First report of Botryosphaeria dothidea causing brown leaf spot on Daimyo oak, Quercus dentata in China

Quercus dentata is a deciduous oak species widely distributed in northern China, with short petioles and dense grayish brown stellate tomentose on the abaxial surface (Lyu et al., 2018). Q. dentata is cold-tolerant (Du et al., 2022), and its broad leaves are used for tussah silkworm rearing, traditional Chinese medicine, kashiwa mochi in Japan, and Manchu delicacy in Northeast China (Wang et al., 2023). In June 2020, a single Q. dentata plant with brown leaf spots was observed in the Oak Germplasm Resources Nursery (N41°82', E123°56') in SYAU, Shenyang, China. From 2021 through 2022, other two nearby Q. dentata plants (six trees in all) became diseased with similar brown spots on their leaves. The small brown lesions with subcircular or irregular shape gradually expanded, and then the entire leaf turned brown. Under magnification, the diseased leaves contain many conidia. To identify the pathogen, diseased tissues were surface sterilized in 2% sodium hypochlorite for 1 min, and washed in sterile distilled water. Lesion margins were plated onto potato dextrose agar and incubated at 28°C in darkness. The aerial mycelium changed color, from white to dark gray, and dark olive green pigmentation was observed on the medium reverse side after 5 days of incubation. The emerging fungal isolates were repurified by the single-spore method. The mean length and width of spores were 20.32 ± 1.90 × 5.2 ± 0.52 μm (n=50). These morphological characteristics resembled the description of Botryosphaeria dothidea (Slippers et al., 2014). For molecular identification, internal transcribed spacer (ITS) region, translation elongation factor1 alpha (tef1-α), and beta-tubulin (tub) were amplified. These new sequences GenBank accession nos. are OQ383627.1, OQ387861.1 and OQ387862.1. Blastn searches showed 100% homology with ITS sequence of B. dothidea strain P31B (KF293892.1) and 98 to 99% similarity with tef and tub sequences of B. dothidea isolate ZJXC2 (KP183219.1) and B. dothidea isolate SHSJ2-1 (KP183133.1). The sequences were also concatenated for phylogenetic analysis (maximum likelihood). Result support isolate SY1 in the same clade as B. dothidea. Based on the multi-gene phylogeny and morphology, the isolated fungus associated with brown leaf spot on Q. dentata was identified as B. dothidea. Pathogenicity tests were performed on five-year-old potted plants. Conidial suspensions (106 conidial/mL) were applied on punctured leaves using a sterile needle and non-punctured leaves. Non-inoculated plants spayed with sterile water served as control. Plants were placed in a growth chamber at 25°C on a 12h fluorescent light/dark regime. Symptoms similar to those from natural infections were observed after 7 to 9 days (non-punctured also infected). No symptoms were found on non-inoculated plants. The pathogenicity test was repeated three times. Fungi re-isolated from inoculated leaves were comfirmed as B. dothidea on the basis of morphological and molecular characterization as described above, fulfilling Koch's postulates. B. dothidea was previously reported as a pathogen causing branch diebacks and twig dieback on sycamore, red oak (Quercus rubra), and English oak (Quercus robur) in Italy (Turco et al., 2006). It has also been reported to cause leaf spot on Celtis sinensis, Camellia oleifera and Kadsura coccinea in China (Wang et al., 2021; Hao et al., 2022; Su et al., 2021). To our knowledge, this is the first report of B. dothidea inducing leaf spot on Q. dentata in China.