First Report of Colletotrichum liriopes Causing Leaf Spot of Rohdea japonica in China

Rohdea japonica, known as Japanese sacred lily, a perennial herb in the family Asparagaceae, is an important ornamental plant in China (Hinkley 2006). In May 2022, at the campus of Jiangxi Agricultural University (28o45′50″N, 115o50′2″E), Nanchang, China, leaf spots occurred on R. japonica with a disease incidence of 95% (285/300 plants). Disease severity ranged from 40-60% of the leaf area on each plant. Symptoms initially appeared as small water-soaked spots, usually at the edges of the leaves. Then, the spots enlarged rapidly and became yellow brown, circular or semicircular. Finally, the dead tissues fell out of the leaf, leaving ragged leaf edges. Small pieces (3×3 mm) cut from the margin of necrotic leaf tissue were surface disinfested in 75% ethanol for 10 s and 0.1% HgCl2 for 20 s. After being rinsed three times with sterile distilled water, tissue was placed on potato dextrose agar (PDA) plates and incubated at 28℃ with a 12 h light-dark cycle. The growing fungal colonies were purified by subculturing hyphal tips, and 12 fungal isolates with similar morphology were obtained. Colonies were flat, smoke-grey with olivaceous green fan-shaped stripes. Conidia were single-celled, hyaline, slightly curved, both ends gradually tapering, measuring 14.9 - 25.3 μm long × 3.9 - 6.2 μm wide (n = 100). Appressoria were solitary, irregular, sometimes ellipsoidal, dark brown, measuring 6.5 - 17.6 μm long × 3.9 - 8.7 μm wide (n = 50). Morphological and cultural characteristics of the isolates matched the descriptions of Colletotrichum liriopes (Damm et al. 2009; Yang et al. 2020). To confirm the pathogens identity, genomic DNA of a representative isolate (WNQ1) was extracted, and the rDNA-ITS, TUB2 and CAL gene were amplified and sequenced using the primers ITS1/ITS4, T1/Bt2b, and CL1C/CL2C (White et al. 1990; Weir et al. 2012), respectively. The sequences were deposited in GenBank under accession number: ON514224 (rDNA-ITS), ON552548 (TUB2) and ON552549 (CAL). BLAST analyses showed 100%, 99.73% and 100% identity with 100% query coverage to the rDNA-ITS, TUB2 and CAL sequence of C. liriopes (MK644098, HM585414 and MN803417, respectively). Phylogenetic analysis using concatenated sequences of rDNA-ITS, TUB2 and CAL placed the isolate WNQ1 in a single clade with the reference strain of C. liriopes CORCK2. To confirm pathogenicity, a conidial suspension (1×106 conidia/ml) of isolate WNQ1 was sprayed on three leaves each of three healthy R. japonica plants wounded with a sterile needle, whereas control plants were wounded in the same way and sprayed with sterile distilled water. All treated plants were placed in a moist incubator at 28℃ with a 12 h photoperiod. Three days later symptoms, similar to those described above appeared. Symptoms did not develop on the control leaves. C. liriopes was recovered from the inoculated leaves, fulfilling Koch's postulates. Anthracnose caused by C. liriopes in R. japonica plants has been reported in the United States and Korea. To our knowledge, this is the first report of anthracnose on R. japonica caused by C. liriopes in China. The disease seriously affected the ornamental value of R. japonica. The result provides the foundation to study the occurrence patterns and control measures of R. japonica anthracnose.