The genus Taxus is the natural material of the anticancer drug paclitaxel (Xiong et al. 2021). Harvesting sources of paclitaxel from the wild has greatly decreased the population of these trees. One of the taxus species, Taxus × media Rehder, a natural hybrid of taxus trees, has a higher paclitaxel content (Zhou et al. 2019). It has been introduced and cultivated in Sichuan, Chongqing, Yunnan, Zhejiang, Jiangxi, and other places in China. In 2021, approximately 20% of T. media (an average 30% of the affected area per tree) showed obvious shoot and leaf blight symptoms in a plantation of taxus trees (about 40 ha of the planting area), located in Sandaoyan county, Sichuan province, China (GPS, 103°94'60″N, 30°84'97″E). Initially, brown necrotic spots appeared on shoots. Gradually, the spots increased in number, expanded to the leaf attached to the branch, and caused wilting of the shoots and leaves. To identify the pathogen, symptomatic samples were randomly collected. Lesion margins of the diseased leaves and barks were surface sterilized for 1 min in 75% ethanol, rinsed with sterile distilled water three times, dried with sterile filter paper, placed on potato dextrose agar (PDA) amended with streptomycin sulfate (50 mg/liter), and incubated at 28°C in the dark. Six purified fungal isolates were obtained. Collected isolates with similar morphology were described as Botryosphaeria spp. (Zhang et al. 2021). The colonies were initially white, gradually became dark gray with dense erial mycelium after 5 days, and formed black pycnidia (Dimensions, 121.3 to 134.6 μm, n = 5) after 16 days. Conidia were fusiform, aseptate, transparent, and thin-walled (23.6 ± 1.2 × 7.27 ± 1.3 μm, n = 50), similar to B. dothidea (Hattori et al. 2021). For pathogenicity testing, ten 2-year-old seedlings of T. media were selected. Fungal cakes of the isolate Tmsdy-2 were applied to the punctured stems of seedlings and covered with Parafilm. Pieces of sterile medium were used as controls. All the seedlings were incubated at 25 ± 2°C, 50% relative humidity, and 16 h of light in a greenhouse. Four days later, the inoculated seedlings developed brown spots and were blighted in 14 days, with symptoms similar to the original diseased plants. The controls remained healthy. The same fungus was reisolated from the infected tissues and subsequently identified by morphological characteristics and DNA sequence analysis. The pathogenicity test was repeated three times with similar results, confirming Koch's postulates. For molecular identification, the DNA of the isolates was extracted using a Quick-DNA Extraction Kit (Tiangen Biotech, Beijing). The ITS, LSU, SSU, TUB2, and TEF 1-α genes were amplified with the primer pairs ITS1/ITS4, LR0R/LR05, NS1/NS4 (Li et al. 2018), Bt2a/Bt2b, and EF1-728F/EF1-986R (Hattori et al. 2021), respectively. The generated sequences were deposited in GenBank with accession numbers OQ179939 (ITS), OQ179940 (LSU), OQ179942 (SSU), OQ268596 (TUB2), and OQ268597 (TEF 1-α). BLAST analyses showed >99.65% identity with previously deposited sequences of B. dothidea in GenBank. Based on the maximum likelihood method, phylogenetic analysis revealed 100% bootstrap support values with B. dothidea. The fungus was identified as B. dothidea based on morphological and multilocus phylogenetic analyses. To our knowledge, this is the first report of B. dothidea causing shoot and leaf blight of T. media in China. These results will contribute to developing control strategies for this disease.
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