Post-harvest Anthracnose of Carambola (Averrhoa carambola) Caused by Colletotrichum fructicola in China.

Abstract

Carambola (star fruit), a popular fruit of Averrhoa carambola in many parts of the world, is considered to have many beneficial nutritional and medicinal effects (Lakmal, K., et al,2021). In March 2020, anthracnose disease was observed on carambola (about 15% of the fruit showed similar symptoms) in multiple local agricultural markets (113°36'E, 23°11'N) of the Yuancun district in Guangzhou, China. Initial symptoms of infected fruit samples appeared as water-soaked, brown lesions. As the disease progressed, numerous acervuli appeared on fruit surfaces. Salmon-colored spore masses were observed on some fruit. To isolate and identify the pathogen, small pieces (3-5 mm2) were excised from the lesion margins of the fruit, which were surface disinfested by 1% NaOCl (60 s), 70% ethanol (30 s) and then washed twice with sterile distilled water. After surface disinfestation, the tissues were cultured on potato dextrose agar (PDA). Pure cultures were obtained by transferring hyphal tips onto fresh PDA. Fungal isolates (YT-5/6/9) were obtained and the strain YT-5 was selected for further study. The colony of strain YT-5 grown on PDA for 7 days appeared to be cottony, white to pale gray with the presence of multiple masses of conidia. Conidia 13.5-20 × 4.8-6.5 μm (n = 50), hyaline, aseptate, straight and cylindrical with rounded ends. Perithecia were thick-walled and globose with a prominent, narrow neck. Asci 37.1-60.2 × 7.1-11.3 μm (n = 25), 4-8 spored, clavate to cymbiform. Ascospores 7.1-17.2 × 4.5-6.5 μm (n = 35), hyaline, large guttulate at the center, slightly curved with rounded ends. Based on the morphological characteristics, the strain was identified as Colletotrichum fructicola (Prihastuti et al. 2009). The molecular identity of the isolates was confirmed by sequencing the internal transcribed spacer (ITS) rDNA region, chitin synthase (CHS-1), actin (ACT), glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and beta-tubulin (TUB2) genes (Prihastuti et al. 2009, Weir et al. 2012). BLASTN analysis of isolate YT-5 sequences, which were deposited in GenBank (ON428449, ON462353, ON886225, ON886224, ON462354) showed 100% identity with those of Colletotrichum fructicola (MW513778.1, MT918417.1, MW426526.1, MN525875.1, MT941526.1), respectively. A phylogenetic tree analysis based on the concatenated sequences confirmed the isolate YT-5 as C. fructicola. Pathogenicity tests were conducted on fresh fruit of carambola with the isolate YT-5. Healthy fruit was surface disinfested and inoculated with 5 mm mycelial discs of the strain YT-5 after being wounded with a needle or unwounded. Control fruit was inoculated with sterilized PDA plugs. All inoculated fruit was incubated at 26°C for 10 days post inoculation. Control fruit remained asymptomatic, whereas inoculated fruit developed symptomatic at the point of inoculation. The pathogenicity test was performed in duplicate. The pathogenic isolate of C. fructicola was successfully re-isolated on PDA from the symptomatic fruit, thus confirming Koch's postulates. C. fructicola has also been reported as a dominant and aggressive causal agent of anthracnose on sandy pear and avocado in China (Zhang et al. 2015; Li et al. 2022). To our knowledge, this is the first study to isolate and characterize C. fructicola causing carambola anthracnose and evaluate its pathogenicity in China, which will provide a better strategy for accurate diagnosis and effective management of anthracnose disease on carambola.