LAUSR

Trifolium repens L. (White clover) a multipurpose legume crop, primarily utilized as a green manure in China. In March 2018, field investigations showed that a leaf spot disease occurred on T. repens in three fields with 50% to 80% incidence (50 plants in each field were investigated) in Nanchong City, Sichuan Province of China. Infected leaves showed symptoms of irregular dark brown spots in the center of leaves or at the leaf margins. Symptomatic leaves were surface sterilized with 3% NaClO for 3 min followed by 75% ethanol for 30 s and then rinsed in sterile water three times. Thereafter, tissue samples from margins of individual lesions were placed on potato dextrose agar and incubated at 25°C in the dark. Four pure cultures (Y3-1; Y3-2; Y3-3; Y3-4) were obtained by single spore isolation. On oatmeal agar medium, a colony reached 57 mm diameter after 9 d in alternating light and dark at 25℃. Fimbriate aerial hypha were flat and compact with pale brown to dark brown color. Conidiogenous cells were hyaline, smooth, ampulliform to doliiform (n = 30), ranging from 4.1 to 10.5 μm long (6.6 ± 1.8 µm) × 2.2 to 5.7 μm wide (4.1 ± 0.8 µm). Conidia were ellipsoidal to cylindrical, hyaline, thin walled, smooth, aseptate, with 2 to 4 polar guttules (n = 50), ranging from 3.2 to 11.3 μm long (6.3 ± 1.1 µm) × 2.1 to 4.1 μm wide (2.8 ± 0.4 µm). Conidial matrix was whitish. Morphologically these isolates resembled species in Boeremia (Chen et al. 2015). Genomic DNA of each culture was extracted from mycelia using the quick and safe method (Chi et al. 2009). The 28S large subunit of nuclear ribosomal RNA (LSU) region, internal transcribed spacer (ITS) region, RNA polymerase II second largest subunit (RPB2), translation elongation factor 1-α (TEF 1-α), β-Tubulin (TUB2) were amplified with corresponding primers (Carbone and Kohn 1999; Liu et al. 1999; Rehner and Samuels 1994; Sung et al. 2007; Vilgalys and Hester 1990; White et al. 1990; Woudenberg et al.2009). Sequences were deposited in GenBank with accession numbers: ON705759 to ON705766, ON734043 to ON734046 and ON841585 to ON841592. Phylogenetic analysis was conducted with combined sequences of the five loci using the maximum likelihood (ML) and the maximum parsimony (MP) methods. The four isolates and the extype strain of B. linicola (CBS 116.76) clustered together with high bootstrap support (BS) values (MLBS = 100; MPBS = 98). All sequences showed 100% identity to those of CBS 116.76, except the ITS region of our isolates (ON705759 to ON705762), which show 99.6% identity to that of CBS 116.76. Based on morphological characteristics and phylogenetic results, our isolates were identified as B. linicola, although the morphological characteristics of CBS 116.76 had not been characterized. To assess pathogenicity, a conidial suspension (approximately 105 CFU/mL) of isolate Y3-1 was sprayed on 1-month-old healthy plants in a greenhouse at 22℃ to 28℃. Plants sprayed with sterilized water were used as negative controls. The test was conducted three times, each with 3 plants. After 10 days, the leaves of the plants showed irregular brown lesions that were similar to the symptoms observed in the field, control plants remained healthy. The pathogen was reisolated and confirmed to be B. linicola, thus completing the verification of Koch's Postulates. Compared to B. exigua, a causal pathogen associated with leaf spot on white clover reported by Wang et al (Wang et al. 2021), B. linicola produced larger conidia, and the two species did not cluster together in the phylogenetic tree. To our knowledge, this is the first report of leaf spot disease caused by B. linicola on Trifolium repens in China.