First Report of Leaf Blight on Chinese Fir (Cunninghamia lanceolata) Caused by Bipolaris setariae in China

Chinese fir (Cunninghamia lanceolata [Lamb.] Hook) is widely cultivated in China, contributing ∼40% to timber production in southern China. In December 2017, a leaf blight disease was found on Chinese fir in the National Forest Park of Kunyushan (E 121°46′, N 37°15′) in Yantai, Shandong. It mainly infected Chinese fir, exhibiting brown to brownish-red with one to three dark brown bands across the leaves. It started at the tips and later covered the whole leaf. Incidence was 9.4%. Symptomatic leaves were collected and surface sterilized. Forty cuttings (∼0.5 × 0.5 cm) from lesion margins were placed on 2% PDA in Petri plates and incubated at 25 ± 2°C for 7 days. Twenty-eight isolates were single spored and stored in the Forest Pathology Laboratory at Nanjing Forestry University. They were grown on PDA for 7 days and exposed to fluorescent light for another 5 days to stimulate sporulation. Conidia of each isolate were individually collected in sterile ddH₂O and used for plant inoculations (1 × 10⁵ spores/ml). Healthy 1-year-old detached leaves collected in the field and 10-month-old seedlings generated from tissue culture were surface sterilized using 75% ethanol and inoculated with 5 µl of the conidial suspension. Sterile ddH₂O was used on control leaves. Inoculated leaves and plants were kept in a moist chamber at 25 ± 2°C under a 12-h photoperiod. The experiment was repeated twice, with at least six replicates for each treatment. At 7 days postinoculation, detached leaves inoculated with isolate XXG7 showed brown necrotic lesions of 1.4 ± 0.6 cm average length. Similar necrotic lesions were observed on seedlings inoculated by XXG7. No lesions were found on plants inoculated by other fungal isolates and ddH₂O. XXG7 was reisolated from the margins of the lesions and used to fulfill Koch’s postulates and for species identification. Colonies of XXG7 grown on PDA were round, with an average mycelial growth rate of 1.6 ± 0.4 cm/day. Aerial mycelium was dense, felted, and olive. The fungus produced black pigments. The vegetative hyphae were septate, branched, light brown to brown, 1.9 to 3.4 μm in width. When the colonies were exposed to fluorescent light for 3 days, conidia and conidiophores were produced. Conidiophores were solitary, brown, rarely branched, multiseptate, flexuous, and geniculate at the upper parts. Conidiophores were 66.8 to 355.6 × 3.5 to 6.7 μm, and the terminal conidiogenous cells were 6.6 to 22.3 μm long (n = 30). Conidia were usually long, oval, straight, smooth, brown to dark brown. Most conidia were 4- to 9-distoseptate and 27.1 to 75.4 × 12.6 to 21.3 μm (mean = 53 × 16.3 μm [n = 60]). These morphological characteristics were similar to Bipolaris setariae (Manamgoda et al. 2014; Shoemaker 1959). The taxonomy was further studied using molecular identification. The ITS region of rDNA, glyceraldehyde-3-phosphate dehydrogenase (GPD) gene, and translation elongation factor-alpha (tef-1α) gene of XXG7 were amplified with primer sets ITS1/ITS4, GDP1/DPG2, and EF1-983/EF1-2218R, respectively (Raza et al. 2019). ITS (MT032396), GPD (MT036970), and tef-1α (MT036971) sequences were deposited in GenBank. The ITS sequence showed 99.8% identity to B. setariae strain LC12047 (MN215632.1); GPD sequence 100% similarity to strain LC12047 (MN264068.1); and tef-1α sequence 99.9% similarity to strain LC12047 (MN263926.1). Phylogenetic analysis using concatenated sequences of ITS, GPD, and tef-1α also showed that XXG7 clustered monophyletically with strains of B. setariae and was supported with a high bootstrap value (73%). Based on morphological and phylogenetic data, XXG7 was identified as B. setariae. B. setariae is an important pathogen on lawn grasses, gramineous crops, and other plants (Manamgoda et al. 2014; Raza et al. 2019). This is the first report of B. setariae causing leaf blight on Chinese fir in China. This discovery will facilitate further studies as well as monitoring and control of the disease in the future.