裙带菜配子体与孢子体的附生微生物群落组成分析

Composition of epiphytic microbial communities in gametophytes and sporophytes of Undaria pinnatifida

中文关键词: 裙带菜  附生微生物  高通量测序  孢子体  配子体

英文关键词: Undaria pinnatifida  Epiphytic microorganisms  High-throughput sequencing  Sporophyte  Gametophyte

中文摘要:

      裙带菜(Undaria pinnatifida)是一种重要的大型褐藻，具有较高的经济和食用价值。藻类的附生微生物既能通过代谢产物调控宿主藻类的生长发育，特定条件下又可能导致病害。了解裙带菜附生微生物群落组成对研究裙带菜与附生微生物间的相互作用、种质资源的有效保存以及防治藻类病害等有重要意义。现有研究大多集中于海带和紫菜，关于裙带菜的附生微生物，特别是不同生活史的对比研究还很少。本研究通过16S rRNA基因高通量测序发现，裙带菜配子体和孢子体的附生细菌群落组成有明显差异，配子体样品中的细菌群落丰度和多样性均大于孢子体。配子体中变形菌门(Proteobacteria)(66.67%)为第一优势菌门，其次为拟杆菌门(Bacteroidetes)(13.48%)和蓝细菌门(Cyanobacteria)(11.13%)，α-变形菌纲(Alphaproteobacteria)(34.58%)为第一优势菌纲，其次为γ-变形菌纲(Gammaproteobacteria)(31.01%)；而孢子体中蓝细菌门(95.67%)占绝对优势，其次为放线菌门(Actinobacteria)(1.65%)和厚壁菌门(Firmicutes)(1.48%)。裙带菜样品经18S rRNA基因测序检测出链形植物(Streptophyta)、纤毛虫门(Intramacronucleata)、担子菌门(Basidiomycota)、顶复亚门(Apicomplexa)、节肢动物门(Arthropoda)、硅藻门(Bacillariophyta)、脊索动物门(Chordata)、腹毛动物门(Gastrotricha)、子囊菌门(Ascomycota)和毛霉菌门(Mucoromycota)，其中，担子菌门、子囊菌门和毛霉菌门属于真菌，孢子体的真核微生物群落丰度大于配子体。本研究确定了裙带菜配子体和孢子体附生微生物群落组成以及不同细菌和真核微生物的相对丰度，结果表明，2个世代之间存在显著差异，为后续研究藻类宿主与微生物之间的相互作用、提高海带目褐藻种质保存技术提供了基础的数据支持。

英文摘要:

      Undaria pinnatifida is a brown alga with high economic value. Its annual production is second only to Saccharina japonica among economically important brown algae in China. Due to climate change and increased cultivation density, the occurrence of diseases in cultivated seaweeds has become more frequent and serious in recent years. Most diseases are directly or indirectly related to the interactions between the seaweed host and the epiphytic microorganisms such as bacteria. There is a close relationship between algae and epiphytic microorganisms. When the phycosphere niche maintains a dynamic balance, the two rely positively on each other growing and developing together. When the balance is disturbed, the structure of the epiphytic microbial community may change, possibly resulting in algal diseases. The absence of certain microbes may also lead to the failure of key biological processes such as the morphogenesis of the host algae. Therefore, understanding the composition of the epiphytic microbial community is of great significance for the study of the interaction between U. pinnatifida and epiphytic microorganisms and the better control of U. pinnatifida diseases. In addition, stock resources of U. pinnatifida are usually conserved in the form of gametophytes, which can persist for a long time under controlled conditions. Hence, understanding the composition of the epiphytic microbial community will also facilitate the development of efficient conservation methods and help remove microbial contaminations when axenic cultures need to be established. The life history of U. pinnatifida consists of the alternation between heteromorphic macroscopic sporophytes and microscopic gametophytes. The stark morphological and physiological differences between these two phases suggest that the composition of epiphytic microbial communities between them is likely different. However, studies on the composition of epiphytic microbial communities, especially comparison studies between sporophytes and gametophytes in U. pinnatifida are limited. The advent of high-throughput sequencing provides robust and efficient tools for studying the composition and relative abundance of the microbial community associated with the algae. In the present study, sporophytes and gametophytes (each with three biological replicates) of U. pinnatifida derived from the cultivated population in Dalian China were selected as the study objects. After DNA extraction and PCR amplification of the v3–v4 region of 16S rRNA gene and the v4 region of 18S rRNA gene, Illumina HiSeq 2500 high-throughput sequencing platform was used to sequence these specific regions. We identified and classified the composition of epiphytic microbial communities of the gametophytes and sporophytes of U. pinnatifida based on the sequencing results. A total of 446 932 effective reads were obtained through 16S rRNA gene sequencing. The raw reads have been submitted to the GenBank database (https://www.ncbi.nlm.nih.gov/) with the accession number PRJNA823903. The bacterial community composition of gametophyte and sporophyte was revealed to be significantly different, and the diversity of the bacterial community in gametophyte samples was higher than that in sporophyte samples. In gametophytes, Proteobacteria (66.67%) was the most dominant phylum, followed by Bacteroidetes (13.48%) and Cyanobacteria (11.13%). At the class level, Alphaproteobacteria (34.58%) was the most abundant, followed by Gammaproteobacteria (31.01%), Bacteroidia (13.16%), and Oxyphotobacteria (11.13%). Cyanobacteria (95.67%) was predominantly detected in sporophytes, followed by Actinobacteria (1.65%) and Firmicutes (1.48%). The distribution of Oxyphotobacteria, Alphaproteobacteria, Bacteroidia, Gammaproteobacteria, Negativicutes, OM190, Acidimicrobiia, Erysipelotrichia, Planctomycetacia, and Verrucomicrobiae were found to be different between gametophyte and sporophyte samples, among which OM190, Acidimicrobiia and Planctomycetacia were unique to gametophytes. The genus-level bacterial groups detected in gametophyte samples were Lewinella (10.06%), Leucothrix (5.99%), Sulfitobacter (4.06%), Bifidobacterium (0.02%), while Bifidobacterium accounted for 1.41% of the bacterial genus of sporophyte samples. There were 57.37% and 95.68% uncultured bacterium in gametophytes and sporophytes, respectively. We obtained a total of 473 770 effective reads through 18S rRNA gene sequencing. A major share (97.22%) of microeukaryotes in gametophytes were unclassified, while in sporophytes, the number was 94.95%. Streptophyta, Intramacronucleata, Basidiomycota, Apicomplexa, Arthropoda, Bacillariophyta, Chordata, Gastrotricha, Ascomycota, and Mucoromycota were detected both in the gametophytes and sporophytes. Among them, Basidiomycota, Ascomycota, and Mucoromycota belong to fungi. The community abundance of sporophyte samples was higher than that of gametophytes. In gametophyte samples, except for Phaeophyceae (88.77%) to which U. pinnatifida belongs, Copepoda of Arthropoda, Mediophyceae of Bacillariophyta, Mammalia of Chordata, Prostomatea of Intramacronucleata, and Liliopsida were dominant, with a proportion of 0.62%, 0.50%, 0.25%, 0.23%, and 0.23%, respectively. In addition to Phaeophyceae (94.49%), Conoidasida of Protozoa, Agaricomycetes of Basidiomycota, Spirotrichea of Intramacronucleata and Mammalia of Chordata were predominant in sporophytes, accounting for 0.91%, 0.83%, 0.51% and 0.24% of OTUs, respectively. Chytridiomycetes, Nassophorea, Colpodea, Tremellomycetes, Sordariomycetes, Conoidasida, Agaricomycetes, Arachnida, and Chromadorea were only detected in the sporophytes, and there was a significant difference in Spirotrichea abundance between gametophytes and sporophytes. In conclusion, the composition of epiphytic microbial communities and the relative abundance of different bacteria and microeukaryotes in the sporophytes and gametophytes of U. pinnatifida were determined through high-throughput sequencing of the amplicons of the v3–v4 region of 16S rRNA gene and the v4 region of 18S rRNA gene. Remarkable differences were revealed between the two life stages, indicating that their growth and development are associated with different microbial communities. The results of this study provide valuable information for sustainable cultivation and stock culture conservation of this important kelp species.