First report of Anthracnose on Asparagus kiusianus caused by Colletotrichum spp. in Japan.

Abstract

Asparagus kiusianus Makino, a native and endangered wild plant of the asparagus genus, is endemic to Northern Kyushu's coastal area in Japan (Ito et al. 2011). In July 2022, symptoms of a disease characterized by stem browning and dark brown patches were observed on wild A. kiusianus plants along the Nijinomatsubara coast (33°26'45.24"N 130°00'10.44"E) in Saga Prefecture, Japan, with an incidence rate reaching 20%. Thirty symptomatic stem segments (approx. 1 cm) were collected for detailed examination. Samples were surface sterilized with 70% ethanol for 30 s, followed by immersion in 5% sodium hypochlorite for another 30 s. After three rinses in sterile water, they were placed on Potato Dextrose Agar (PDA) and incubated in the dark at 25°C for 7 days. Five representative isolates were identified based on morphology and named as AKW 22.207, AKW 22.208, AKW 22.212, AKW 22.302 and AKW 22.306. For AKW 22.207, AKW 22.208, and AKW 22.302, the colonies were flat and orange, with the center and surrounding areas displaying a black coloration. The conidia were slightly curved, hyaline, unicellular, aseptate, and fusiform with blunt ends, measuring 11.5 to 23.6 μm in length and 2.2 to 3.1 μm in width (n = 20). For AKW 22.212, the colony was fluffy and grayish-brown, with a dark brown reverse side. Extracellular pigment caused a reddish-brown discoloration in the PDA medium. The conidia were falcate, hyaline, unicellular, aseptate, and fusiform, tapering gradually to each end, measuring 17.6 to 24.4 μm in length and 1.8 to 3.0 μm in width (n = 20). For AKW 22.306, the colony initially appeared white, then developed gray aerial mycelium and produced yellow and black acervuli. The conidia were hyaline, unicellular, aseptate, and cylindrical in shape, measuring 13.7 to 17.8 μm in length and 3.8 to 5.3 μm in width (n = 20). Fungal DNA extraction was performed, using the DNeasy Plant Mini Kit (manufactured by QIAGEN). PCR amplification targeted the ITS, ACT, and GAPDH regions using the primers ITS1 / ITS4 (White et al. 1990), ACT-512F / ACT-783R (Carbone et al. 1999) and GDF / GDR (Templeton et al. 1992), respectively. The obtained sequences were deposited in GenBank (ITS, LC790306 to LC790310; ACT, LC790312 to LC790316; GAPDH, LC790317 to LC790321). A phylogenetic tree was constructed using RAxML 8.2.12 software, which identified one isolate as Colletotrichum fructicola (AKW 22.306), one as C. guizhouense (AKW 22.212), and three as C. liriopes (AKW 22.207, AKW 22.208 and AKW 22.302). Six healthy A. kiusianus plants were inoculated, five with different isolates and one as a control. Stems were washed, wounded, and inoculated with isolate discs, secured with bandages and parafilm. A sterile PDA disc was used for the control. Plants were incubated at 25°C under a 14-hour light/10-hour dark cycle. The experiment was repeated twice for reproducibility. After one week, stem browning occurred, and all Colletotrichum isolates were successfully re-isolated. The morphology and DNA sequences of the re-isolated fungi were matched the original isolates, thereby fulfilling Koch's postulates. This study marks the first isolation of Colletotrichum spp. from naturally infected A. kiusianus plants in the wild, demonstrating their role as causative agents of anthracnose in this species. In Japan, anthracnose disease of A. officinalis caused by C. gloeosporioides and C. spaethianum has been reported (Moriwaki et al. 2021). However, the Colletotrichum spp. identified in this study are distinct from the previously reported species.