First Report of Yellow Leaf Spots on Maize Caused by Colletotrichum siamense in Yunnan Province, China.

The southwest maize planting area is the third largest maize-producing region in China, including the entire provinces of Sichuan, Yunnan and Guizhou, parts of Guangxi and Hunan provinces. In June 2022, yellow leaf spot symptoms were observed commonly on maize in southern Yunnan province, including Pu'er City, Xishuangbanna Dai autonomous prefecture and Honghe Hani & Yi autonomous prefecture. The disease incidence on maize in Pu'er ranged from 10% to 20% from June to August. The initial symptoms appeared as needle-like spots scattered on the leaf surface with obvious yellow haloes, with a diameter ranging from 0.2 to 2 mm and were quite similar to maize Curvularia leaf spot. But the lesion size did not expand significantly and without reddish or dark brown margins. In July 2023, 30 diseased leaves were collected in Pu'er City, Yunnan Province. Leaf tissues (3×3 mm) were cut from the infected margins, surface disinfested with 75% ethanol for 30 s, 2% sodium hypochlorite for 2 min, and rinsed three times with sterile water, then placed on PDA at 25℃. Forty-eight isolates with the morphological characteristics of Colletotrichum ssp. were obtained by single-spore isolations (isolation frequency 42.5%). The fungal colonies on PDA were dense with white mycelia on the edges, and yellowish-white on the reverse side. The conidia were transparent, cylindrical, smooth-walled, and 6.8 to 17.5 × 3.8 to 6.5 μm. Two isolates (YNH-1 and YNH-2) were used for DNA extraction. The ribosomal internal transcribed spacer (ITS), actin (ACT), calmodulin (CAL), glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and β-tubulin 2 (TUB2) regions were amplified by PCR. The PCR primers in this study were as described previously (Weir et al. 2012). The sequences of both isolates were 100% identical, and all sequences showed >98% identity with Colletotrichum siamense in the GenBank. The sequences were deposited in GenBank (ITS, PP237394; ACT, PP265410; CAL, PP265411; GAPDH, PP265412; TUB2, PP265413). A phylogenetic tree was constructed by MEGA_v. 11.0.13 with the Maximum Likelihood (ML) method. The isolate YNH-1 and YNH-2 clustered with C. siamense DAR 76934 (97% bootstrap support) in the same branch. Pathogenicity tests were performed on the susceptible maize variety B73. Twelve healthy maize seedlings were inoculated with a conidial suspension (1×106 conidia/ml) of isolate YNH-1. All the seedlings were kept in an incubator at 26℃, with a 90% humidity and a 12 h light/dark cycle. After 5 days, yellow spots appeared on the leaves of the plants. The symptoms on inoculated leaves were similar to those observed in the field after 10 days, whereas no symptoms appeared in the control. The pathogen C. siamensis was re-isolated from the infected leaves, which fulfilled the Koch's postulates. C. siamense can cause leaf diseases on a wide range of hosts. It has been reported causing anthracnose on tea (Camellia sinensis) (Wang et al. 2016) and wax apple (Syzygium samarangense) (Yao et al. 2023) in Yunnan Province, China. To our knowledge, this is the first report of C. siamense causing yellow leaf spots on maize in China as well as a new host record for C. siamense causing leaf disease. However, how C. siamense spreads among different host plants in the region is still unknown. This study provides important information for epidemiological study and comprehensive management of yellow leaf spot on maize.