First Report of Walnut Anthracnose Caused by Colletotrichum fructicola in China

Walnut (Juglans regia L.) is a valuable woody nut and oil tree planted worldwide. Recently, walnut production has been limited by walnut anthracnose due to early-fruiting varieties, such as ‘Xiangling,’ a major variety, dwarf pruning, dense planting, and rainy summers in China. Walnut leaf spot disease in China has been attributed to Colletotrichum fioriniae (Zhu et al. 2015). In late July 2014, 20 walnut fruits with anthracnose symptoms were collected from two orchards in Jinan, Shandong, China. Symptoms on fruit were 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 and oozed slimy pink conidial masses. On leaves, lesions were circular or irregular, water soaked, brown or black, and expanded along the veins. Later, the central lesions became necrotic and perforated. Necrotic tissues (3 to 4 mm²) of the fruits were sterilized with 1% NaClO for 60 s, rinsed with sterile distilled water, and placed on potato dextrose agar (PDA). Pure cultures were obtained by monosporic isolation, and isolate (SQ-12) was deposited into the China’s Forestry Culture Collection Center (CFCC 51553). On PDA, colonies were gray, cottony, pale white to pale gray, producing strong brownish pigmentation near the center on the reverse. The daily growth rate was 15.2 mm/day at 28°C under a 12 h light/dark cycle. Conidia were 1-celled, colorless, oblong, subcylindrical, attenuated with blunt ends, 15.2 to 15.6 × 4.7 to 4.9 μm (= 15.4 ± 0.2 × 4.8 ± 0.1, n = 45). Most conidia germinated and developed one pleurogenous, 1-celled appressorium. Appressoria were medium to dark brown, obovoid to ellipsoid or irregular, 7.8 to 8 × 5.6 to 5.8 μm (= 7.9 ± 0.1 × 5.7 ± 0.1, n = 45). The morphological characteristics fit the descriptions of C. fructicola (Prihastuti et al. 2009). The identity of isolate SQ-12 was further studied by phylogenetic analysis of the ribosomal internal transcribed spacer (ITS1-5.8S-ITS2) gene, and partial sequences of actin (ACT), glyceraldehyde-3phosphate dehydrogenase (GAPDH), and chitin synthase (CHS-1). The ITS, ACT, GAPDH, and CHS-1 sequences (KX913950, KX913947, KX913956, KX913953) were compared with sequences in Q-Bank. The similarity of isolate SQ-12 was 100% with ITS C. fructicola ex-type culture ICMP18581 and ACT C. fructicola isolate ICMP12568. SQ-12 GAPDH and CHS-1 sequences showed 99% with C. fructicola isolate ICMP18581. A neighbor-joining phylogenetic tree was generated based on combining all sequenced loci in MEGA7. Isolate SQ-12 fell in the C. fructicola clade with 97% bootstrap support. To complete Koch’s postulates, 20 healthy 1-month-old ‘Xiangling’ fruits and 12 fully expanded leaves were inoculated using a point inoculation with a drop of conidial suspension (10⁶ conidia/ml) and dH₂O as a control. All inoculated leaves and fruits were placed in sterile tissue culture bottles containing two layers of wet paper towels at 28°C under a 12 h light/dark cycle. All fruits and leaves developed anthracnose symptoms similar to those observed in the field while the controls did not show any symptoms after 7 days. The same fungus was reisolated from the lesions. To our knowledge, this is the first report of C. fructicola as a pathogen of walnut anthracnose. The result provided crucial information for epidemiologic studies and management of this disease.