First report of Colletotrichum godetiae causing anthracnose fruit rot on Prunus pseudocerasus in Zhejiang province, China.

Abstract

Prunus pseudocerasus (L.) G. Don is an economically important crop, with 8,420 hm2 of harvested area and 35,000 tons in 2020 (https://www.fao.org/faostat/zh/#data/QCL), and is one of the favorite fruits among consumers. A severe fruit rot disease of P. pseudocerasus cultivar "HeiZhenZhu" was observed in an orchard in Pujiang county (119°42'E, 29°21'N), Zhejiang province in April 2022. Sixty-three plants from a survey of about 200 plants showed anthracnose symptoms, with a disease incidence of more than 30%. Ten diseased fruits were collected from eight different trees. The naturally affected fruits during color changing stage showed initial light brown necrotic lesions, later, the lesions were depressed, and dark brown, the fruits were rotten, and pink conidial masses were produced (Fig. 1 A-F). Conidia were transferred using a sterilized needle into sterile water, diluted to approx. 10 conidia/µL, and spread onto potato dextrose agar (PDA) (Thermo Scientific™) (containing cephalosporin 50 µg/mL). After 24 h, single colonies were transferred, and six single-spore isolates were obtained from different plants. When the strains were grown at 26°C for 7 days, the colonies on PDA were flat with entire margin, surface medium gray-green to white, reverse salmon, light gray-green to white (Fig. 1 G). The conidia of the representative strain YTTJ-JHGS5 were unicellular, hyaline, smooth-walled, cylindrical to fusiform with one end round and one end acute, or both acute ends, 14.4 to 18.9 µm (mean = 16.4 ± 0.9 µm, n = 30) × 4.4 to 5.8 µm (mean = 5.1 ± 0.3 µm, n = 30) (Fig. 1 I). Conidiogenous cells were hyaline, smooth-walled, cylindrical to clavate, 6.5-16.8 µm × 2.6-5.3 µm (n = 30), opening 1.1-2.0 µm (Fig. 1 H). Appressoria were single, light brown to medium brown, elliptical or irregular in shape, the outline entire or undulate, 9.2 to 12.1 µm (mean = 9.2 ± 1.1 µm, n = 30) × 4.6 to 6.7 µm (mean = 5.6 ± 0.4 µm, n = 30) (Fig. 1 J). The morphological characteristics of YTTJ-JHGS5 were consistent with the Colletotrichum acutatum species complex (Damm et al. 2012). To further identify the species, the internal transcribed spacer (ITS), actin (ACT), beta-tubulin (TUB), chitin synthase (CHS-1), calmodulin (CAL) and glyceraldehyde-3-phosphate dehydrogenase (GPD) genes were sequenced (Weir et al. 2012; O'Donnell et al. 2000). The thirty-six sequences had been deposited in GenBank (ITS: ON155427-ON155432; ACT: ON191542-ON191547; CAL: ON191548-ON191553; CHS-1: ON167522-ON167527; GPD: ON191554-ON191559; TUB2: ON191560-ON191565, respectively). A BLAST search of these sequences (ITS, ACT, CHS-1, GPD, and TUB2) showed 99% identity with the sequences of ex-holotype C. godetiae CBS133.44 (ITS: JQ948402; ACT: JQ949723; CHS-1: JQ949063; GPD: JQ948733; TUB2: JQ950053) (Damm et al. 2012); the sequences of CAL genes of these strains showed 99% identity with the sequence of C. godetiae VV-087 (CAL: MK416001) (Varjas et al. 2020). The Bayes phylogenesis showed that six strains and C. godetiae CBS133.44 were clustered in a robust branch (Bayesian posterior probability = 1). Based on the morphological characteristics and phylogenesis, these strains were identified as C. godetiae. To fulfill Koch's postulates, in April 2022, live plant pathogenicity tests were performed in the field, and color-changing-stage fruits of five-year-old trees were disinfected with 75% alcohol and air-dried for 10 min to volatilize excess alcohol. A conidial suspension (50 mL of 1 × 106 conidia/mL) was sprayed on fifteen fruits for each plant, fifteen fruits sprayed with sterilized water served as control. The experiment was repeated four times, each repeat contained ten trees. The daily average temperature and daily average RH in the orchard were 22°C and 67%, respectively. After 6 days, the fruit surfaces were depressed, dark brown, later, the lesions were expanded, and pink conidial masses were observed (Fig. 1, L). Control fruits remain healthy (Fig. 1, K). C. godetiae was re-isolated from the lesions of inoculated fruits. C. godetiae has been reported on Ceanothus sp., Fragaria × ananassa and so on worldwide (Farr and Rossman 2022), in addition, C. fructicola infected cherry (Zhao et al. 2022). To our knowledge, this is the first report of C. godetiae causing anthracnose fruit rot on Prunus pseudocerasus in China. This disease occurs mainly on immature fruits and leads to yield loss in the field, therefore it is necessary to take preventive measures in advance.