RESUMO
Amorphophallus konjac is a crop in the family Araceae, which is widely cultivated in Hunan, Yunnan, and Guizhou in China. A. konjac flour is highly valuable economically as a product for weight reduction. In June 2022, a new leaf disease was discovered in an understory A. konjac plantation in Xupu County, Hunan Province, China, where 2,000 hm2 of A. konjac had been planted. Approximately 40% of the total cultivated area exhibited symptoms. The disease outbreaks occurred during warm and wet weather (May to June). In the early stages of infection, small brown spots appeared on the leaves and then gradually spread into irregular lesions. There was a light yellow halo around the brown lesions. In severe cases, the whole plant gradually turned yellow and died. Six symptomatic leaf samples were collected from three different fields in Xupu County to isolate the causal agent. They were rinsed with sterile water, and the lesions were cut off. The lesions were rinsed in 3% hydrogen peroxide for 30 s followed by a 90 s treatment in 75% alcohol. They were then rinsed five times in sterile water, placed on water agar plates and incubated for 2-3 days at 28°C. After the mycelium had grown, they were transferred to potato dextrose agar (PDA) plates and incubated for 3-5 days at 28°C. In total, ten isolates were obtained, and seven of them were Colletotrichum (isolation frequency 70%). Three representative isolates (HY1, HY2, and HY3) were selected for further study. This fungus grew as circular white colonies, which then became grey. The older colonies looked like cotton and had dense aerial hyphae. The conidia were cylindrical, lacked a septum, and were thin-walled. They measured 14.04 to 21.58 × 5.89 to 10.40 µm (n=100). To further confirm its fungal identity, the fungus was amplified and sequenced using six genetic regions, including ß-tubulin (TUB2), actin (ACT), the internal transcribed spacer (ITS), glyceraldehyde 3-phosphate dehydrogenase (GAPDH), calmodulin (CAL) and chitin synthase (CHS). The universal primers BT2a/TUB2R, ACT512F/ACT783R, ITS4/ITS5, GDF/GDR, CL1C/CL2C, CHS79F/CHS345R were used for amplification (Weir et al. 2012), sequenced by the Sanger chain termination method, and submitted to GenBank (TUB2: OQ506549, OQ506544, OP604480; ACT: OQ506551, OQ506546, OP604482; ITS: OQ457036, OQ457498, OP458555; GAPDH: OQ506553, OQ506548, OP604484; CAL: OQ506552, OQ506547, OP604483; CHS: OQ506550, OQ506545, OP604481). An analysis of a joint phylogenetic tree that was constructed using the six genes showed that the three isolates clearly clustered with Colletotrichum camelliae (syn. Glomerella cingulata f. sp. camelliae) strain ICMP 10646 (GenBank: JX010437.1, JX009563.1, JX010225.1, JX009993.1, JX009629.1, JX009892.1) and HUN1A4 (GenBank: KU252173.1, KU251646.1, KU251565.1, KU252019.1, KU251838.1, KU251913.1). HY3 was used as a representative strain for the pathogenicity test on the leaves of A. konjac from the whole plant. PDA blocks that were 6 × 6 mm and had been cultured for 5 d were placed on the leaf surface, and sterile PDA blocks were used as the control group. The climate chamber was maintained at 28°C at all times, and 90% relative humidity was maintained. The pathogenic lesions appeared after 10 days of inoculation. The pathogen that was re-isolated from the diseased tissues had the same morphological characteristics as HY3. Thus, Koch's postulates were fulfilled. C. camelliae has been shown to be the primary pathogenic fungus responsible for anthracnose in tea (Ca. sinensis (L.) O. Kuntze) (Wang et al. 2016) and camellia oleifera (Ca. oleifera Abel.) (Li et al. 2016). Anthracnose caused by Colletotrichum gloeosporioides has been reported on A. konjac (Li. 2021). However, to our knowledge, this is the first report in China and worldwide that C. camelliae causes anthracnose on A. konjac. This research lays the foundation for future research to control this disease.
