First Report of Anthracnose Disease on avocado (Persea americana) caused by Colletotrichum fructicola in China.

Abstract

In September 2019, anthracnose-like symptoms were observed on fresh avocado fruits cv. "Hass", which were imported from Peru (Mission Produce, Inc.) and purchased at Ganfuyuan store, Guangzhou, China. After being stored for 5 days at room temperature, initial black specks developed into larger brown or black lesions on fruits, and salmon-colored conidial mass in the lesions were observed. To isolate and identify the pathogen, small pieces (5 mm × 5 mm) were excised from the lesion margins of the fruits, which were surface sterilized by 1% NaOCl (1 min), 70% ethanol (30 s) and then washed twice with sterile distilled water (SDW). After sterilization, the tissues were cultured on Potato Dextrose Agar (PDA) to obtain the pure strain NYG. The colonies grown on PDA for 7 days appeared to be cottony, white to pale gray with the presence of a conidial mass. Conidia 11.7-19 × 3.6-6.4 μm (n = 50), hyaline, aseptate, straight and cylindrical with rounded ends, Appressoria 6.2-11.9 × 4.5 × 7.4 μm (n = 50), brown to dark brown in different shapes. Perithecia were thick-walled and globose with a prominent, narrow neck. Asci 31.5-55 × 6-12.5 μm (n = 15), 6-8 spored, clavate to cymbiform. Ascospores 5-18 × 4.5-6 μm (n = 25), hyaline, large guttulate at the centre, slightly curved, rounded ends. Based on the morphological characteristics, the strain NYG was identified as Colletotrichum fructicola (Prihastuti et al. 2009). The identity of the strain was confirmed by means of multi-locus gene sequencing. The genomic DNA was extracted using Ezup Column Fungi Genomic DNA Purification Kit (Sangon Biotech Co., Ltd., China). The internal transcribed spacer (ITS) rDNA region, actin (ACT), chitin synthase (CHS-1), calmodulin (CAL), glyceraldehyde-3-phosphate dehydrogenase (GAPDH) partial genes (Templeton et al. 1999, Carbone et al. 1999) were amplified and sequenced, which were deposited in GenBank (OL413493, OL517766, OL517768, OM141126, OL517767). BLASTN analysis revealed that DNA sequences of the isolate showed 100% identity with those of C. fructicola (MT476840.1, MK208862.1, MZ965245.1, JX009665.1, MN982434.1), respectively. A phylogenetic tree analysis based on the concatenated sequences confirmed the isolate as C. fructicola. Pathogenicity was tested by infecting the fresh healthy avocado fruits with the isolated strain NYG. The fruits were surface sterilized, three unwounded and wounded avocados fruits were respectively inoculated with 10 l of conidial suspension (1×106 conidia/ml) by the drop inoculation method. Control fruits were inoculated with SDW containing Tween 20 (1 μl/ml H2O), respectively. All inoculated fruits were incubated at 25°C in the dark. Anthracnose symptoms were observed on the wounded and unwounded fruits after 3 to 5 days post inoculation, respectively. No symptoms were observed in the control on both the wounded and unwounded fruits. The pathogenicity test was performed in duplicate. The inoculated fungus was reisolated from the infected fruits and confirmed as C. fructicola, thus confirming Koch's postulates. C. fructicola represents an important fungal pathogen in several plantations worldwide (Farr et al. 2020), for example, the avocado fruits in Mexico (Dionicio, et al. 2018) and New Zealand (Hofer et al. 2021). This is the first report of anthracnose caused by C. fructicola on imported avocado fruits in China. The results of this study can not only help establish effective quarantine measures against anthracnose disease for imported avocado fruits in China, but also provide important reference to prevent the spread of this disease on China's domestic avocados.