First Report of Colletotrichum nymphaeae Causing Walnut Anthracnose in China

Walnut (Juglans regia L.) is a high quality woody nut and edible oil tree with a planting area of about 5,000,000 hectare in China. Walnut anthracnose is a serious disease, infecting approximately 50% of the fruits and causing a great yield losses (Wang et al. 2016). In 2019 to 2020, walnut fruits with anthracnose symptoms were collected from walnut orchards in province of Hubei, Sichuan procinve and Chongqing municipality, China. Symptoms on fruits were circular or subcircular or irregular shaped, with brown to black water soaked and sunken lesions. The black lesions enlarged and amalgamated into large necrotic areas. The older spots in the center became blackish with acervuli causing the complete mummification of the fruit, and orange conidial masses appeared under wet conditions. Necrotic tissues of the fruits were sterilized in 75% ethanol solution for 30 s, then sterilized in 4% sodium hypochlorite for 1min, and washed 3 times with sterile distilled water. The tissues were put on potato dextrose agar (PDA) and incubated at 25℃. Pure cultures were obtained by single-spore culturing method and the isolate HBBK4-4 was deposited into the China's Forestry Culture Collection Center (CFCC 57388). On PDA, the colonies were cottony, white to pale gray with aerial mycelium on the upper side and pink with black spots on the reverse. The mycelial growth rate was 9.6 mm/day at 25°C. Conidia were 1-celled, colorless, smooth-walled, straight, cylindrical to cylindrical-clavate with acute ends, 12.5 to 18.2 × 3.9 to 5.4 μm (mean 15.3 ± 3.7 × 4.9 ± 0.6 μm, n = 40). Most conidia germinated and developed one pleurogenous, 1-celled appressorium. Appressoria were single, medium brown, smooth-walled, ovate to ellipsoid, 5.4 to 7.8 × 5.4 to 7.8 μm (mean 6.7 ± 0.6 × 6.3 ± 0.5 μm, n = 30). These morphological characteristics were in concordance with published descriptions of Collectotrichum acutatum species complex. To further confirm the identity, internal transcribed spacer (ITS), beta-tubulin (TUB2), chitin synthase 1 (CHS-I) and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) genes were amplified and sequenced (Damm et al. 2012). The ITS (OM189549) and TUB2 (OM273642) sequences of isolate HBBK4-4 showed 100% similarity, and GAPDH (OM249791) and CHS-1 (OM273641) sequences showed 98.7% and 99.6% similarity with C. nymphaeae CBS100064 respectively. A maximum likelihood phylogenetic tree was generated based on combining all sequenced loci in MEGA5. 18 isolates including HBBK4-4 fell in the C. nymphaeae clade with 96% bootstrap support. To verify Koch's postulates, six isolates were used for pathogenicity test, and 20 healthy fruits and 15 fully expanded leaves for each isolate were inoculated with 5-mm-diameter mycelial plugs. Controls consisted of detached premature fruits inoculated with a PDA plug without the fungus. Six days after inoculation, all fruits and leaves developed anthracnose symptoms similar to those observed in the field, while the controls remained healthy. The pathogenicity tests were repeated twice with the same results. The morphology of the reisolated fungi was consistent with the inoculated one, fulfilling Koch's postulates. The isolate HBBK4-4 was identified as C. nymphaeae, based on the description by Damm et al. (2012). The species C. nymphaeae has been previously reported to cause severe anthracnose on walnut in France (Da Lio et al., 2018), Brazil (Savian et al., 2019) and Italy (Luongo et al., 2022). To our knowledge, this is the first report of C. nymphaeae as a pathogen of walnut anthracnose in China. The result provided crucial information for epidemiologic studies and management of this disease.