First report of Botryosphaeria dothidea as a causal agent to stem rot disease on plumcot trees in Korea

Botryosphaeria dothidea (B. dothidea) is a fungal pathogen commonly associated with stem canker, dieback, and rot disease in a variety of woody plants worldwide (Dong and Guo, 2020). In Korea, B. dothidea was reported to cause a disease problem to serval crops such as apple and blueberry (Kim, 1995; Choi, 2011). In early 2020, a typical symptom resembling the stem rot disease was spotted to occur at a plumcot cultivation area around Wanju (35.827870, 127.030380) province, Korea. At the early stage of infection, a small blister appeared on the plumcot branch and stem. However, as the blister extended, a light brown canker was observed appeared on the infected area and in some cases a sticky sap oozed from the branch bark crack. If not managed or treated properly, all leaves beyond the infection site will turn brown, wilt, and the whole plumcot tree eventually dies. A survey in the affected area showed that approximately 5% of the plumcot trees were infected which cause up to 10% reduction in total production. To identify the causal agent, symptomatic tissues were excised and surface sterilized with 70% ethanol for 30 sec followed by 1% NaClO for 30 sec before rinsing with sterile water, thrice. The samples were then dried with a piece of filter paper and later air-dried before being placed on a potato dextrose agar (PDA). The PDA plates were then incubated at 25°C for 5 days with 12 hours light/dark cycles period. Among several fungal isolates obtained, four were selected for further analyses. Morphological identification revealed that the fungal conidia were hyaline, ovoid, fusiform (type that rarely form a septum) and unicellular with an average size of 18 - 20 μm × 4.5 -5.5 μm (n = 50). These morphological characters have a strong resemblance to B. dothidea that described by Slipper et al., (2004). For molecular identification, Internal transcribed spacer (ITS), beta-tubulin (β-tubulin) and elongation factor 1 alpha (EF-1α) were amplified and sequenced using universal primer pairs ITS1/ITS4 (White et al., 1990), Bt2a/Bt2b (Glass and Donaldson, 1995) and EF1/EF2 (O’Donnell et al. 1998) respectively. Alignment analysis showed that ITS (LC602817), β-tubulin (LC602820) and EF-1α (LC602821) sequences were 99-100% identical to the orthologous genes identified in B. dothidea infecting soybean in China [MW130133 (identity 537/536 bp), MW147482 (identity 394/394 bp) and MW147481 (identify 250/250 bp) respectively] (Chen et al. 2021). However, phylogenetic analysis of concatenated ITS, β-tubulin and EF-1α genes sequence established the identity of these isolate as B. dothidea. Due to the 100% identical at the molecular level, isolate NIHHS 20-262 was selected as a representative for further analysis. For the pathogenicity test, fungal mycelium (via PDA plug) was used as a source of inoculum for both intact and detached plumcot stems trials. For the intact trial, mycelium was inoculated on the wounded spots of ten plumcot stems that grew at the NIHHS trial farm. Ten days post-inoculation (dpi), disease symptoms i.e. stem colour turn from greenish to dark brown was observed at the inoculated sites. For the detached trial, mycelium was inoculated on the wounded spots of ten detached plumcot stems. The inoculated stems were kept in a closed container to maintain 90% humidity before incubated at 25ºC in the dark. Interestingly, on the detached stems, disease symptoms (greenish colour turn to dark brown) were observed to appear seven days early compare to intact stems. A sterile PDA plug replacing fungal mycelium served as a negative control and the result shows no symptoms were observed on either intact or detached control stems. For consistency purposes, pathogenicity tests on intact stems were performed on three different plumcot trees, whereas three biological replicates for detached stems. Isolation and re-identification of two colonies from the infected sites (intact and detached stems) were attempted and the results obtained were identical to the original isolate, thus fulfilling Koch’s postulates. Local farmers described this disease as a “certain death disease” in plumcot. Therefore, accurate identification of B. dothidea as the causal agent is critical for effective disease management to minimise qualitative and quantitative losses in the plumcot industry. Although has been reported to cause dieback disease in blueberry in Korea (Choi, 2011), to our knowledge, this is the first study to report B. dothidea causing stem rot diseases on the plumcot trees in Korea.