First Report of Anthracnose Caused by Colletotrichum fructicola on Loropetalum chinense in China.

Abstract

Loropetalum chinense Oliv. (Hamamelidaceae), an evergreen shrub or small tree, is widely used as a bonsai and landscape plant because of its aesthetic value. In addition, it has medicinal value, its leaves, flowers and roots can be used to treat diarrhea, coughs, hemorrhaging and burn (Zhang et al. 2013). In October 2020, leaf spot symptoms were observed on L. chinense distributed in Meiling Scenic Spot of Nanchang, Jiangxi Province, China (28.78°N, 115.83°E). We surveyed about 500 m2 of the mountain area which holds about 60 trees of L. chinense, with an incidence up to 20%. Initially, small gray-brown spots with dark brown edges appeared on the leaf surface, becoming large circular or irregular brown necrotic lesions over time. To isolate the pathogen, ten leaves of infected tissues were cut into 4 mm2 pieces, and surface disinfected with 75% ethanol for 30s and 1% hypochlorite for 1 min, rinsed three times with sterile water, plated on potato dextrose agar (PDA), and incubated at 25°C in the dark for 5 to 7 days. Five isolates with similar morphological characteristics were obtained. Conidia were hyaline, aseptate, smooth-walled, cylindrical with obtuse to slightly rounded ends, and their dimensions varied from 8.57 to 15.80 × 1.56 to 4.65 μm (n = 20). The morphological characteristics of the isolates matched the descriptions of Colletotrichum gloeosporioides species complex (Cai et al. 2009; Weir et al. 2012; Liu et al. 2015). For molecular identification, two representative isolates (JAUCC L003 and JAUCC L004) were selected for genomic DNA extraction, and the internal transcribed spacer (ITS), partial calmodulin (CAL), chitin synthase (CHS-1), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), and actin (ACT) were amplified, using primer pairs ITS1/ITS4, CL1C/CL2C, CHS-79F/CHS-345R, GDF1/GDR1, and ACT-512F/ACT-783R, respectively (White et al. 1990; Weir et al. 2012; Yang et al.2020). The sequenced loci (GenBank accession nos. ITS: MZ054401, MZ054400; CAL: MZ056750, MZ056754; CHS-1: MZ056751, MZ056755; GAPDH: MZ056752, MZ056756; ACT: MZ056753, MZ056757) exhibited up to 99% homology with corresponding sequences of C. fructicola Prihast., L. Cai & K.D. Hyde strains (GenBank accession nos. LC494271, MN525833, MN525854, MN525875 and MN525814). Concatenated sequences of the five genes were used to conduct a phylogenetic analysis using Maximum-Likelihood (ML) method in MEGA7. The isolates were identified as C. fructicola based on morphology and a multigene phylogenetic analyses. Pathogenicity of one representative isolate (JAUCC L003) was tested indoor by inoculating the top wounded leaves of six healthy L. chinense plants, rather than inoculating unwounded leaves due to the low incidence. Three leaves from each of three plants were punctured with flamed needles and sprayed with a conidial suspension (1 × 106 conidia/ml), and three leaves from each of other three plants were wounded and inoculated with mycelial plugs (5 × 5 mm3). Mock inoculations were used as controls with sterile water and PDA plugs on three leaves each. Treated plants were incubated in an artificial climate box at 25 °C, 90% relative humidity, with a 12-h photoperiod. All leaves inoculated with conidial suspension and mycelial plugs produced similar symptoms as described above 10 days postinoculation, whereas the mock inoculated plants remained asymptomatic. The fungus isolated from inoculated leaves was identical to the original pathogen on account of morphological and molecular data, confirming Koch's postulates, but not from the mock inoculated plants. Anthracnose disease caused by C. fructicola has been reported affecting numerous plants worldwide, including cotton, coffea, grape, citrus, mango, apple, pear, and cassava, among others (Guarnaccia et al. 2017, Oliveira et al. 2018). To our knowledge, this is the first report of C. fructicola causing anthracnose on L. chinense in China. This disease is significant concern in horticulture due to its impact on the aesthetics of ornamentals used in landscape plantings.