First Report of Stem Rot Caused by Botryosphaeria dothidea and Phomopsis velata on Forsythia suspensa in China

Forsythia suspensa (Thunb.) Vahl (Oleaceae) is a well-known traditional Chinese medicine. It exhibits antioxidant activity and exerts antibacterial, antiviral, and antiemetic effects (Li and Chen 2005). From May 2020 to October 2021, a disease was observed on field-grown forsythia plants in Lingbao City, Henan Province, China (110°33′25.74″E, 34°30′19.34″W). The diseased plants were characterized by stem rot, retarded growth, a declined fruit quality, and in extreme cases, death of F. suspensa. Approximately 3.0% to 5.0% individuals exhibited stem rotten in the main branches. On average, 60% of the branches of infected individual trees were affected by this disease. During the initial infection stage, the bark of the plants was raised and curled, and the xylem and phloem of the stems turned brown color, whereas in the late stage of the infection, the outer bark had dried and become detached, and the inner xylem and phloem had blackened. Upon infection, the growth of plants was reduced, and the main branches became desiccated as the disease progressed. We randomly selected five diseased branches from five plant fields, the bark tissues (about 25 mm²) of which were surface-sterilized in 75% ethanol for 30 s, treated with 1% NaClO for 5 min, rinsed five times with sterile water, and placed on potato dextrose agar (PDA). After incubating 3 days, 20 clones were observed, and two representative strains (FSJF11 and FSJF13, three replicates for each) was selected for intensive study. Samples of these strains have been deposited in Institutes of Traditional Chinese Medicine, Henan University. On PDA, the colonies of FSJF11 were initially white and fluffy in appearance, later turning gray, and finally black. The vigorously growing hyphae were branched and septate. However, no spores was observed during culture. FSJF13 colonies were rapidly growing, initially white in color and later turning gray. After culturing for 20 days, black conidia appeared and yellow conidial horns were released. The alpha conidia were elliptical, slightly pointed at both ends, and each end possessed an oil ball (6.40±0.60 × 1.86±0.25 μm). The beta conidia were slender, linear, and hook shaped with a slightly curved end (28.92±2.81 × 0.96±0.14 μm). DNA of the isolates was extracted using a Fungal Genome DNA Extraction Kit (Sangon Biotech, Shanghai), and selected genes were amplified using the primer pairs ITS1/ITS4 (Tian et al. 2018), LROR/ LR5, and NS1/NS4 (Aiello et al. 2020). Sequences have been deposited in GenBank (ITS: MW834579 and MW834580; LSU: MW829566 and MW829567; SSU: MW834582 and MW834583). The lengths of the amplified ITS, LSU and SSU sequences were 491, 759, and 1013 bp for FSJF11, respectively, and these in FSJF13 were 543, 927, and 901 bp, respectively. The ITS, LSU, and SSU sequences of FSJF11 were found to have sequence identities of 99.19%, 100%, and 100% with those of Botryosphaeria dothidea stains AY259092, EU673243, and Eu673174, respectively, and a phylogenetic tree was constructed based on the concatenated sequences (ITS, LSU, and SSU) revealed that FSJF11 and B. dothidea formed a clade with 96% support. A BLAST search of the Genbank database revealed that the ITS sequence of FSJF13 showed 99.81% identity with that of Phomopsis velata (MN183778). Given that no LSU or SSU sequences of this species are currently available, we constructed a phylogenetic tree based solely on ITS sequences, which revealed that FSJF13 and P. velata formed a clade with 99% support. Based on the morphological and molecular characteristics(Qi et al. 2007), the isolates of FSJF11 and FSJF13 were identified as B. dothidea and P. velata, respectively. Healthy branches of F. suspensa were wounded in vitro after inoculating active fungal cake of B. dothidea or P. velata (diameter = 5 mm) on the bark, and control branches were treated with PDA. In total, each branch was inoculated via four holes were inoculated on each branch, and three branches were used for each treatment. The inoculation sites were covered with a piece of wet absorbent cotton and then wrapped with plastic film, and the branches were incubated at 26 °C. The branches continued to grow after removal of the cotton and the film on the fourth day. All inoculated points on the branches showed lesions similar to those observed in the field, whereas the control branches were asymptomatic. The pathogenicity rates of FSJF11 and FSJF13 (three replicates for each) were 66.67% and 83.33%, respectively. Both species were re-isolated from the symptomatic branches respectively, thereby fulfilling Koch’s postulates. To the best of our knowledge, this is the first report of B. dothidea and P. velata causing branches rot in F. suspensa. The findings of this study will contribute to developing effective strategies for the control of this newly emerging plant disease.