First Report of Botrytis californica Causing Gray Mold on Blueberry in China.

Abstract

Blueberry (Vaccinium corymbosum) is a perennial shrub which grows in acid soil. Its cultivation area has recently expanded rapidly due to its unique flavor and high nutritional value (Silver and Allen 2012). In June 2021, gray mold symptoms (8 to 12% incidence) were observed on harvested fruits of the blueberry cultivar 'Lanmei 1' during storage in Jiangning (31°50'N, 118°40'E), Nanjing, China. The infection started with wrinkles, atrophy, and depressed spots on the fruit surfaces, ultimately resulting in fruit rot. To determine the causal agent, diseased fruits were sampled and rinsed with sterile water (Gao et al. 2021). Small fragments (5 × 5 × 3 mm) of decayed tissues were excised and plated onto acidified potato dextrose agar (PDA) (containing 4 ml of 25% lactic acid per liter). Plates were incubated at 25°C for 3 to 5 days, and edges of fresh culture were transferred onto new plates. This procedure was repeated thrice to obtain pure cultures. Two isolates (BcB-1 and BcB-2) were obtained. Their colonies appeared whitish to gray, with an average daily growth rate of 11.3 ± 0.6 mm (no. of plates = 30). Conidiophores were long and erect, 2560.9 to 4885.3 μm × 10.7 to 13.0 μm in size. Conidia were one-celled, elliptical to ovoid, nearly hyaline, and 9.6 to 12.5 μm × 6.7 to 8.9 μm in size. Sclerotia were gray to black in color and round or irregular in shape. These morphological features were identical to those of Botrytis spp. (Amiri et al. 2018). To further identify the isolates, we amplified four genetic markers including internal transcribed spacer region (ITS), heat-shock protein 60 (HSP60), glyceraldehyde-3-phosphate dehydrogenase (G3PDH), and DNA-dependent RNA polymerase subunit II (RPBII) (Saito et al. 2014; Walker et al. 2011). Sequences of BcB-1 and BCB-2 were deposited in GenBank under accession nos. OP721062 and OP721063 for ITS, OP737384 and OP737385 for HSP60, OP746062 and OP746063 for G3PDH, and OP746064 and OP746065 for RPBII, respectively. BLAST analysis indicated that these sequences shared high identities (99 to 100%) with those of other B. californica isolates. Phylogenetic analysis showed that BcB-1 and BcB-2 clustered with several reference isolates in the B. californica clade. To confirm their pathogenicity, fresh blueberry fruits were surface sterilized with 0.5% sodium hypochlorite, rinsed with sterile water, air-dried, then wounded thrice per fruit using a sterile needle at the equator. Twenty wounded fruits were sprayed with 10 ml conidial suspension (1 × 105 conidia/ml) of each isolate onto the fruit surface. Twenty fruits treated with sterile water were used as controls. Inoculated or non-inoculated fruits were incubated at 25°C with 90% relative humidity. The pathogenicity test was performed twice. After 5 to 7 days, all inoculated fruits developed disease symptoms similar to those observed on the original fruits, while non-inoculated control fruits were asymptomatic. Re-isolated pathogens from the inoculated fruits exhibited identical morphological characteristics to those of BcB-1 and BcB-2. Their identity as B. californica was also verified based on their ITS sequences. Previously, B. californica has been reported to cause gray mold on blueberry in the Central Valley of California (Saito et al. 2016). To our knowledge, this is the first report of B. californica causing gray mold on post-harvest blueberry fruits in China. These results can provide the basis for future studies on the occurrence, prevention, and control of this disease.