【作者】 刘敏；

【导师】 王启明；

【作者基本信息】 河北大学 ， 生物学， 2024， 博士

【摘要】 酵母菌(Yeasts)是指一类主要以单细胞形态存在的真菌,它通过芽殖或裂殖的方式进行无性繁殖,并且在有性生殖阶段不会产生子实体。换而言之,酵母菌实际上是一类单细胞的真核微生物。目前,已经发现并命名的酵母菌约2000种,242个属,然而,这个数字只是自然界酵母菌物种多样性的冰山一角,仅占酵母菌总量的1%。可能超过99%的酵母菌种类,就像许多真菌种类一样,尚未被发现,仍待被挖掘。酵母菌分为子囊菌酵母(Ascomycetous yeasts)和担子菌酵母(Basidiomycetous yeasts)。酵母菌在自然界中广泛存在,可以从水生、大气和陆地生境中分离得到。然而对于从大型真菌(macrofungi),俗称蘑菇(mushroom)上分离酵母菌的相关研究较少。本研究系统地对大型真菌相关酵母菌进行研究,并对分离到的酵母菌进行常规和分子方法相结合的鉴定,为新种地位的确立、系统发育分析和描述提供了重要的数据支撑。在此基础上本研究详细探讨了河北和北京地区大型真菌相关酵母菌的物种多样性,研究结果对该地区后续酵母菌资源调查和物种多样性的研究具有重要的指导作用。本研究通过单基因(ITS、D1/D2 LSU)和多基因(18S,TEF1、RPB1和RPB2)序列分析,对已发表的两个新种进行鉴定、系统发育分析、二代测序和基因组分析。主要结果如下:1.大型真菌的采集本研究提供了一种以分离酵母菌为目的的大型真菌野外处理方法,并对75个(河北地区16个和北京地区59个)生物多样性较丰富的区域进行了大型真菌的采集,共获得713份大型真菌相关样本,其中河北地区387份,北京地区326份。样本依据形态学、生态学和生物地理学等特征,基因序列等信息进行了鉴定。2.酵母菌的分离本研究对采集到的样本,利用稀释涂布法进行酵母菌分离,共分离得到1559株酵母菌。选取其中具有代表性的菌株360株(河北地区173株,北京地区187株)进行后续的形态学观察和系统发育分析。3.酵母菌的鉴定及其系统发育分析本研究对代表性菌株,进行了常规和分子鉴定。为河北和北京地区酵母菌的系统发育和物种多样性研究提供了重要的基础数据。(1)根据ITS和D1/D2 LSU的核酸序列比对分析,将与已知种模式菌株同源率大于99%的菌株鉴定为相应的种。在本研究中共有295株菌被鉴定为76个已知种,分布在9纲15目18科42属。(2)根据ITS和D1/D2 LSU的核酸序列比对分析,将与已知种模式菌株同源率小于99%的菌株鉴定为新种。在本研究中共有65株酵母菌菌株被鉴定为20个新种(包括1个疑难种),分布在3纲5目7科11属。我们对这些菌株进行了详细的生理生化和核酸序列分析,通过比较分离菌株与已知种的生理生化差异和碱基差异,共确定20个新种(包括1个疑难种),分别为Candida sp.nov.1、Candida sp.nov.2、Candida sp.nov.3、Candida sp.nov.4、Clavispora sp.nov.、Kazachstania sp.nov.、Kodamaea sp.nov.、Magnusiomyces sp.nov.、Metschnikowia sp.nov.、Nakazawaea tricholomae sp.nov.、Nakazawaea sp.nov.2、Starmerella sp.nov.1、Starmerella sp.nov.2、Tetrapisispora sp.nov.、Teunomyces sp.nov.1、Teunomyces sp.nov.2、Teunomyces sp.nov.3、Wickerhamomyces corioli sp.nov.、Wickerhamomyces sp.nov.2和Wickerhamomyces sp.nov.3,并对20个新种进行了规范和详细的描述,极大地丰富了我国的酵母菌资源,为后续研究提供了参考。4.已发表新种的多基因系统发育分析和基因组分析(1)从河北省承德市围场县五道沟采集到的口蘑(Tricholoma gambosum)和大盖皮伞(Marasmius maximus)子实体中分离得到2株酵母菌,菌株编号为20–27–1和20–28。通过对ITS、D1/D2 LSU和SSU区域序列以及两个蛋白编码基因RPB1和TEF1的多基因序列分析,结果显示这两株菌株与Nakazawaea ernobii和Nakazawaea holstii亲缘关系密切,ITS、D1/D2 LSU、TEF1和RPB1的相似度分别为97.2–97.1%、99.3–98.7%、91.9–92.5%和84.6%。在生理生化上,这两个菌株在melibiose、inulin和DL–lactic acid的同化方面不同于Nakazawaea ernobii和Nakazawaea holstii。表型和系统发育分析表明,这两个菌株代表了Nakazawaea属的一个新种,并将其命名为Nakazawaea tricholomae sp.nov.(Fungal Names:FN 571492)。(2)从河北省承德市丰宁满族自治县采集到云芝(Trametes versicolor)和小皮伞(Marasmius siccus)的子实体中分离得到2株酵母菌,菌株编号为19–39–3和19–40–2。通过对D1/D2 LSU、SSU和TEF1多基因系统发育分析,确定这些菌株属于Wickerhamomyces属,与Wickerhamomyces alni和Candida ulmi亲缘关系最近,D1/D2LSU、SSU和TEF1的相似度分别为93.3–93.4%、92.6–94.2%、和90.7–94.5%。此外,新种与相关的种之间存在明显的生理生化差异。在这些菌株中没有观察到有性繁殖,因此将这个新发现的物种命名为Wickerhamomyces corioli sp.nov.(Fungal Names:FN571744)。(3)对Nakazawaea tricholomae sp.nov.和Wickerhamomyces corioli sp.nov.进行了二代测序和基因组分析。基因组分析主要包括BUSCO分析(Benchmarking Universal Single–Copy Orthologs)用于评估基因组组装和注释的完整性、VFDB(Virulence factor Database)毒力因子预测、CAZy(Carbohydrate–Active enzymes Database)碳水化合物活性酶预测、PHI–base分析(Pathogen Host Interactions Database)用于病原与宿主互作的分析。研究结果不仅为Nakazawaea和Wickerhamomyces属提供了高质量的参考基因组信息,并为生物信息学分析奠定了坚实的基础,这将有助于种质资源的保护和遗传改良。5.大型真菌相关酵母菌的物种多样性分析基于酵母菌的鉴定结果,探讨了酵母菌的物种多样性和分布。得出以下结论:(1)供试的360株酵母菌鉴定为96个种,分布于44个属,其中已知种76个,未知种20个(新种19个和疑难种1个)。未知种的菌株共计65株,分布于11个属。这一结果显示,大型真菌这类基物蕴含丰富的酵母菌资源。