Taxus media Rehder is a high-value tree species and can be used for landscape afforestation and medicinal purposes considering its beautiful shape and secondary metabolite paclitaxel. In May 2019, branch blight of T. media was observed in plantations in Hangzhou, Zhejiang province, China. The lesions were brown, necrotic and sunken, and the branches were withering. Plants from five plantations were surveyed, and the disease incidence was 60%. Necrotic tissues from 5 plans from each plantation were cut into small pieces (5×5 mm), surface disinfected with 75% alcohol for 30 s and 2% sodium hypochlorite solution for 2-3 min, washed twice with sterile water, then dried on sterile filter paper. Branch pieces were aseptically transferred to potato dextrose agar (PDA) plates and incubated at 25℃ in the dark for 3 days. Five purified fungal cultures were obtained and the representative isolate (LA-01) that grew slightly more vigorously under the same condition was selected for morphological characterization and DNA sequence comparison. The colony was initially white and cottony, with circular growth, undulated edge, wavy surface and pale beige on the back. The conidia were long spindle-shaped, straight or slightly curved, 21-27 × 6-8 μm (n=50) in overall shape. They contained 5 cells with those at the apex and base hyaline. The second and third cells from the apex were darker (brown) than the fourth cell from the apex (pale brown). Two or three (most frequently observed) hyaline appendages were apparent at the apex, 16-31 μm in length. Morphological characteristics of the obtained isolate were consistent with those in the genus of Neopestalotiopsis (Solart et al. 2018). DNA of LA-01 was extracted from fresh mycelia using Minibest Universal Genomic DNA Extraction Kit (Takara, Toyoto, Japan) according to the manufacturer's instructions. The partial regions from the internal transcribed spacer (ITS), β-tubulin gene (TUB2), and translation elongation factor subunit 1-a gene (TEF1) were amplified and sequenced using universal primer pairs ITS5/ITS4, BT2A/BT2B, and EF1-526F/EF1-1567R (Maharachchikumbura et al. 2012), respectively. The resulting nucleotide sequences were individually subjected to BLAST searches in GenBank. The nucleotide sequences of ITS (MZ148633, 554 bp), TUB2 (MZ286974, 473 bp ), TEF1 (MZ286975, 1030 bp) of the isolate LA-01 showed 99.64% similarity to ITS sequence (MW512858.1), 99.35% similarity to TUB2 sequence (MH423961.1), 98.18% similarity to TEF1 sequence (KU096881.1). The multi-locus phylogenetic tree was constructed based on the combined ITS-TUB2-TEF1 sequences using MEGA 7 using the neighbor-joining method, indicating that LA-01 represents Neopestalotiopsis clavispora. Inoculation was done on branches of ten T. media plants in the field to verify the pathogenicity of LA-01. Two healthy branches of each plant were surface sterilized with 75% ethanol allowed to dry, and two small wounds on each branch were obtained by puncturing with a sterilized needle. One wound was inoculated with 5-mm-diameter PDA discs excised from 7-day-old cultures, and wrapped with parafilm; the other inoculated with sterile PDA plugs as control, and wrapped with parafilm. Disease symptoms developed on all N. clavispora-inoculated wounds 3 days after inoculation. The yellow brown lesions became apparent 20 days later, whereas the control wounds remained asymptomatic. The fungus N. clavispora was reisolated from the diseased lesions using the same methods above, fulfilling Koch's postulates. Previously, the species was reported to cause leaf spot disease on another Taxus tree species T. chinensis in China (Wang et al. 2019). This is the first report of T. media branch blight caused by N. clavispora worldwide. Special attention should be paid to the damage it may cause to T. media producers, and precautionary monitoring on T. media population will be a significant part of the conservation efforts in China.
Keywords: Neopestalotiopsis clavispora; Taxus media; branch blight; morphological characterization; multi-locus phylogenetic tree.