First report of Colletotrichum fructicola causing Anthracnose on the leaf of flowering cherry (Prunus serrulata 'Sekiyama') in China.

Abstract

Flowering cherry (Prunus serrulata) is a notable garden tree that blooms in early spring. In June 2023, an anthracnose disease was identified on 60% of P. serrulata 'Sekiyama' plants in the Fenghuanggou Scenic Area (0.2 ha.) in Nanchang City, Jiangxi Province (28.37° N, 116.01° E). At early disease stages, brown, small spots (0.5 to 1 mm) appeared on the upper surface of leaves. Subsequently, a chlorotic halo developed around each lesion. Then necrotic lesions were enlarged, the leaves were perforated. Ten symptomatic leaves were collected from 5 trees, rinsed under running water for 3 min, sectioned into 5×5 mm pieces, disinfected in 75% ethanol for 30 sec and rinsed three times with sterile water. Tissue pieces were placed on potato dextrose agar (PDA) and incubated at 27 °C for three days. Among the 15 isolates, 5 isolates with similar morphology were obtained from the 10 infected tissue pieces. Colonies were off-white with cotton-like hyphae, with the reverse of the culture became brownish. Conidia were hyaline, smooth-walled, cylindrical, aseptate, and had wide rounded ends and were measured 0.91 to 1.41 μm × 2.11 to 3.18 μm (n=28). To identify these isolates, ITS1/ITS4 (Gardes and Bruns, 1993) primers were first used to amplify the ITS sequence, and it was found that the five isolates had 100% homology to the genus Colletotrichum (Tang et al., 2022). One isolate (ZJ1-1) was selected for subsequent verification, and partial chitin synthase (CHS), calmodulin (CAL) and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) genes were amplified with the primers CHS-79F/CHS-345R (Carbone and Kohn, 1999), CL1C/CL2C (Weir et al., 2012) and GDF/GDR (Templeton et al., 1992), respectively. Sequences were deposited in GenBank as accession numbers PP481926 (CHS), PP481927 (CAL), PP481925 (GAPDH), and PP077091 (ITS). Blast analysis demonstrated that the sequences were 99% to 100% identical with those of Colletotrichum fructicola isolate ICMP18646 (JX009874, JX009674, JX010032, JX010173). A phylogenetic tree was produced using CHS-CAL-GAPDH-ITS concatenated sequences in MEGA 11.0 and found that ZJ1-1 was assigned to the C. fructicola clade with 95% bootstrap support. According to its morphological characteristics and molecular biology, the strain was identified as C. fructicola ( Kang et al., 2023). The fungus ZJ1-1 was inoculated onto healthy 3-year-old P. serrulata 'Sekiyama' trees. 3 trees were wounded and inoculated with 10 μL spore suspension (1.0×106 conidia/mL) of ZJ1-1, and other 3 trees were inoculated with sterile ddH2O to act as the control. The inoculated plants were incubated in a greenhouse with 27 ℃ and 95% RH. Each plant was inoculated on three leaves, with three replicates. Symptoms of anthracnose appeared on the third day following inoculation, but no symptoms appeared on the trees maintained as controls. C. fructicola was re-isolated (named 003) from the inoculated leaves and confirmed by the methods described above, whereas no fungus was isolated from the control plants. To our knowledge, C. fructicola caused anthracnose has been reported on Paeonia lactiflora, Prunus avium and Prunus sibirica (Kang et al., 2023; Zhou et al., 2023; Han et al., 2023), but this is the first report of C. fructicola causing leaf spot disease on flowering cherry in China. C. fructicola can induce leaf defoliation of flowering cherry in severe cases, and further investigations are required to ascertain the potential pathogenicity of other Colletotrichum sp.