LAUSR

Avocado (Persea americana Mill.) is an economically important fruit crop in China. In September 2018, symptoms of leaf spots were frequently observed causing severe defoliation in an avocado plantation (19°56′ N; 109°49′ E) in Danzhou (Hainan province) in China. On this plantation (about 2.5 ha), 70% of the trees showed the same symptoms. Initially, symptoms on young leaves appeared as small (1 to 2 mm) yellow circular lesions, which then grew into circular to irregular brown to dark-brown lesions (3 to 5 mm), usually with an obvious yellowish margin and slightly raised on the adaxial side of the leaves. Later on, lesions coalesced and formed large necrotic areas. When mature (old) leaves were infected, the lesion was not raised. For pathogen isolation, 10 symptomatic leaves were sampled from different parts of the field. Leaves with typical symptoms were washed with tap water. Small pieces of tissue (4 × 4 mm) were cut from the border of symptomatic and healthy tissue, surface sterilized in 75% ethanol solution (v/v) for 30 s followed by 2% sodium hypochlorite for 3 min, rinsed three times in sterile distilled water, plated on potato dextrose agar (PDA), and incubated at 28°C in constant light. Twenty samples were plated, and one fungus grew from 95% of the samples after 4 days. Purified strains of this fungus were transferred onto new PDA medium and were incubated for 7 days at 28°C, in constant light. The colony (78 mm in diameter) on PDA had abundant hair-like or fine fuzzy aerial hyphae. And the color of the colony was gray to grayish brown. Conidia were cylindrical, straight to slightly curved, pale brown, 46 to 307 µm (average 180 µm; n = 80) × 5 to 16 µm (average 7 µm; n = 80). These morphological characteristics resemble Corynespora sp. (Berk. & M.A. Curtis) C.T. Wei (Ellis et al. 1971). The ACT, EF-1α, and TUB genes were amplified and sequenced with the primer pairs ACT-512F/ACT-783R, EF1-728F/EF-986R (Carbone and Kohn 1999), and tub-F1/tub-R2 (Blair et al. 2008), respectively. A maximum likelihood phylogenetic analysis based on ACT, EF-1α, and TUB (accession nos. MN956378, MN737730, and MN737729) sequences using MEGA 7.0 revealed that the isolate was placed in the same clade as Corynespora cassiicola with 99% bootstrap support. For the pathogenicity test, the conidial suspension (1 × 10⁵ spores/ml) of YLBB1125 isolate was prepared by harvesting conidia from a 15-day-old culture on PDA. Conidia were sprayed onto young leaves of six potted plants. Two additional trees sprayed with sterile distilled water served as controls. After 7 days, typical symptoms of brown to dark-brown spots were observed on all inoculated leaves. Mature and old leaves of six healthy plants were wound-inoculated with 5-mm-diameter mycelial plugs obtained from 7-day-old cultures. The same number of plants treated with PDA plugs served as controls. After 4 days, the inoculation sites with the mycelium showed brown spots with a yellowish margin and a gray mold layer on the abaxial side of leaves. All leaves on control plants remained asymptomatic. All plants were covered with plastic bags for 4 days to maintain high humidity and then maintained in a greenhouse at 28°C with a 12-h/12-h light/dark cycle. C. cassiicola was specifically reisolated and identified from the inoculated (spores and mycelial plugs) symptomatic leaves, thus confirming Koch’s postulates. This is the first report of C. cassiicola as a pathogen causing leaf spot on avocado, although it has been reported previously as an endophyte (Dingley et al. 1981).