First Report of Colletotrichum aenigma Causing Anthracnose on Bromus japonicus in Tianjin, China.

Japanese brome (Bromus japonicus Thunb.) is a weed commonly found in roadsides, floodplain wetlands, and farmlands. During September 2020 and 2021, a leaf spot disease was observed on B. japonicus in greenhouses of Baodi district, Tianjin, China (117°15'E, 39°47'N). More than 10% of the weeds were infected. Initial irregular brown spots on leaf apices continued to expand until adjacent spots coalesced. Eventually, severely infected leaves became yellow, thinner, drier and withered. Small patches (3×3 mm) were cut from symptomatic leaves, sterilized with 75% ethanol for 30s, rinsed three times with sterile water and incubated on Petri dishes with potato dextrose agar at 25°C in darkness for 7 days. Three isolates, with uniform morphology were selected for further analysis. Colonies were cottony with entire edges and aerial white mycelia; and average growth rate of 4.5 mm/day. The upper side was pale white, and the reverse side was grayish-green. Conidia were aseptate, hyaline, subcylindrical with rounded ends, 8.6 to 18.7×4.4 to 8.3 μm (n = 50). Appressoria were dark brown, oval or irregular shaped with a few lobes, 5.7 to 9.4×4.5 to 7.8 μm (n = 50). Total genomic DNA of isolates was extracted with Fungal DNA Kit (GBCBIO, Guangzhou, China). Primers for sequences of internal transcribed spacer (ITS) regions, glyceraldehyde-3-phosphate dehydrogenase (GAPDH), β-tubulin (TUB2), and calmodulin (CAL) genes were amplified and sequenced (Weir et al., 2012). After aligned and trimmed, the sequences of TJBDA1, TJBDA2, and TJBDA3 were identical. TJBDA1 representative isolate sequences were deposited in GenBank ITS OP247554 with 99.83% (576/577) similarity to MT476809, GAPDH OP414834 with 99.59% (244/245) similarity to MT501009, TUB2 OP414836 with 100% (703/703) to MT501053, and CAL OP414835 with 100% (601/601) to MT500921. Maximum likelihood trees based on concatenated sequences of the four genes were constructed using MEGA7.0. The results showed that the strains isolated from B. japonicus were closely related to C. aenigma with 99% bootstrap support. Pathogenicity tests were conducted on 3-leaf stage B. japonicus seedlings. Conidial suspension of TJBDA1 (1×106 conidia/ml) brushed from a 7-day-old culture of the fungus were sprayed on 9 B. japonicus seedlings. Control plants were sprayed with sterile water. All treatments were replicated four times. The treatment plants were placed in an incubator (25°C, relative humidity > 80%, 12-h photoperiod). Typical leaf spot symptoms resembling ones in the fields were observed on inoculated leaves after 7 days, but control leaves remained symptomless. The fungi reisolated from diseased leaves were morphologically and molecularly identical to the inoculated isolatescompleting Koch's postulates. According to morphological, pathological characteristics and multilocus phylogenetic analysis, the isolated strains from B. japonicus were identified as C. aenigma. To our knowledge, this is a new host record for C. aenigma causing anthracnose on B. japonicus in China. Currently, B. japonicus has evolved a high level of resistance to herbicides in some regions of China (Li et al, 2022) and C. aenigma caused serious disease to B. japonicus. We hope to discover a biocontrol method against weed on non-host cultivated plants through the production of secondary metabolites by C. aenigma.