中国黄河流域壳囊孢属的分类和系统学研究

壳囊孢属(Cytospora Ehrenb.)真菌是一类重要的林木病原菌，在世界范围内广泛分布，可侵染85余种木本植物引起腐烂病和枝枯病。其中以桉树、苹果树和杨柳树腐烂病最为常见，导致树势衰弱甚至死亡，造成严重的经济损失和生态破坏。壳囊孢属己报道110个种，其中我国记载31个种。由于传统壳囊孢属真菌的分类主要依据其寄主植物种类，而该类真菌寄主专一性弱，且有性型与无性型的对应关系不明确，加之大部分种存在模式标本丢失、菌株缺乏，以及种的记载缺乏详细的形态描述和DNA数据分析等原因，导致该属同物异名现象极为普遍。同时该属对应白孔座壳菌属(Leucostoma)、黑腐皮壳属(Valsa)、小黑腐皮壳属(Valsella)和拟蕉孢壳属(Valseutypella)等多个有性阶段，目前这些属所含种类己被提议作为壳囊孢属的同物异名处理。这种不完善的分类学体系和现状，对我国该类病原菌的准确鉴定和病害的正确识别都带来了很大困难，并直接影响植物病害的预防与控制。因此，需要以广泛的调查和大量采集新鲜标本为基础，通过结合详细的形态学观察和多基因系统发育分析重新校正该属在我国重点地区的分类学研究，建立相应的壳囊孢属的分类系统。我国黄河流域地区土地资源丰富，气候特征明显，是重要的森林资源分布和生态保护区域，也是寄主主导性林木病害常发区域。本论文收集我国黄河流域14科25属45种寄主上的林木病害标本684份，分离菌株700株。通过真菌子实体、孢子及相关结构、培养物的形态特征，并结合2个核糖体基因和4个编码蛋白质基因，利用最大节约法(MP)，最大似然法(ML)和贝叶斯分析(BI)三种不同的分析方法构建系统树，分析其亲缘和进化关系。主要结果如下：1.确定了壳囊孢属种水平上的形态学分类依据。根据无性型子实体形态将我国壳囊孢属真菌划分为6个形态学类型。类型Ⅰ为有黑色界限圈，单腔室单孔口；类型Ⅱ为有黑色界限圈，多腔室单孔口；类型Ⅲ为有黑色界限圈，多腔室多孔口：类型Ⅳ为无黑色界限圈，单腔室单孔口；类型Ⅴ为无黑色界限圈，多腔室单孔口；类型Ⅵ为无黑色界限圈，多腔室多孔口。每种类型再根据培养形态、分生孢子及相关结构的大小和有性型形态划分到种。子实体产生喙状结构和寄主种类也有一定分类意义，可作为次要分类依据。2.记述了我国黄河流域的22种壳囊孢属真菌，包括Cytospora berberidis、 C. gigalocus、 C. gigaspora 和 C. pruinopsis、 C. rostrata、 C. sibiraeae 和 C. sophoricola等7个新种，明确了其地理分布特点。其中以该属模式种金黄壳囊孢(C. chrysosperma)分布最为普遍。3.分析了壳囊孢属多基因系统发育关系。发现内转录间隔区(ITS)，核糖体大亚基片段(LSU)，肌动蛋白基因(act)、 RNA合成酶Ⅱ第2亚基基因(rpb2)、转录延长因子基因(tef-1α)和β微管蛋白基因(tub)等具有很好的种间变异性和种内稳定性，可以作为用于壳囊孢属快速分子鉴定和DNA条形码的候选基因。4.揭示了我国杨柳、核桃、榆、国槐寄主上壳囊孢属病原菌的物种多样性。研究发现杨柳树腐烂病的壳囊孢属病原菌有Cytospora chrysosperma、 C. nivea、 C. populina 和 C. rostrata。核桃枝枯病症状多样，分别对应四种壳囊孢属病原菌：C.atrocirrhata、C. chrysosperma、 C. gigalocus和C.sacculus。引起榆树腐烂病的壳囊孢属病原菌为：C. carbonacea、 C. chrysosperma、 C. pruinopsis 和 C. ribis。国槐枝枯病的不同表观症状由三种壳囊孢属病原菌引起：C. chrysosperma、 C. sophorae Bres.和 C. sophoricola.。本研究还报道了Cytospora chrysosperma可侵染杨属、柳属、榆属、槐属、火炬树、红瑞木和白蜡7种寄主，其中榆属、槐属、火炬树、红瑞木和白蜡为新纪录寄主。

Fungi of the genus *Cytospora* Ehrenb. are important forest pathogens with a worldwide distribution, which can infect more than 85 woody plant species and cause canker and branch blight. Among them, canker on eucalyptus, apple trees, willows and poplars is the most common, leading to tree decline and even death, causing severe economic losses and ecological damage. A total of 110 species of *Cytospora* have been reported worldwide, among which 31 species have been documented in China. Traditionally, the classification of *Cytospora* fungi was mainly based on their host plants. However, this genus has weak host specificity, the correspondence between sexual and asexual morphs is unclear, and most species suffer from missing type specimens, lack of available strains, insufficient detailed morphological descriptions and lack of DNA sequence data for most documented species. All these factors lead to a high prevalence of synonymous names within the genus. Meanwhile, the genus corresponds to several sexual morph genera including *Leucostoma*, *Valsa*, *Valsella* and *Valseutypella*, and species in these genera have been proposed as synonyms of *Cytospora*. This imperfect taxonomic system and current status pose great difficulties for the accurate identification of these pathogens and correct recognition of the diseases in China, and directly affect the prevention and control of plant diseases. Therefore, it is necessary to re-calibrate the taxonomic research of this genus in key regions of China based on extensive surveys and large-scale collection of fresh specimens, combined with detailed morphological observations and multi-gene phylogenetic analyses, so as to establish a corresponding taxonomic system for *Cytospora*. The Yellow River Basin in China is rich in land resources with distinct climatic characteristics, serving as an important area for forest resource distribution