First Report of the Colletotrichum siamense species complex causing Anthracnose on Photinia × fraseri Dress in China.

Abstract

Photinia × fraseri Dress is mainly distributed in the southeast and east of Asia and North America and has been widely cultivated in China. In summer 2018, an anthracnose disease of P. × fraseri Dress was found in a park in Nanjing City, China. Disease leaves showed small, round, light reddish brown spots in the early stage of infection that gradually expanded into round spots, with light gray in centers and brown edges. Fresh lesions were cut into 2-3 mm2 sections, sterilized with 75% ethanol for 30 s, followed by 1% NaClO for 90 s, washed with sterile water 3 times, and placed on potato dextrose agar (PDA) with 0.1 mg/mL ampicillin at 25°C. Colonies of a representative strain "HDSN2-1" were white to greenish grey, and the daily growth rate was 9.5 to10.5 mm/day. Aerial mycelium was grayish white, dense, and cottony, with visible conidial masses at the inoculum point. Conidia were one-celled, smooth-walled, hyaline, with obtuse to rounded ends, with a size of 12.8 to 18.4 × 4.5 to 6.8 µm. Appressoria were one-celled, brown, thick-walled, ellipsoidal, and 7.3 to 10.3 × 5.4 to 6.97µm. The morphological characteristics of HDSN2-1 matched those of the Colletotrichum gloeosporioides species complex (Weir et al. 2012). For further identification, DNA was extracted from HDSN2-1 mycelium and the internal transcribed spacer (ITS) region and partial sequences of β-tubulin (TUB2), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), chitin synthase (CHS-1) and calmodulin genes(CAL) were amplified by PCR, and sequenced with primers ITS1/ITS4, TubF1/TubR1, GDF/GDR, CHS-79F/CHS-345R, and CAL1C/CAL2C, respectively(Weir et al. 2012). The sequences were deposited in GenBank [Accession nos: MN889417, MN894596, MN894597, MN894598, MN894599]. A phylogenetic tree was constructed using the maximum likelihood/span>method with concatenated sequences (ITS, TUB2, GAPDH, CHS and CAL) (Zhu et al. 2019). Analyses conducted in MEGA7 placed HDSN2-1 in the C. siamense clade, which includes ex-type ICMP 18578. Pathogenicity of HDSN2-1 was verified on leaves from 7 healthy 8-year-old P. × fraseri and inoculated with either 5-mm mycelial plugs from the edge of 5-day old cultures on PDA or 10 μL of spore suspension (106 conidia/mL),15 healthy plants（8-year-old）were used in 5 repetitions (5 for control, and 10 for the pathogenicity test) in the same way. Controls were treated with PDA plugs or sterile dH2O. The leaves were incubated at 25 ℃ and the inoculated plants were kept in a greenhouse (relative humidity > 85%, 25 ± 1°C). Symptoms were not observed on control plants. Fungal isolates from the symptomatic plants showed the same morphological characteristics with HDSN2-1. C. siamense is a common fungal pathogen of many plants. For example, it was previously reported infecting apples and citrus fruits ( Abirammi et al. 2019). This is the first report of anthracnose of P. × fraseri caused by C. siamense in China.