First Report of Colletotrichum fructicola Causing Anthracnose on Canna indica L. in Guangxi, China.

Canna indica L., a well-known wetland plant (Lei et al. 2023), was found with leaf spots in a planting area (∼667 m²) in Tiandong, Guangxi, China, in June 2022. 5500 plants were affected by this disease. Symptoms began as yellow lesions, and then developed brown sub-ellipsoid spots with yellow borders, then gradually expanded and encompassed the entire leaves until leaves wilted. 18 diseased leaves were collected and cut into small pieces (3 ×3 mm) from the brown margins. The pieces were moistened with 75% ethanol for 10 seconds, disinfected with 2% NaClO for two minutes and rinsed with sterile water three times. Pieces were placed on potato dextrose agar (PDA) and incubated at 28°C for four days. 15 isolates with similar morphological characterizations were isolated and purified (about 68% isolation frequency) from 18 diseased leaves. Three isolates (CI1-1, CI1-2 and CI1-3) were selected for further morphological and molecular identification. Fungi mycelia on PDA were grayish white initially, and became dark gray after seven days. Conidia were hyaline, guttulate, unicellular, cylindrical, and averaged 15.09 × 5.72 μm. To confirm the identification, genomic DNA was extracted from mycelium of the three isolates, and the partial internal transcribed spacer (ITS) regions, intergenic region of apn2 and MAT1-2-1 (ApMAT), fragments of actin (ACT), glyceraldehydes-3-phosphate dehydrogenase (GAPDH), chitin synthase (CHS-1), and β-tubulin (TUB2) genes were amplified, sequenced and submitted to GenBank (ITS: OR501461 to OR501463; ApMat: OR684455-OR684457; ACT: OR765956-OR765958; GAPDH: OR779527-OR779529; CHS-1: OR797622-OR797624; TUB2: OR820537-OR820539). The sequences of the three isolates were 99%-100% identical (ApMat, 882/882 bp; ACT, 228/230 bp; GAPDH, 278/280 bp; CHS-1, 298/299 bp and TUB2, 298/299 bp) with those of Colletotrichum fructicola isolate ICMP18581 (JQ807838, FJ907426, JX010033, JX009866 and JX010405) (Liu et al. 2015). Compared with C. fructicola isolate ICMP18581 (JX010165), the ITS sequence identities were 94% (556/594 bp). A Maximum Likelihood phylogenetic tree was constructed by using MEGA v. 10.1.5 based on the concatenation of multiple sequences. Based on these results, the three isolates were identified as C. fructicola. Pathogenicity tests of three isolates were conducted on nine one-year-old seedlings. Three leaves per plant (six sites per leaf) were inoculated with the adjusted conidial suspension of each isolate. Ten μl suspension (10⁶ conidia/ml) was dripped on each inoculation site without wounding. Three additional plants were inoculated with sterile water as negative controls. All plants were covered with plastic bags sprayed with sterile water, and cultured in a light incubator at 28°C, with 14:10 h light/dark cycle. After five days, dark-brown spots (0.1-1.4cm×0.2-1.6cm) appeared on the leaves of experimental groups, while no lesions were found in the controls. The pathogen was reisolated from the symptomatic leaves and confirmed as C. fructicola based on molecular and morphological methods, fulfilling Koch's hypothesis. C. fructicola has been reported in various ornamental plants (Silva-Cabral et al. 2019, Guarnaccia et al. 2021, Sun et al. 2020). This is the first report of C. fructicola causing anthracnose on C. indica in China, according to literature analysis. The findings will help growers to prevent and control this pathogen, and improve the landscape effect.