First Report of Colletotrichum fructicola Causing Anthracnose on Punica granatum in China.

Abstract

Punica granatum L. (Pomegranate), a deciduous shrub, is widely cultivated as a fruit tree and decorative plant in China. Its flowers, leaves, roots and fruit bark also has been widely used for the treatment of different types of human disease because of the high anti-inflammatory and antibacterial activitiy (Tehranifar et al. 2011). In October 2022, leaf spot symptoms were observed on P. granatum leaves in a landscaped area on the campus of Jiangxi Agricultural University (28.75°N, 115.83°E), Nanchang, Jiangxi Province, China. A survey of 40 P. granatum of 300 m2 found that up to 20% of the foliage was infected. Infection normally starts at the tip or edge of the leaves, with the initial symptoms of lesions usually being small dark brown spots (0.8 to 1.5 mm) that gradually expand into irregular spots with grayish white central parts, and brown margins (2.3 to 3.8 mm). Ten freshly infected leaves from three different plants were collected and cut into small slices, disinfected with 75% ethanol for 30 seconds followed by 5% NaClO for 1 minute, rinsed 3 times with sterile water, and then plated on potato dextrose agar (PDA) and incubated in the dark at 25°C. After 7 days, all incubated samples produced similar morphology of aerial mycelium pale grey, dense, and cottony. Conidia were hyaline, smooth-walled, cylindrical, aseptate and measuring 12.28 to 21.05 × 3.51 to 7.37 µm (n = 50). Morphological characteristics were consistent with those of Colletotrichum gloeosporioides species complex (Weir et al. 2012; Park et al. 2018). For molecular identification, we used two representative isolates (HJAUP CH005 and HJAUP CH006) for genomic DNA extraction and amplification, using primers for ITS4/ITS5 (White et al. 1990), Bt2a/Bt2b, GDF1/GDR1, ACT-512F/ACT-783R and CL1C /CL2C (Weir et al. 2012), respectively. The sequenced loci (GenBank accession nos. ITS: OQ625876, OQ625882; TUB2: OQ628072, OQ628073; GAPDH: OQ628076, OQ657985; ACT: OQ628070, OQ628071; CAL: OQ628074, OQ628075) exhibited 98 to 100% homology with corresponding sequences of C. fructicola strains (GenBank accession nos. OQ254737, MK514471, MZ133607, MZ463637, ON457800, respectively). A phylogenetic tree was constructed using the maximum-likelihood method in MEGA7.0 for the sequences of five concatenated genes (ITS-TUB2-GAPDH-ACT-CAL). Our two isolates clustered together with three strains of C. fructicola with 99% bootstrap support values in the bootstrap test (1000 replicates). The isolates were identified as C. fructicola based on morpho-molecular approach. The pathogenicity of HJAUP CH005 was tested indoors by inoculating the wounded leaves of four healthy P. granatum plants. Four leaves from each of two healthy plants were punctured with flamed needles and sprayed with a spore suspension (1 × 106 spores/ml), and four wounded leaves from each of other two plants were inoculated with mycelial plugs (5 × 5 mm3), respectively. Mock inoculations with sterile water and PDA plugs on four leaves each were used as controls. Treated plants were incubated in a greenhouse at high relative humidity, 25°C, and a photoperiod of 12 h. After 4 days, typical anthracnose symptoms similar to natural infection appeared on the inoculated leaves, whereas the control leaves remained asymptomatic. Based on morphological and molecular data, the fungus isolated from the inoculated and symptomatic leaves was identical to the original pathogen, confirming Koch's hypothesis. Anthracnose caused by C. fructicola has been reported to affect numerous plants worldwide, including cotton, coffee, grapes and citrus (Huang et al. 2021; Farr and Rossman 2023). This is the first report of C. fructicola causing anthracnose on P. granatum in China. This disease seriously affects the quality and yield of the fruit and should be of wide concern to us.