and ecological conservation, as well as a frequent occurrence area of host-dominant forest diseases. In this study, a total of 684 forest disease specimens and 700 isolated strains were collected from 45 host species belonging to 25 genera under 14 families in the Yellow River Basin of China. Morphological characteristics of fungal fruiting bodies, spores and related structures, and cultures were observed, combined with two ribosomal RNA genes and four protein-coding genes. Phylogenetic trees were constructed using three different analytical methods: Maximum Parsimony (MP), Maximum Likelihood (ML) and Bayesian Inference (BI), to analyze their phylogenetic relationships. The main results are as follows: 1. The morphological classification criteria for species level of *Cytospora* were determined. Fungi of *Cytospora* in China were divided into six morphological types based on the morphology of their asexual fruiting bodies: Type I: with black boundary zone, unilocular and single ostiole; Type II: with black boundary zone, multilocular and single ostiole; Type III: with black boundary zone, multilocular and multiple ostioles; Type IV: without black boundary zone, unilocular and single ostiole; Type V: without black boundary zone, multilocular and single ostiole; Type VI: without black boundary zone, multilocular and multiple ostioles. Each type was further assigned to species based on cultural morphology, size of conidia and related structures, and sexual morph morphology. The presence of rostrate structures on fruiting bodies and host plant species also have certain taxonomic significance and can be used as secondary classification criteria. 2. A total of 22 *Cytospora* species in the Yellow River Basin of China were documented, including seven new species: *Cytospora berberidis*, *C. gigalocus*, *C. gigaspora*, *C. pruinopsis*, *C. rostrata*, *C. sibiraeae* and *C. sophoricola*, and their geographical distribution characteristics were clarified. Among them, the type species of the genus *Cytospora chrysosperma* has the widest distribution. 3. The multi-gene phylogenetic relationships of *Cytospora* were analyzed. It was found that the internal transcribed spacer (ITS), large subunit of ribosomal RNA gene (LSU), actin gene (act), RNA polymerase II second largest subunit gene (rpb2), translation elongation factor 1-α gene (tef-1α) and β-tubulin gene (tub) exhibit good interspecific variability and intraspecific stability, and can be used as candidate genes for rapid molecular identification and DNA barcoding of *Cytospora*. 4. The species diversity of *Cytospora* pathogens on willows, poplars, walnut trees, elms and Chinese scholar trees in China was revealed. Studies found that the pathogens causing canker on willows and poplars include *Cytospora chrysosperma*, *C. nivea*, *C. populina* and *C. rostrata*. The symptoms of walnut branch blight are diverse, corresponding to four *Cytospora* pathogens: *C. atrocirrhata*, *C. chrysosperma*, *C. gigalocus* and *C. sacculus*. The pathogens causing elm canker are *C. carbonacea*, *C. chrysosperma*, *C. pruinopsis* and *C. ribis*. Different apparent symptoms of Chinese scholar tree branch blight are caused by three *Cytospora* pathogens: *C. chrysosperma*, *C. sophorae* Bres. and *C. sophoricola*. This study also reported that *Cytospora chrysosperma* can infect seven host genera: *Populus*, *Salix*, *Ulmus*, *Sophora*, *Rhus typhina* (staghorn sumac), *Cornus alba* (red osier dogwood) and *Fraxinus chinensis* (Chinese ash), among which *Ulmus*, *Sophora*, *Rhus typhina*, *Cornus alba* and *Fraxinus chinensis* are newly recorded hosts.