First report of Botryosphaeria dothidea as the causal agent of a new fruit rot disease of pepper in China.

Abstract

Pepper is an important and widely cultivated economic vegetable in the world (Yin et al., 2021). In June 2021, approximately 25% to 33.3% of the pepper plants had rot disease symptoms in Zhuanghang Comprehensive Experimental Base (30.894829 °N, 121.391374 °E), Fengxian district, Shanghai city, China. Water-soaked spots appeared on fruits that increased in size and leading to smelly fruit decay. To isolate the pathogen, three pepper samples with severe symptoms were collected. The samples were surface disinfected with 70% ethanol for 30 sec, 10% chlorine bleach for 10 min, rinsing with sterile water for three times and the rot tissues were cut and dried on sterile filter paper. The dried paper was later placed on potato dextrose agar (PDA) medium and incubated at 28°C (Tang et al., 2021). After 2-3 days, four types of colonies with different colony appearances were observed, in which only one can induce fruit rot phenotype (data not shown). Four isolates were cultured for molecular identification in each type. ITS1/ITS4, T1/βt-2b and EF1-526F/EF1-1567R primers were used to amplify the internal transcribed spacer region (ITS), the beta-tubulin (TUB2) and the translation elongation factor I alpha (EF1-α) genes, respectively (Chen et al., 2018) and corresponding sequences from the isolates were analyzed with BLAST. Sequences of the isolate which can induce pepper decay were submitted to GenBank under the accession numbers of OM663701 (ITS), OM720127 (TUB2) and OM720128 (EF1-α). The results showed that the pathogen had 99% sequence homology to most strains of Botryosphaeria dothidea (B. dothidea) and displayed the highest sequence similarity to strain LBSX-1 (ITS: KF55123), strain JGT01 (TUB2: MW202404) and isolate CZA (EF1-α: MN025271). Based on molecular characterization, the isolate was identified as B. dothidea isolate SH01. A phylogenetic tree was constructed using Maximum Parsimony (MP) methods by MEGA7, and showed that SH01 was closely related to isolate CMW9075. To confirm the pathogenicity, five healthy pepper fruits were surface sterilized, 500μl of conidial suspension (1×103 conidia/ml) were injected into pepper (sterilized distilled water as control). Six days post inoculation (dpi), fruit rot symptoms appeared and the pepper decayed at 12 dpi. Four days post inoculation with mycelium plugs (from a 4-day-old culture on PDA, PDA plugs as control), hyphae were observed in the inoculation site and B. dothidea was re-isolated from the symptomatic areas, thus fulfilling Koch's postulates (Back et al., 2021, Chen et al., 2020). The pepper rotted severely at 7 dpi. The colonies of SH01 were pale to white and gradually turned into gray in 4-6 days. Conidia of the pathogen were unicellular, aseptate, hyaline and fusiform to fusoid, with dimensions of 19.7-23.5 μm × 3.8-5.2 μm (average = 21.9 μm × 4.8 μm, n = 50). Hyphae were transparent, branched and composed of multiple cells. The characteristic was consistent with the descriptions of B. dothidea (Vasic et al., 2013). B. dothidea belongs to Botryosphaeriaceae, causing widespread diseases in many plant species, commonly associated with cankers and dieback of woody plants and economic crops, such as plumcot trees (Back et al., 2021), eucalyptus (Yu et al., 2009) and soybeans (Chen et al., 2020) in China and Korea. Our findings reported for the first time that B. dothidea (SH01) can induce the pepper rot disease and future work on its pathogenesis may provide strategies for disease control against this fungus.