(2)分离得到了子囊菌酵母260株,分布在31个属,20个新种。分离得到了担子菌酵母100株,分布在12个属,0个新种。子囊菌酵母占有较大优势。(3)来源于河北地区的173株酵母菌,鉴定为65个种,分布于36个属,其中51个已知种,14个新种(26株)。来源于北京地区的187株酵母菌,鉴定为45个种,分布于28个属,36个已知种,8个新种(38株)和1个疑难种(1株)。从结果中很容易看出,河北地区酵母菌的多样性和均匀度更高,分离到新种的机率较大,为70%。(4)河北地区酵母菌的优势属是Candida、Teunomyces、Wickerhamomyces、Starmerella和Pichia;河北地区酵母菌的优势种是Vanrija humicola、Wickerhamomyces corioli和Yamadazyma mexicana。北京地区酵母菌的优势属是Candida、Hanseniaspora、Lachancea、Rhodotorula和Pichia;北京地区酵母菌的优势种是Rhodotorula mucilaginosa、Wickerhamomyces corioli、Candida palmioleophila和Saccharomyces cerevisiae。河北和北京地区的共有属有20个。本研究对该地区后续酵母菌资源调查和物种多样性的研究具有非常重要的学术价值。更多还原

【Abstract】 Yeasts refer to fungi that mainly exist in single cell form,asexually reproduce through budding or fission,and do not produce fruiting bodies during sexual reproduction.In brief,yeasts are single celled fungi.At present,up to 2000 species and 242 genera of yeasts have been discovered.However,this number is only the tip of the iceberg in the diversity of yeast species in nature,accounting for only 1% of the total yeasts population.More than 99% of yeast species,like many fungal species,are unknown and yet to be discovered.Yeasts are divided into ascomycetous yeasts and basidiomycetous yeasts.Yeasts can be isolated from aquatic,atmospheric,and terrestrial habitats.Yeasts are widely present in nature,whereas there is relatively little research on isolating yeasts from macrofungi,which are commonly known as mushrooms.This study is the first systematic study on yeasts isolated from macrofungi and identified the yeasts using a combination of conventional and molecular methods.These research results provide important data support for the establishment of new species status,phylogenetic analysis,and description.This study also explored the species diversity of yeasts isolated from macrofungi in Hebei and Beijing areas,and the results have very important instruction meaning for the research on resource survey and species diversity of yeasts in the area.This study conducted single-gene and multi-gene phylogenetic analysis,second-generation sequencing,and genomic analysis on two novel species,further clarifying their status and phylogenetic positions.This not only provides high-quality reference genomic information for the Nakazawaea and Wickerhamomyces genera,but also lays the foundation for subsequent bioinformatics analysis,which will contribute to the protection and genetic improvement of germplasm resources.The main results are as follows:1.Collection of macrofungiThis study found a field treatment method for macrofungi aimed at isolating yeasts.A total of 713 samples were collected from 75 regions(16 in Hebei and 59 in Beijing)with high biodiversity.Among them,387 samples were collected in the Hebei area,and 326 samples were collected in the Beijing area.The sample was identified based on morphological,ecological and biogeographical features,as well as genetic sequence information.2.The isolation of yeasts1559 yeast strains were collected from 713 samples.Among them,857 yeast strains were isolated from 387 samples in the Hebei area,and 702 yeast strains were isolated from 326 samples in the Beijing area.We selected 360 representative strains(173 in Hebei and 187 in Beijing)for subsequent morphological observation and phylogenetic analysis.3.Identification and phylogenetic analysis of yeastsThis study conducted routine and molecular identification on 360 yeast strains and the results provide important basic data for the phylogenetic and species diversity analysis of yeasts.