Blueberry is an important economic crop and its cultivation has been greatly promoted in China. During a survey of blueberry diseases in the summer of 2014, a new leaf spot disease was observed in blueberry (Vaccinium corymbosum) in commercial fields in Chengdu (30°42' N; 103°48' E) and Yaan (30°13' N; 103°06' E), Sichuan Province, China. The disease mainly caused premature defoliation of seedlings that greatly influenced the growth of seedlings. First red dots, then reddish brown, circular to irregular-shaped, 0.2 to 1.5 cm-diameter leaf spots appeared in approximately 80 to 90% of blueberry leaves. Spots frequently showed a zonate pattern with light centers and dark margins. Leaves began to abscise when lesions covered >50% of the leaf area. Lesions were more frequent in sites with poor ventilation and high temperature and humidity. Leaf spots on abaxial sides of leaves were covered with dark gray mildew layers. Abundant conidiophores and conidia were observed on lesions using a compound microscope (ECLIPSE 80i, Nikon, Japan). Conidiophores were upright, brown, unbranched, single or in clusters, 1 to 12 septate, 100 to 500 µm × 4 to 11 µm. Conidia formed singly or in chains, obclavate to cylindrical, straight to slightly curved, with 7 to 22 pseudosepta, 75 to 242 µm × 5 to 12 µm, with conspicuous thickened hilum. A fungus was isolated from the lesion tissue and its single-spore isolate was cultured on potato dextrose agar (PDA) to produce gray and villiform colonies which secreted sepia pigment into the agar after one week cultivation at 27°C. Conidia were shorter in culture (54 to 145 µm long and 3 to 9µm wide) than in lesions. Colony morphology and microscopic characteristics conformed to the description of Corynespora cassiicola (Berk. & M.A. Curtis) C.T. Wei (Ellis. 1957). The internal transcribed spacer (ITS), ²-Tubulin, and translation elongation factor EF-1a gene of one representative isolate were amplified and sequenced with the corresponding primer pairs of ITS1 (5'-TCCGTAGGTGAACCTGCGG) and ITS4 (5'-TCCTCCGCTTATTGATATGC), Bt2a (5'-GGTAACCAAATCGGTGCTGCTTTC) and Bt2b (5'-ACCCTCAGTGTAGTGACCCTTGGC), and EF728F (5'-CATCGAGAAGTTCGAGAAGG) and EF986R (5'-TACTTGAAGGAACCCTTACC) (Li et al. 2013), respectively. A BLAST search of the three genes (GenBank Accession Nos. KP211426, KP735615, and KP735616) showed 99 to 100% similarity to the sequences (HM145960, AB539197, and KC748010) of C. cassiicola from Patchouli, Papaya, and Ramie, respectively (Chen et al. 2010; Shimomoto et al. 2011). Pathogenicity tests of three isolates from different regions were conducted by inoculating 7-day-old mycelia-bearing agar plugs (5-mm diameter) of C. cassiicola grown on PDA to the surface disinfested leaves in 2-year-old blueberry (O’Neal) seedlings. Ten healthy leaves for each seedling were inoculated as one treatment in this experiment, while sterile PDA plugs were used as the control. The assay was conducted four times. Subsequently, seedlings were kept in moist chambers in 27°C in a lighted incubator with 12/12 h light/dark. Disease symptoms were observed within 2 days in the inoculated leaves, whereas noninoculated leaves did not develop foliar symptoms. Eventually, the pathogen was reisolated from inoculated leaves onto PDA. To our knowledge, this is the first report of leaf spot caused by C. cassiicola in blueberry in Sichuan, China. Because blueberry is commonly cultivated in greenhouses, which are conducive to C. cassiicola infection, corynespora leaf spot is a potential threat to the blueberry industry in China. These findings lay a foundation for further study of the pathogenicity mechanism of this fungus, and for more effective disease prevention. © The American Phytopathological Society.
