First Report of Botryosphaeria dothidea Causing Fruit Rot of Rellowhorn (Xanthoceras sorbifolium) in China

Yellowhorn (Xanthoceras sorbifolium Bunge) is a woody deciduous shrub that produces capsular fruits with seeds rich in oil and it has also been widely cultivated for biodiesel production in northern China (Venegas-Calerón et al. 2017; Wang et al. 2017). Beginning in June 2015, a rot of yellowhorn that severely reduced fruit production was observed in Weifang City, Shandong Province, China. Typical symptoms were black-brown spots in a radial pattern in the initial stages and thereafter as sunken necrotic lesions that often enlarged and merged, rendering these fruits unusable. Mycelia and fruit bodies were observed on lesions under the moist circumstance. Thirty infected fruits were collected randomly from 30 trees of the three orchards and 90 samples were cut into 6-mm pieces, surface-sterilized, plated onto potato dextrose agar (PDA), and incubated at 25°C in the dark. The colonies then appeared white, aerial mycelia turned dark gray after 8 days, and black pycnidia, circular or irregular, which aggregated mono- to multilocularly in one stroma, appeared after 18 days. Conidia were one-celled, hyaline, aseptate, thin walled, ellipsoidal to fusiform, and measured 16.8 to 30.4 × 3.5 to 8.0 μm (avg. 25.5 × 5.4 μm). The morphological characteristics fit the descriptions of Fusicoccum anamorph of Botryosphaeria dothidea (Moug.) Ces. & De Not. (Phillips et al. 2005; Slippers and Wingfield 2007). The genomic DNA was extracted from four representative isolates (SDXF2, SDXF3, SDXF4, and SDXF6) and amplicons of 18S rRNA gene and elongation factor 1-α gene were obtained using primers ITS1 and ITS4 (Lawrence et al. 2017) and EF1 and EF2 primers (Phillips et al. 2005), respectively. Amplicons were sequenced and compared in GenBank using a BLAST analysis. The ITS sequences (accession nos. MG282090, MG282091, MG282092, and MG282093) had 100% similarity with the sequences of B. dothidea (KR260804, LC317473, MG020754, and JF441086, respectively) and the EF1-α sequences (MG149784, MG421004, MG421005, and MG421006) matched 100% with the sequences of B. dothidea (GU251224, GU251224, HM176507, and HM176506, respectively). A phylogenetic analysis based on ITS and EF1-α sequences placed the four obtained isolates within the same clade containing the B. dothidea isolates PD122, CMW9075, and CMW8000. Pathogenicity tests were carried out in mid-June 2016. Fifty healthy fruits were collected from orchards, rinsed in tap water, and then surface-sterilized in 10% sodium hypochlorite for 30 s. Ten fruits were inoculated with 5-mm mycelial discs from a 5-day-old culture of each representative strain, and 10 healthy fruits were inoculated with uncolonized PDA discs as controls. All fruits were incubated at 25°C and 100% relative humidity. After 10 days, all inoculated fruits showed similar disease symptoms as those observed on yellowhorn fruits in the orchards. No symptoms developed on control fruits. The pathogen was reisolated only from the inoculated fruits and its identity confirmed with both morphology and using molecular tools. These results indicated that the pathogen of yellowhorn fruit rot is B. dothidea. To our knowledge, this is the first report of B. dothidea causing fruit rot of yellowhorn in China, and apparently even worldwide.