First Report of Leaf Brown Spot Caused by Epicoccum sorghinum on Digitaria sanguinalis in China

Abstract

Digitaria sanguinalis (L.) Scop. is a cosmopolitan malignant weed that infests autumn harvest crops and vegetable fields. In September 2017, leaf spots were observed on more than 70% of leaves from D. sanguinalis in a paddy bund located in Chaohu city, Anhui. The spots were primarily circular or elliptical, straw colored, and had brown well-defined margins. Small pieces of symptomatic leaf tissue were surface sterilized with 0.5% NaClO for 2 min, rinsed three times with sterile water, plated on quarter-strength potato dextrose agar (PDA) medium, and cultured in the dark at 28°C. The fungal colonies were transferred to PDA medium after 2 days of cultivation. The colonies were villose and regular and initially white to pale yellow. The colonies then turned gray and had chlamydospore formation at the colony surface. The chlamydospores were gray and nearly spherical, unicellular or multicellular, ranging from 11 to 27 × 10 to 23 μm. The mycelium could secrete pigment and caused the medium to eventually turn crimson. Scratched fungal colonies grown on PDA medium that were irradiated under an 8-W ultraviolet lamp for 12 h at a distance of 30 cm could be induced to produce numerous pycnidia. The pycnidia were brown, spherical, had a cylindrical or coniform ostiole, and ranged from 110 to 183 × 80 to 143 μm. The conidia were hyaline, ellipsoidal, unicellular, aseptate, and ranged from 4.6 to 5.4 × 1.9 to 2.3 μm. These morphological characteristics were consistent with the characteristics described for Epicoccum sorghinum (Sacc.) Aveskamp (Zhou et al. 2018). The internal transcribed spacer (ITS) regions of rDNA, actin (ACT), and β-tubulin (TUB) genes were amplified with the primers ITS1/ITS4, ACT512F/ACT783R, and TUB2FD/TUB4RD (Aveskamp et al. 2009; Carbone and Kohn 1999; White et al. 1990), respectively. The sequence results were submitted to GenBank (ITS, MK039445; ACT, MK044832; TUB, MK044833). BLAST analysis showed that the sequences of the three genes showed 99 to 100% identity with the existing sequences of E. sorghinum in GenBank (MF948994.1, MF987526.1, and MF987525.1). Based on these morphological and molecular analyses, this pathogen was identified as E. sorghinum. Pathogenicity tests were performed on a five-leaf-stage D. sanguinalis plant. For inoculation with mycelium discs, 5-mm-diameter mycelium discs that were cut from the edge of a 5-day-old E. sorghinum colony were inoculated on 10 healthy leaves from D. sanguinalis. Each leaf was inoculated with one disc. Sterile PDA disks of the same size were inoculated as a control. In addition, mycelium suspensions were sprayed on potted D. sanguinalis plants (25 to 30 plants per pot). Control plants were mock inoculated with sterile water, and five pots were used for each treatment. The tests were performed under laboratory conditions of 25 ± 2°C, 90% relative humidity, and 12-h light/dark photoperiod. Obvious lesions were observed 24 h after mycelium disc inoculation, whereas control leaves remained asymptomatic for the next 3 days. Severe symptoms developed on whole plants that were sprayed with the mycelium suspension 5 days later, whereas control plants remained healthy. The same E. sorghinum strain was consistently reisolated from inoculated symptomatic leaves, confirming Koch’s postulates. To our knowledge, this is the first report of E. sorghinum causing leaf brown spots on D. sanguinalis in China. This pathogen could be considered as a potential biocontrol fungus to control D. sanguinalis pending further study.