(1)Based on the ITS and D1/D2 region of LSU sequence analysis,strains with percent identity higher than 99% with known type strains were identified as corresponding species.In this study,a total of 295 strains were identified as 76 known species,distributed within 42 genera.(2)Based on phylogenetic trees and ITS,D1/D2 region of LSU sequence analysis,we identified strains with percent identity less than 99% with known type strains as novel species.A total of 65 strains have been identified as 20 novel species(including 1 difficult species),distributed within 11 genera.A total of 20 new species(including 1 difficult species)were proposed by comparing the physiological,biochemical,and gene sequence base differences between isolated strains and existing species,namely Candida sp.nov.1,Candida sp.nov.2,Candida sp.nov.3,Candida sp.nov.4,Clavispora sp.nov.,Kazachstania sp.nov.,Kodamaea sp.nov.,Magnusiomyces sp.nov.,Metschnikowia sp.nov.,Nakazawaea tricholomae sp.nov.,Nakazawaea sp.nov.2,Starmerella sp.nov.1,Starmerella sp.nov.2,Tetrapisispora sp.nov.,Teunomyces sp.nov.1,Teunomyces sp.nov.2,Teunomycessp.nov.3,Wickerhamomyces corioli sp.nov.,Wickerhamomyces sp.nov.2 and Wickerhamomyces sp.nov.3.Standardized and detailed descriptions were provided for 20 new species,greatly enriching China’s yeast resources and providing a reference for subsequent research.4.Multi-genic identification and genomic analysis of novel species(1)Two yeast strains designated as 20–27–1 and 20–28 were isolated from the fruiting bodies of Tricholoma gambosum and Marasmius maximus,respectively,which were collected in Wudaogou,Weichang county,Chengde area,Hebei Province,China.The multi–locus analysis of the sequences of the ITS,D1/D2 LSU,and SSU regions,together with partial sequences of two protein–coding genes RPB1 and TEF1 indicates that the two strains are closely related to Nakazawaea ernobii and Nakazawaea holstii,showing the similarity values of 99.3–98.7%,97.2–97.1%,91.9– 92.5% and 84.6% in ITS,D1/D2 LSU,TEF1,and RPB1,respectively.Physiologically,the two strains are different from Nakazawaea ernobii and Nakazawaea holstii in the assimilation of melibiose,inulin and DL–lactic acid.Both the phenotypic and phylogenetic analyses indicate that those two strains represent a novel species in the genus Nakazawaea,for which the name Nakazawaea tricholomae f.a.,sp.nov.(Fungal Names: FN 571492)is proposed.