First Report of Anthracnose Disease Caused by Colletotrichum truncatum on the Velvetleaf (Abutilon theophrasti) in China

Abstract

Velvetleaf (Abutilon theophrasti), an annual plant in the family Malvaceae, is an important source of fiber and medicine (Flora of China 2007; Su et al. 2010). Dark brown lesions were observed on the leaves of A. theophrasti in the Herbal Garden of Jilin Agricultural University (125.23°E, 43.47°N), Jilin Province, China, in July 2015 and 2016, respectively. The disease incidence was about 20%. The lesions appeared initially as small, light brown spots on the leaves. These enlarged gradually and coalesced with others to form large spots having irregular edges. Finally, the central portions of the lesions broke open leaving small holes on the leaves. A small piece of tissue was cut from the lesion margin of 30 leaves and then surface sterilized in 75% ethanol for 30 s followed by 1.5% NaClO for 1 min, washed three times with sterilized distilled water, and then dried on sterilized filter paper. Each tissue piece was incubated on potato dextrose agar at 25°C in the dark for 7 days. Colonies formed and were light olive (after 3 days) and then became dark olive (after 7 days). The growth rate (colony radius) ranged from 3.5 to 5.2 mm per day. Conidia were hyaline, aseptate, both ends were bent slightly, and measured 21.9 to 28.0 (25.4 ± 1.4) μm in length and 3.2 to 5.8 (4.3 ± 0.6) μm in width. The hyphae were hyaline, septate, and branched. Appressoria were light to dark brown, solitary, with the outline roundish to ellipsoidal, and 7.3 to 9.8 × 4.8 to 6.3 μm in size. Setae were dark brown, two to five septate, and measured 79.2 to 162.6 μm in length. The morphological characteristics corresponded to Colletotrichum truncatum more clearly and directly (Damm et al. 2009). To further confirm the identity of the pathogen, DNA was extracted from one single-spore isolate, CT12. The following loci were amplified according to Weir et al. (2012): the internal transcribed spacer (ITS) region of ribosomal DNA and the glyceraldehyde-3-phosphate dehydrogenase (GAPDH), β-tubulin-2 (TUB2), and chitin synthase (CHS-1) genes. The numbers of base pairs matching were 548/550 for ITS, 225/228 for GAPDH, 241/242 for CHS-1, and 683/683 for TUB2 compared with the sequences of C. truncatum in GenBank (KX621963 for ITS, MK359986 for GAPDH, MK118057 for CHS-1, and MH925229 for TUB2). Maximum likelihood analysis of the combined four gene sequences and reference sequences of Colletotrichum spp. from GenBank and the results of the phylogenetic tree showed that CT12 belonged in C. truncatum with 100% bootstrap support. Sequences of the representative isolate CT12 were deposited in NCBI GenBank (MG993325 [ITS, 558 bp], MH038172 [GAPDH, 225 bp], MH038171 [TUB2, 683 bp], and MH038173 [CHS-1, 242 bp], respectively). Conidial suspension (10⁶ conidia/ml) of CT12 was sprayed on 10 healthy, 3-month-old velvetleaf leaves, and sterilized distilled water was sprayed on 10 healthy 3-month-old velvetleaf leaves as a control. The experiment was conducted three times. All plants were incubated in a controlled incubator with a 12-h photoperiod and 90% relative humidity at 25°C. After 5 days, necrotic lesions occurred on the inoculated leaves but not on the control plants. The same fungus was reisolated from the infected leaf tissues. C. truncatum has been reported to cause anthracnose on soybean, pepper, tomato, eggplant, muskmelon, chickpea, grapes, and other plants (Damm et al. 2009; Diao et al. 2014). To our knowledge, this is the first report of C. truncatum causing anthracnose on velvetleaf in China. The identification of the pathogen will provide a theoretical basis for the identification and control of the disease and reduce economic losses.