Lycium barbarum Is a New Host of Botryosphaeria dothidea Associated with Soft Rot Disease in China

Goji berry (Lycium barbarum) is a plant of the Solanaceae family that is cultivated in the Chinese provinces of Xinjiang, Ningxia, Gansu, and Qinghai, and its fruit is used as a traditional Chinese medicine (Yossa Nzeuwa et al. 2019). In July 2019, fruit rot was observed at an incidence of 20 to 25% on the Goji berry at a fruit market in Yinchuan, Ningxia, China. The fruit symptoms began as slightly shriveled areas on fruit peel, with noticeable softening of the infested portion of the tissue, followed by rotting and a sour odor. To isolate the pathogen, ten symptomatic tissues were randomly collected from different boxes, surface-sterilized for 30 s with 75% ethanol, followed by 0.1% mercuric chloride, then rinsed in sterile distilled water three times and plated onto PDA. The plates were incubated at 25°C in the dark for 7 days. Five purified fungal isolates from different fruit were obtained and single-spores. Emergent fungal colonies were white with 1 to 3 mm white margins and abundant aerial hyphae, 1 to 6 mm high, that became dark gray after 4 to 5 days. Conidia were hyaline, unicellular, fusiform, and measured 19.3 to 28.2 μm× 3.8 to 6.4 μm (n=50). All the morphological characteristics were consistent with Botryosphaeria spp. (Slippers et al. 2004). Five representative isolates, BJN1-BJN5, were selected for molecular identification. Total genomic DNA of the isolates was extracted with a Plant/Fungi DNA Isolation Kit. Translation elongation factor 1-alpha (EF1) gene and internal transcribed spacer (ITS) regions were amplified with primers EF1-728F/986R (Carbone and Kohn 1999) and ITS1/ITS4, respectively. The sequencing results of the five isolates were consistent, and those of the isolate BJN1 we deposited in the NCBI GeneBank database for EF1 (MK733274) and ITS (MK359291). A BLAST search of the GenBank database indicated that the EF1 and ITS sequences had 100% and 99% similarity, respectively, to B. dothidea ex-type strain (AY236898 and KF766151). A phylogenetic tree was constructed using maximum parsimony methods in MEGA11 and BJN1 isolate clustered with the reference sequence of B. dothidea. Pathogenicity tests were performed, inoculating healthy fruit with both mycelial plugs (7 days old) and conidial suspension (1 × 106 conidia/ml), repeated three times. Mycelial plugs of five isolates (BJN1-BJN5) growing on PDA with a colony diameter of 4 mm were placed on the sterilized surface of 20 Goji berry fruit. Sterile PDA plugs were placed on 12 healthy fruit as a control. In a second test, conidial suspensions of five isolates were sprayed on the surface of 20 healthy fruit and sterilized distilled water was used as a control. The inoculated fruits were maintained in an artificial climate chamber at 25°C and 80% to 85% relative humidity with a 12-h photoperiod for 7 days. The development of soft rot, similar to that observed on the original samples, was observed on inoculated fruit while control fruits remained asymptomatic. The pathogen was reisolated from infected fruit and confirmed as B. dothidea based on morphological characteristics and molecular sequences. To our knowledge, this is the first report of B. dothidea causing postharvest fruit rot of Goji berry, and this pathogen has been reported to cause fruit rot in Kiwifruit (Li et al. 2016) and Yellowhorn (Liu et al. 2018). This study provides information on a new postharvest fruit rot of Goji berry in China that has the potential to cause economic losses.