First report of Colletotrichum fructicola causing anthracnose on Phoebe sheareri in China

Abstract

Phoebe sheareri (Hemsl.) Gamble is a high-value timber tree species with a wide range of uses. It can also be applied for landscape afforestation considering its beautiful shape and luxuriant branches. Previous studies disclosed that Neofusicum parvum can cause ulceration and necrosis on the main stems of P. sheareri (Chen et al. 2019), and branch wilt of P. zhennan (Zhu et al. 2019). In September 2019, anthracnose was observed from P. sheareri leaves in Lishui, Zhejiang province, China. The diseased leaves are characterized by dark brown lesions. The infection usually starts from the leaf tip or edge, then the infected leaves turn yellow, wither and fall finally. The infection sometimes occurrs on the small twigs, causing the whole branch to wither. Plants from 15 plantations were surveyed, and the disease incidence was about 30%. A total number of 15 freshly infected leaves were collected and cut into small pieces (5×5 mm), sterilized in 75% ethanol solution for 30 s, and washed with sterile water three times. They were further sanitized in 2% sodium hypochlorite solution for 2 to 3 minutes and dried in sterile filter paper after being washed with sterile water another three times. Leaf pieces were transferred to potato dextrose agar (PDA) plates and incubated at 25℃ in the dark for 3 days. The fungal cultures were purified and the typical isolate (TJ01) was selected for further morphological characterization and DNA sequence comparison. The strain was initially grayish white, villous, and posteriorly grayish green, and produced orange red spore clumps. The spores are single celled after maturation, oblong, colorless, and the size ranges from 8.5-19.5 × 4.5-5.5 μm. Morphological characteristics of the obtained isolate are consistent with those in the genus of Colletotrichum. DNA of the isolate was extracted and sequence data obtained and compared with those downloaded from GenBank. The internal transcribed spacer (ITS), intronic glyceraldehyde-3-phosphate dehydrogenase (GAPDH), actin (ACT), beta-tubulin (TUB2), and calmodulin (CAL) genes were partially amplified and sequenced using the primer pairs ITS-1 + ITS-4, GDF + GDR, ACT-512F + ACT-783R, T1 + Bt2b, CL1C + Cl2C, respectively (Fu et al. 2019). The resulted nucleotide sequences were individually subjected to BLAST searche in GenBank. The nucleotide sequences of ITS (MZ088144), GAPDH (MZ133607), ACT (MZ133608), TUB2 (MZ133609), CAL (MZ133610) of the isolate showed 99.83% similarity to ITS sequence (MN829453), 98.24% similarity to GAPDH sequence (MN525875), 98.94% similarity to ACT sequence (MK341539), 100% similarity to TUB2 sequence (MG657352), and 100% similarity to CAL sequence (MN525839). The phylogenetic tree was constructed using MEGA 7 based on the tandem of five genes (ITS-GAPDH-ACT-TUB2-CAL). The result revealed that the isolate resides in the clade of C. fructicola species. Inoculation was done on leaves of ten P. sheareri plants in the field to verify its pathogenicity. Three healthy leaves of each plant were surface sterilized with 75% ethanol and dried up. The leaves were punctured using a sterilized needle, inoculated with 5-mm-diameter PDA plugs excised from 7-day-old cultures, and wrapped with parafilm. Nine pieces of healthy leaves inoculated with sterilized PDA plugs were served as controls. Disease symptoms developed on all the C. fructicola-inoculated leaves 5 days after inoculation, and a yellow brown lesion became apparent 16 days later, whereas the control leaves remained asymptomatic. C. fructicola was reisolated from the lesions, but not from the control leaves, fulfilling the Koch’s postulates. This fungus is a well-known pathogen and has led to anthracnose on many plant species globally. However, our study represents the first report of C. fructicola causing anthracnose on P. sheareri worldwide and its potential threat should be evaluated.