First Report of Anthracnose Caused by Colletotrichum ciggaro on Lonicera macranthoides Hand.-Mazz. in China.

Lonicera macranthoides Hand.-Mazz. is a traditional medicinal plant that is cultivated in Hunan, Yunnan, and Guizhou Provinces in China. In June 2020, a new leaf spot disease was observed on this plant in Longhui County, Shaoyang City, Hunan Province, China, where 14,000 hm2 of L. macranthoides had been planted. About 20% of the total cultivated area exhibited symptoms. Brown spots appeared on the leaves during the early stage and gradually expanded into irregular lesions, which became necrotic and dry. The whole plant withered and died in severe cases. To isolate the pathogen, the infected leaves were collected from different fields and washed with flowing sterile water. The small lesions were then cut and surface sterilized with 75% alcohol for 45 s followed by a 3 min treatment in 3% sodium hypochlorite. The lesions were rinsed five times in sterile water, incubated on potato dextrose agar (PDA) plates and cultured for 3-5 d at 28℃. In total, eleven isolates were obtained, and eight of them were Colletotrichum (isolation frequency 73%). Three representative isolates (JYH1, JYH2, and JYH5) were selected for further study. The fungus grew as circular white colonies, which then became grey. The older colonies looked like cotton and had dense aerial hyphae. The conidia were aseptate, transparent, cylindric, and thin walled, which measured 11.54 to 22.64 × 3.55 to 4.75 µm (n=100). Six genetic regions were amplified and sequenced to further confirm the identity of fungus. They included ß-tubulin (TUB2), the internal transcribed spacer (ITS), actin (ACT), chitin synthase (CHS), calmodulin (CAL) and glyceraldehyde 3-phosphate dehydrogenase (GAPDH). The sequences were submitted to GenBank (ITS: OQ746331; ON954583; OQ746334; TUB2: OQ772278; ON960155; OQ772279; CHS: OQ772280; ON960156; OQ772281; ACT: OQ772282; ON960157; OQ772283; CAL: OQ772284; ON960158; OQ772285; GAPDH: OQ772286; ON960159; OQ772287). The construction of a 6-gene joint phylogenetic tree analysis showed that the three isolates unambiguously clustered with Colletotrichum kahawae subsp. ciggaro strains C022-1 (GenBank: KJ001120.1, KJ001124.1, KJ001109.1, KJ001102.1, KJ001106.1, KJ001113.1) and R019 (GenBank: JN715847.1, KC860023.1, KC859980.1, KC859954.1, KC859972.1, KC859997.1), which was recently reclassified as C. cigarro (Cabral et al. 2020). Three representative isolates were used for the pathogenicity test on the young leaves of the whole plant. A sterile pin was used to prick the leaf epidermis, and 6 × 6 mm mycelial blocks that had been cultured on PDA for 7 d were placed on the leaf wounds. The controls were treated in the same manner except that sterile blocks of PDA were used. There were three replicates per treatment. All the plants used in the experiment were maintained at 28°C in a climate chamber. There was a 12 h photoperiod, and the chamber was kept at 80% relative humidity. Dark brown spots appeared at the sites of inoculation on the plants after 5 days. All the strains that were re-isolated from the lesions shared the same morphological characteristics and had the same type of colonies as the pathogen Colletotrichum ciggaro. Thus, Koch's postulates were fulfilled. C. ciggaro had been shown to cause anthracnose on Olea europaea L. (Weir et al. 2012), Mangifera indica L. (Ismail et al. 2015), Citrus reticulata L. (Perrone et al. 2016) and Areca catechu L. (Zhang et al. 2020). To our knowledge, this is the first report of C. ciggaro causing anthracnose on L. macranthoides in China and worldwide. This research provides a basis for further research to control epidemics of this disease.

First Report of Anthracnose Caused by Colletotrichum camelliae on Amorphophallus konjac in Hunan Province of China.

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.