First Report of Colletotrichum siamense causing anthracnose on Podocarpus macrophyllus in Sichuan Province of China.

Abstract

Podocarpus macrophyllus (Thunb.) D. Don is a perennial evergreen tree of the Podocarpaceae family, which is widely used in landscape, medicine and forest interplanting (Qin et al. 2021). In August 2020, approximately 10% of the leaves have expressed symptoms of anthracnose in the campus of Sichuan Agricultural University (E103°51'35.88″, N30°42'30.41″). The lesions were light brown small sunken spots on the leaf tip in the early stage, then spread along the petiole to expanded into larger, irregular gray-white lesions in the late stage, with sparse black dots arranged above. The edge of the lesion was obvious with a fine smooth dark brown line. Samples taken from the lesions were surface disinfected for 3 min in 4% sodium hypochlorite, rinsed in sterile water and plated on potato dextrose agar (PDA), Eight single-spore cultures isolates from 10 samples were obtained and subcultured. After five days at 25°C in the dark, the mycelium of a representative culture LJS1 covered the entire plate surface (9 cm diameter). Hyphae were initially white at first, and turned pale grayish in the later stage. After about 10 days, a large number of pink conidial mass were formed around the center. Conidia 14.7 - 18.6 μm (mean 16.2 μm) in length and 4.4 -7.1 μm (mean 5.8 μm) in width (n = 100), nonseptate, cylindrical, two ends round or one end slightly acute. Conidial appressoria 5.7 - 9.3 μm (mean 7.8 μm) in length and 4.4 - 7.9 μm (mean 6.2 μm) in width (n = 50), clavate, ovoid to slightly irregular. Based on these characteristics, isolates were tentatively identified as Colletotrichum siamense complex (Sharma et al. 2013). Pathogenicity tests were conducted by spraying a conidial suspension of LJS1 (1 × 107conidia/ml) to 10 wounded and 10 non-wounded leaves from P. macrophyllus plants. Two areas of cuticle on either side of the midrib of each leaf were wounded by lightly scratching with a needle prior to inoculation (Du et al. 2020). As a control, distilled water was sprayed onto an equal number of wounded and non-wounded leaves. All inoculated and control plants were incubated in greenhouse (about 25 ± 2°C). Lesions similar to those observed in the field appeared on all wounded inoculated leaves within eight days after inoculation, whereas the non-wounded inoculated leaves and the controls remained symptomless. Reisolations of the putative pathogen were confirmed through morphological characteristics and the representative culture LJS1 were confirmed to be the pathogenic agents. The internal transcribed spaces (ITS), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), gene spacer region between Apn2 and Mat1-2(ApMat) genes were sequenced (Sharma et al. 2013) and deposited in GenBank (accession numbers OK036793, OK067325 and OK086086 respectively). These sequences were highly identical to those of C. siamense Prihastuti, L. Cai & K.D. Hyde (culture LF 139): accession numbers KJ955087.1 (99%), KJ954788.1 (99%), KJ954503.1 (99%), respectively. Based on the morphology and our multi-locus approach, the pathogen was convincingly identified for the first time as C. siamense. However, there are no reports of C. siamense causing anthracnose on P. macrophyllus worldwide. The identification of the causal agent of the disease made clear the pathogen causing anthracnose on P. macrophyllus, and provide theoretical basis for the diagnosis and treatment of disease.