Leaf Spot of Hedychium coronarium Caused by Colletotrichum gloeosporioides in China

Hedychium coronarium (white ginger) is widely cultivated for garden decoration and folk medicine. Since 2020, symptomatic leaves showed brown necrosis and yellow borders on H. coronarium in the field (approximately 200 m2) at Southwest University, Rongchang District, Chongqing City. Small brown-yellow spots gradually enlarged and caused withering in severe cases with a mortality rate of around 10%. Disease incidence and severity varied from 55 to 65% and from 30 to 40%, respectively. Infected tissues (5 mm in diameter) were cut from lesion margins, surface sterilized in 70% ethanol for 10 s and 0.1% acidic mercuric chloride for 3 min, followed by rinsing in sterile water three times, and then were cultured onto potato dextrose agar (PDA) at 25 °C. Five isolates were transferred onto fresh PDA and purified by single-spore culturing. The colonies were initially white and turned hoary, and the diameter reached 32.95 to 38.37 mm × 32.42 to 38.61 mm after 3 days of incubation. Pale gray abundant fluffy aerial mycelia were arranged irregularly and densely. Hyphae were septate and branched, 2 to 5 µm in width. Conidiophores were pale brown, septate, branched, cylindrical to ampulliform. Conidia were hyaline, smooth-walled, cylindrical with obtuse ends, and 8.1 to 13.3 μm × 2.4 to 5.8 μm (n = 50) in size. Appressoria were medium brown to dark brown, aseptate, in irregular shape, solitary or in groups, and measured 1.5 to 12.5 μm × 2.1 to 13.3 μm. Morphological characteristics of isolates agreed with the description of Colletotrichum (Liu et al. 2015). Genomic DNA was extracted from fungal colonies incubated on PDA for 7 days following the instructions from the PlantGen DNA Kit (CWBIO, China). The internal transcribed spacer (ITS) region, and fragments of glyceraldehyde-3-phosphate dehydrogenase (GAPDH), actin (ACT), beta-tubulin (TUB2) and large subunit ribosomal DNA (LSU rDNA) genes were amplified by primer pairs ITS1/ ITS4, GDF1/GDR1, ACT512F/ACT783R (Naz et al. 2017), T1/Bt2b (Glass et al. 1995) and LROR/LR7 (Castlebury et al. 2002), respectively. The sequence of representative isolate CG-H (GenBank accession nos. OM010355, OM238213, OM238214, OM045778 and OM010358 for ITS, GAPDH, ACT, TUB2 and LSU rDNA, respectively) exhibited 99 to 100% identity to Colletotrichum gloeosporioides. A multi-locus phylogenetic tree with concatenated sequences of ITS, GAPDH, ACT and TUB regions was constructed using the maximum likelihood method by MEGA7, which revealed that strain CG-H was grouped with C. gloeosporioides. To confirm the pathogenicity, six healthy H. coronarium plants were surface sterilized, and conidial suspension (1 × 106 conidia/mL) was sprayed onto the leaves. Six plants were inoculated with sterile distilled water as controls. All the plants grew in a greenhouse at 25 °C under 12 h/12 h photoperiod. The experiment was repeated four times. Yellow lesions appeared after 7 days of inoculation, irregular-shaped brown spots were formed and slightly sunken within 14 days, and the whole leaf gradually became withered in 50 days. All inoculated plants exhibited leaf spot symptoms while the control plants remained asymptomatic. C. gloeosporioides was re-isolated from lesions of leaves and identified by morphology and sequence analysis, fully confirming Koch's postulates. This is the first report of C. gloeosporioides associated with H. coronarium leaf spot in China and worldwide. Further studies will be conducted on the sensitivity of C. gloeosporioides to various fungicides.