(2)Two yeast strains,designated as 19–39–3 and 19–40–2,obtained from the fruiting bodies of Trametes versicolor and Marasmius siccu collected in Yunwu Mountain Forest Park,Fengning Manchu Autonomous county,Chengde area,Hebei Province,China,have been identified as a novel asexual ascomycetous yeast species.Analyzing the D1/D2 LSU,SSU,and TEF1 sequences through phylogenetic analyses,it was determined that these strains belonged to the genus Wickerhamomyces,with Wickerhamomyces alni and Candida ulmi as the closest relatives.The two strains are closely related to Wickerhamomyces alni and Candida ulmi,showing similarity values of 93.3–93.4%,92.6–94.2%,and 90.7–94.5% in D1/D2 LSU,SSU,and TEF1,respectively.Additionally,distinct biochemical and physiological variances were observed between the new species and its related counterparts.No sexual reproduction was observed,in these strains,leading to the proposal of the name Wickerhamomyces corioli f.a.,sp.nov.for this newly discovered species.The Fungal Names number of Wickerhamomyces corioli is FN 571744.(3)Second–generation sequencing and genomic analysis were performed on Nakazawaea tricolomae sp.nov.and Wickerhamomyces corioli sp.nov.Genomic analysis mainly includes BUSCO,VFDB,CAZy,and PHI analysis.The research results not only provide high–quality reference genomic information for the Nakazawaea and Wickerhamomyces genera but also lay a solid foundation for targeted bioinformatics analysis in the future,which will contribute to the protection and genetic improvement of germplasm resources.5.Species diversity of yeasts associated with macrofungiBased on the identification results of yeasts,the species diversity and distribution of yeasts were discussed,and the following conclusions were drawn:(1)The 360 strains of yeasts tested are 96 species,distributed in 44 genera,including 76 known species and 20 unknown species(19 new species and 1 difficult species).There are 65 strains of new species distributed in 11 genera.This result indicates that substrates such as macrofungi have abundant yeast resources.(2)260 strains of ascomycetous yeasts were isolated,and distributed in 31 genera and20 new species.100 strains of basidiomycetous yeasts were isolated,and distributed in 12 genera.Ascomycetous yeasts has a significant advantage.(3)173 strains of yeast in the Hebei area,with a total of 65 species distributed in 36 genera,including 51 known species and 14 new species(26 strains).187 strains of yeast in the Beijing area,with a total of 45 species distributed in 28 genera,including 36 known species,8 new species(38 strains),and 1 difficult species(1 strain).It is easy to see from the results that the diversity of yeasts in the Hebei area is better,and the probability of isolating new species is higher,at 70%.(4)The dominant genera in the Hebei area are Candida,Teunomyces,Wickerhamomyces,Starmerella and Pichia.The dominant species in Hebei area are Vanrija humicola,Wickerhamomyces corioli and Yamadazyma mexicana.The dominant genera in the Beijing area are Candida,Hanseniaspora,Lachancea,Rhodotorula,and Pichia.The dominant species in the Beijing area are Rhodotorula mucilaginosa,Wickerhamomyces corioli,Candida palmioleophila,and Saccharomyces cerevisiae.This study has very important guiding significance for the follow-up investigation of yeast resources and species diversity in the region.更多还原