First Report of Colletotrichum truncatum Causing Anthracnose of Green Foxtail (Setaria viridis) in China.

Green foxtail (Setaria viridis (L.) Beauv.), belonging to the family Gramineae, is a monocotyledonous plant that is distributed in tropical and subtropical regions of the world. S. viridis is one of the most abundant weeds in corn, soybean, rice and other major crops in China, which competes with crops for light, moisture and nutrients, leading to yield losses. In September 2021, an unknown leaf spot disease was observed on the leaves of S. viridis in many greenhouses of Xinkou town, Xiqing district, Tianjin, China (116.95729, 39.09088), under cloudy and high humid conditions after a week of rain. Over 60% of the weeds were observed with leaf spots in 28 greenhouses of XinKou town. The characteristics of the disease were observed and investigated. Initial symptoms were brown spots of 1 to 5 mms, longitudinal elliptic, round, or spindle-shaped lesions on leaves of S. viridis. These spots continued to spread shortly after the onset of the symptoms. At the late-stage disease, the spots' edges were dark brown and irregular. Eventually, the center of the spots turned grayish-white and became thinner and drier until fracture. To investigate the disease, symptomatic weed leaves were separated and small patches with infected spots were cut out. Diseased tissues (3×3 mm) were disinfected with 75% alcohol for 30s  35s, rinsed three times with sterile distilled water and then placed on potato dextrose agar (PDA) at 25°C with a 12-h photoperiod for 7 days in incubators (RXZ-280C, Ningbo, China). With the pathogen growing on the PDA, three mycelia with uniform morphology were observed, which were named SVCT-01, SVCT-02, and SVCT-03, respectively. These mycelia were transferred and cultured for daily observation. The color of these mycelia on PDA appeared gray at first, which eventually turned to grayish black with numerous black microsclerotia, setae, and a few aerial mycelia after 7 days. The setae were 75 to 120 ×3.5 to 5 μm, with elliptic to claviform appressoria. Conidia were hyaline, falcate, unicellular, 16 to 25 × 2.6 to 3.8 μm (n=50). All characteristics of isolates were consistent with the description of Colletotrichum truncatum (Sutton, 1992). Pathogenicity testing was conducted on 3-leaves-stage S. viridis seedlings. Conidial suspension (106 conidia/mL) of isolates were sprayed on 20 S. viridis seedlings with the suspension of each isolate was sprayed on 10 seedlings. Ten seedlings sprayed with sterilized distilled water were used as the control. Three replicates were performed on each treatment. The treatment plants were maintained in the incubator (25°C, relative humidity > 80%, 12-h photoperiod). Typical leaf spot symptoms were observed on inoculated leaves after 7 days, the control leaves remained symptomless. The fungus reisolated from the lesions of diseased leaves were morphological and molecularly identical to the inoculated isolates. The results echo with Koch's postulates，suggesting that the obtained isolates SVCT-01, SVCT-02 and SVCT-03 are potential pathogen in Setaria viridis. To confirm the species' identity, total genomic DNA of isolates were extracted using a Fungal DNA Kit (GBCBIO, Guangzhou, China). Sequences of internal transcribed spacer (ITS), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), actin (ACT), and β-tubulin (TUB2) regions were identified via PCR (Guerber et al, 2003; Weir et al, 2012). The sequences of SVCT-01, SVCT-02 and SVCT-03 showed more than 99% homology with Colletotrichum truncatum strains CBS:151.35 (GenBank Accession No. GU227862, GU228254, GU227960, GU228156) (Damm, 2009). The sequences of SVCT-01 were deposited in GenBank as a representative isolate under the accession numbers OL629177, OL627527, OM040388, OM040389. Maximum likelihood trees based on concatenated sequences of the four genes were constructed using MEGA7.0. The results showed that the strains isolated from Setaria viridis were closely related to Colletotrichum truncatum with 100% bootstrap support. According to morphological, pathological characteristics, and multilocus phylogenetic analysis, the isolated strains (SVCT-01, SVCT-02 and SVCT-03) from S. viridis were identified as Colletotrichum truncatum (Weir et al, 2012). Colletotrichum sp. is a significant plant pathogen that was previously reported causing anthracnose on Setaria sp. Up to now, it has been reported that C. graminicola has infected nine species of Setaria sp. Such as Setaria glauca in New Zealand (Pennycook, 1989) and Setaria pumila in Zimbabwe (Lenne, 1990). In 1979s, C.graminicola was obtained from Setaria lutescens in China (Tai, 1979). To our knowledge, this is a new host record for Colletotrichum truncatum causing anthracnose on S. viridis in China. Colletotrichum truncatum spread rapidly and caused serious disease to Setaria viridis. We hope to discovery a biocontrol method against weed on non-host cultivated plants through the production of secondary metabolites by C. truncatum.