红豆杉属是抗癌药物紫杉醇的天然物质(Xiong等。2021)。从野外收获紫杉醇的来源大大减少了这些树的种群。紫杉属物种之一,Taxus×mediaRehder,紫杉树的天然杂种,具有较高的紫杉醇含量(Zhou等人。2019)。已在四川引种栽培,重庆,云南,浙江,江西,以及中国的其他地方。在2021年,大约20%的T.media(平均每棵树的受影响面积的30%)在紫杉树的种植园(约40公顷的种植面积)中显示出明显的芽和叶枯病症状,位于三道岩县,四川省,中国(GPS,103°94\'60″N,30°84\'97“E)。最初,芽上出现棕色坏死斑点。渐渐地,斑点数量增加,扩展到附着在树枝上的叶子,并导致枝条和叶子枯萎。为了识别病原体,随机收集有症状的样本.将病叶和树皮的病变边缘在75%乙醇中表面灭菌1分钟,用无菌蒸馏水冲洗三次,用无菌滤纸干燥,置于用硫酸链霉素(50毫克/升)修正的马铃薯葡萄糖琼脂(PDA)上,并在黑暗中在28°C下孵育。获得了六种纯化的真菌分离物。收集的具有相似形态的分离株被描述为肉毒杆菌。(张等人。2021)。殖民地最初是白色的,5天后逐渐变成深灰色,具有致密的菌丝体,并形成黑色比尼迪亚(尺寸,121.3至134.6μm,16天后n=5)。分生孢子是梭形的,无菌,透明,薄壁(23.6±1.2×7.27±1.3μm,n=50),类似于B.dothidea(Hattori等人。2021)。对于致病性测试,选择10个2年生的T.media幼苗。将分离物Tmsdy-2的真菌饼施用于刺穿的幼苗茎上,并用Parafilm覆盖。无菌培养基片用作对照。所有幼苗均在25±2°C下孵育,相对湿度50%,和16小时的光在温室里。四天后,接种的幼苗在14天内出现棕色斑点并枯萎,症状与原始患病植物相似。对照组保持健康。从感染的组织中重新分离出相同的真菌,然后通过形态学特征和DNA序列分析进行鉴定。致病性测试重复三次,结果相似,确认科赫的假设。对于分子鉴定,分离物的DNA使用Quick-DNA提取试剂盒(天根生物技术,北京)。ITS,LSU,SSU,用引物对ITS1/ITS4,LR0R/LR05,NS1/NS4扩增TUB2和TEF1-α基因(Li等人。2018),Bt2a/Bt2b,和EF1-728F/EF1-986R(Hattori等人2021),分别。产生的序列存放在GenBank中,登录号为OQ179939(ITS),OQ179940(LSU),OQ179942(SSU),OQ268596(TUB2),和OQ268597(TEF1-α)。BLAST分析显示与先前在GenBank中保藏的B.dothidea序列>99.65%的同一性。基于最大似然法,系统发育分析显示,B.dothidea具有100%的自举支持值。根据形态学和多基因座系统发育分析,将真菌鉴定为B.dithidea。据我们所知,这是T.media在中国首次报道B.dothidea引起的芽和叶枯病。这些结果将有助于开发这种疾病的控制策略。
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.