Davidia involucrata Baill. (D. involucrate), also known as dove tree, is listed as the first class national protected plant in China and the only extant member of the Davidiaceae family (Fu & Jin 1992). Referred to by the terms 'living fossil' and 'giant panda' owing to its evolutionary status as a Tertiary relic and its native distribution, D. involucrate exhibits substantial ornamental and academic value (Fang & Song 1975; Wu et al. 2004). A small rounded head inflorescence beneath its big white bracts have a unique charm to catch people's attention, thus they were cultivated in many areas of the world as an ornamental plant (Claßen-Bockhoff & Arndt 2018). In September 2021, dove trees in Meigu country (N 28°33', E 103°14'), Sichuan Province, China were found to appear symptoms of leaf blight of unknown origin. This blight disease incidence was 90% in a survey of 30 D. involucrata trees. Early symptoms appeared as circular, necrotic tissue that developed into circular or irregular spots (FigS1. A). Five leaves exhibiting typical symptoms of this form of leaf blight were excised from the margin between diseased and healthy tissue. These pieces were then treated for 40 s with 75% ethanol for surface sterilization, followed by treatment with 5% NaClO for 2 min, rinsed then plated on potato dextrose agar (PDA) medium supplemented with carbenicillin and ampicillin (each 50 μg/mL), and incubated in the dark for 4 days at 28°C. Pure cultures were then prepared by transferring hyphal tips from the edges of these colonies onto fresh PDA plates, with isolate LW11 being selected as a representative isolate for causal pathogen characterization. These cultured colonies were initially white before turning grayish-black over time (FigS1. B). Conidia were single-celled, black, spherical or oblate and ranged from 10 - 16.0 μm in diameter (mean = 12.5 ± 0.43 μm, n = 40) (FigS1. C), with conidia being present at the tip of conidiophores on hyaline vesicles. These morphological traits were found to align well with those of Nigrospora oryzae (Wang et al. 2017). To confirm this tentative identification, gDNA was extracted from isolate LW11, followed by the amplification of the internal transcribed spacer (ITS) region, beta-tubulin (TUB2), and translation elongation factor 1-alpha (TEF1) sequences with the respective ITS1/ITS4 (White et al., 1990), Bt-2a/Bt-2b (Glass & Donaldson 1995), and TEF1-728F/EF1-986R primer pairs (Carbone et al. 1999). Fragments of 577 bp, 442 bp, and 309 bp were obtained. A maximum likelihood bootstrapping approach (1000 bootstrap replicates) was used to construct a phylogenetic tree based on a combination of the ITS, TUB, and TEF1-α sequences, indicating that isolate LW11 clustered with other N. oryzae isolates (FigS2). The ITS, TUB, and TEF1-α sequences from isolate LW11 were deposited in GenBank with the accession numbers OL659284, OL685345, and OL685347, respectively. The pathogenicity test was performed by detached D. involucrate leaves with mycelial plugs, with the other half instead being inoculated using pure agar plugs as a negative control. Following incubation for 5 days, black lesions were evident on leaves inoculated with mycelial plugs (FigS1. F; FigS1, DE) but not on control leaves (FigS1. F). This report is the first to our knowledge of D. involucrata leaf blight by N.oryzae in China or anywhere else in the world. Further research is thus needed to better manage the spread of this disease with the goal of protecting this living fossil species.
Keywords: Davidia involucrate; Leaf Blight; Nigrospora oryzae.