymptomatic (Fig. S2). The same fungus was reisolated from these lesions with a frequency of 100%. Consequently, the pathogen responsible the disease in C. chinensis Bunge was identified as C. gloeosporioides. To the best of our knowledge, this is the first report of C. gloeosporioides causing leaf blight on C. chinensis Bunge in China. This study provides valuable insights for implementing targeted measures to control leaf blight on C. chinensis Bunge and lays a foundation for the prevention and treatment of the disease.