First report of Leaf Spot Associated with Boeremia exigua on White Clover in China.

White clover (Trifolium repens L.) is an important perennial legume forage widely cultivated in China (Zhang et al. 2016). In April 2018, severe necrotic lesions on leaves were observed in a cultivated white clover field in Chongqing, China. Approximately 90% of plants in the field were affected. Leaf spots were amphigenous, dark-brown, elliptic to subcircular, with diameter ranging between 1 to 12 mm, well defined by brown margins and yellow halos. Severely infected leaves became withered and abscised. Stems and flowers were not affected by the disease. Symptomatic leaves were surface sterilized with 70% ethanol for 30 s followed by 0.1% HgCl2 treatment for 3 min, and rinsed in sterile water three times. Thereafter, tissue samples from margins of individual lesions were placed on potato dextrose agar (PDA) amended with 50 mg/L of chloramphenicol and incubated at 25℃ in the dark. An olivaceous gray fungal colony was consistently isolated (90.5% isolation frequency). After 15 days of incubation, subglobose, black pycnidia developed in the cultural medium. Conidia were hyaline, ellipsoid to oblong, nonseptate(n = 50), ranging from 4.0 to 7.5 μm long (5.6 ± 2.3µm) × 2.0 to 3.8 μm wide(2.8 ± 1.0 µm). On the basis of its morphological characteristics, the fungus was identified as Boeremia sp. (Aveskamp et al. 2010). To confirm the identity, the internal transcribed spacer region (ITS), large subunit ribosomal RNA (LSU), partial actin (ACT), RNA polymerase II second largest subunit (rpb2) and beta-tubulin (tub2) genes were amplified with primers ITS1/ITS4, LR0R/LR7, ACT-512F/ACT-783R, RPB2-5F2/fRPB2-7cR, and Btub2Fd/Btub4Rd, respectively, in eight representative isolates and sequenced(Aveskamp et al. 2009; Chen et al. 2015). BLAST results showed 100% identity of the ITS (506/506 nucleotides), LSU (966/966 nucleotides), ACT (244/244 nucleotides) and tub2 (297/297 nucleotides) sequences with those of B. exigua (KY419536, MK398746, EU880878, and MK514090) and 99.83% identity of those of the rpb2 (593/594 nucleotides) sequence with B. exigua (KT389572).Based on morphology and DNA sequence analysis, the associated fungus was identified as B. exigua. Representative sequences of one isolate (BT2-1) were deposited in GenBank (MN826339, MN836592, MT265217, MT265218 and MT265219). In a pathogenicity test, ten 2-month-old potted white clover plants were spray-inoculated with a spore and mycelial suspension (approximately 105 CFU/mL) and the control plants were inoculated with sterile distilled water. Plants were incubated in a greenhouse at 20 to 24°C under natural light and enclosed in plastic bags for the first 3 days to maintain high humidity. After 10 days, typical dark-brown lesions similar to those seen in naturally infected leaves developed on the inoculated leaves and not on the control plants. B. exigua was reisolated from the lesions, thus completing Koch's postulates. There is some evidence that B exigua is capable of invading seedling root tissue of white clover and causing necrotic lesions on roots (Skipp and Christensen,1982). However, to our knowledge, this is the first report of leaf spot on T. repens caused by B. exigua. This disease severely reduces forage quality and yield. Proper identification of the causal organism is essential in formulating management strategies.