First Report of Anthracnose Caused by Colletotrichum aenigma on Actinidia arguta in China

Abstract

Actinidia arguta (Sieb. & Zucc.) Planch. ex Miq. is a fruit tree being newly cultivated worldwide and is a widely distributed wild fruit tree in China (Zhao 2002). Owing to their fruits rich in vitamin C, A. arguta enjoys the labels “world treasure” and “fruit king.” In September 2017, anthracnose symptoms were observed on the fruit of a 6-year-old A. arguta plantation in Dandong, Liaoning Province, China. Symptoms began with light brown round spots that developed into slightly sunken brown or black-brown lesions on infected pericarp; 15 to 20% of the trees exhibited typical anthracnose symptoms. For characterization, 5-mm² pieces of symptomatic tissue were surface sterilized with 1% NaOCl for 2 min, triple rinsed with sterile water, dried on sterilized filter paper, and cultivated on acidified potato dextrose agar (PDA) medium at 25°C to obtain the pathogenic fungus. Three isolates were cultured on PDA at 25°C under a 12-h/12-h light/dark cycle for 7 days. The average resulting colony diameter was 6.80 ± 0.80 cm. Colonies were round with smooth edges and white aerial mycelia. The surfaces were fluffy and white, and the colonies’ undersides were pale yellow to white. Perithecia and ascospores were produced on PDA. The ascospores were cylindrical, both ends blunt, with slight depressions in the middle, and no setae or conidial discs. Conidial spores were smooth and transparent, cylindrical or stick type, both ends blunt, with small particles inside, evenly distributed, 16.1 to 19.7 × 5.0 to 8.1 µm (average = 17.8 ± 1.03 × 6.01 ± 0.72, n = 50). Appressoria were brown to dark brown, usually scattered or irregular, 7.2 to 11.4 × 4.3 to 8.2 µm (average = 9.29 ± 1.08 × 6.33 ± 0.86, n = 20). Based on morphological and cultural characteristics, the isolates were identified as part of the Colletotrichum gloeosporioides species complex (Deng et al. 2017; Phoulivong et al. 2010; Weir et al. 2012). To confirm the species identification, six DNA regions of the randomly selected strain AACAWY203DRT5 were amplified by polymerase chain reaction and sequenced: rDNA internal transcribed spacers regions (ITS), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), calmodulin (CAL), chitin synthase (CHS) (Gan et al. 2017), actin (ACT) (Liang et al. 2017), and β-tubulin (TUB2). A BLASTn algorithm-based search revealed that all sequences (GenBank accession nos. MH476561 for ITS, MH476562 for GAPDH, MH476563 for ACT, MH476564 for CAL, MH476565 for CHS, and MH476566 for TUB2) had 99 to 100% sequence identities with Colletotrichum aenigma (NR_120140 for ITS, JX009913 for GAPDH, KX885151 for ACT, KU251793 for CAL, KX513884 for CHS, and KY820891 for TUB2). Based on these results, the isolate was identified as C. aenigma. To confirm pathogenicity, 18 A. arguta fruits were surface disinfested by immersion in 1% NaOCl for 1 min, triple rinsed with sterile water, and then dried on sterilized filter paper. Inoculations were performed by inoculating 10 μl of a conidial suspension (10⁶ spore/ml) on the fruit surface. As controls, nine fruits were mock inoculated with sterile distilled water. All fruits were maintained at 25°C, about 90% relative humidity and with natural light. After 1 week, symptoms started to develop only on inoculated fruit, whereas control fruit did not develop symptoms. The fungus was only consistently reisolated from the diseased fruits, thus fulfilling Koch’s postulates. To our knowledge, this is the first report C. aenigma causing anthracnose of A. arguta in China. The disease leads to lower fruit quality and decreased yield, and research is needed on management options to minimize losses.