First Report of Anthracnoseon on Annona squamosa L. Caused by Colletotrichum fructicola in China.

Abstract

Annona squamosa L. is a tropical fruit, cultivated in various provinces of China, such as Guangxi, Taiwan, and Yunnan. This fruit has good edible and medicinal value. In November 2021, anthracnose on A. squamosa L. fruits was observed in a plantation in Longzhou (22°11´N, 106°51´E), Guangxi, China. The incidence rate of the plantation was 13.6%. The symptoms included gray-black to black spots, initially small and blackish brown, which expanded into large irregular spots(Fig.1A-B). To isolate the pathogen, symptomatic fruits were collected and small pieces (5 mm2) were cut from the lesions, surface sterilized in 75% ethanol for 15 seconds, followed by 2% AI sodium hypochlorite solution for 3 minutes, rinsed three times with sterile water, and incubated on PDA at 28°C. A total of 23 isolates with identical morphology were obtained and 3 representative isolates (FLZ-1 to FLZ-3) were selected for further characterization. The fungal colonies presented concentric rings on PDA plates, initially white, later turning gray green and producing light orange conidia(Fig.1C). The mature conidia were cylindrical, unicellular, with dimensions of 11.09-20.02 μm × 4.06-6.54 μm, usually containing 2 oil droplets with a size of 3.21-8.36 μm × 2.50-4.55 μm, and L/W ratio of 1.11-2.09(n=100) (Fig.1D). These morphological features were aligned with those of Colletotrichum spp. (Weir et al. 2012). To confirm the identity of the isolates, DNA was extracted and specific gene regions were amplified and sequenced using the following primer sets: ITS (ITS1 and ITS4), GAPDH (GPD1 and GPD2), ACT (ACT-512F and ACT-783R), TUB (T1 and Bt2b), CHS-1 (CHS-79F and CHS-354R), and ApMat (AM-F and AM-R) (Wang et al. 2024). Sequences were submitted to GenBank (Accession nos. PP968176 to PP968178 for ITS, PQ122814 to PQ122816 for GAPDH, PQ122808 to PQ122810 for ACT, PQ169643 to PQ169645 for TUB2, PQ122811 to PQ122813 for CHS-1, and PQ152017 to PQ152019 for ApMat). Maximum likelihood phylogenetic analysis using combined sequences of ITS, GAPDH, ACT, TUB2, CHS-1 and ApMat in MEGA X confirmed the isolates as Colletotrichum fructicola (Fig.2), marking the first report of this pathogen on A. squamosa L. in China. Pathogenicity tests were conducted on wound and nonwound healthy A. squamosa L. fruits to confirm the pathogenicity of the isolated C. fructicola. The fruits were wounded using a sterile needle. Each wounded and nonwounded fruit was inoculated with a 10 µL conidial suspension (1×106 conidia/mL) drop at corresponding sites. Control fruits were inoculated with sterile water. Five fruits per treatment were used and the experiment repeated three times. The fruits were placed in an incubator at 28°C and 85% RH. Anthracnose symptoms developed on the inoculated fruits within 5 days (Fig.1E-F), while control fruits remained symptom-free (Fig.1G-H). C. fructicola was successfully reisolated from the symptomatic fruits, fulfilling Koch's postulates and confirming its role as the causal agent of the disease. According to previous reports, C. fructicola also causes anthracnose on Psidium guajava (William R. O et al. 2024), leaf spots on apple (Moreira Rafaele R et al. 2019), and Kadsura coccinea (Jiang et al.2021). This study identifies C. fructicola as the pathogen causing anthracnose on A. squamosa L. and no cases have yet been discovered in the field in China. This report will raise awareness among growers about the anthracnose disease of A. squamosa L. and develop effective disease control strategies.