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一株海洋类棘孢木霉化学成分与生物活性研究
邹积雪
Subtype博士
Thesis Advisor季乃云
2022-05-09
Training institution中国科学院烟台海岸带研究所
Degree Grantor中国科学院大学
Keyword类棘孢木霉 次生代谢产物 生物活性 木霉菌剂 促生作用 Trichoderma asperelloides Secondary metabolite Biological activity Trichoderma agent Growth promotion
Abstract木霉是一类来源丰富且具有重要农用价值的真菌类群。木霉不仅能防治植物病原菌,而且能在植物根表面定殖,对植物的生长具有促进作用。随着绿色农业思想的普及,人们越来越迫切地需要环境友好型的农用微生物菌剂。海洋木霉是指来源于海洋的木霉属真菌,由于海洋独特的环境特点,使得海洋木霉本身及其次生代谢产物具有重要的研究意义。 类棘孢木霉(Trichoderma asperelloides) RR-dl-6-11是一株分离自松节藻的内生真菌,对该菌株进行了批量发酵,并提取其次生代谢总产物。利用硅胶柱色谱、反相硅胶柱色谱、Sephadex LH-20凝胶柱色谱、制备型薄层层析和高效液相色谱等手段分离单体化合物,通过核磁共振谱、红外光谱、质谱、圆二色谱和X射线单晶衍射等波谱技术确定单体化合物的结构。共分离鉴定了44个单体化合物,包括18个新化合物(1–18),其中1个罕见的proharziane二萜(1)、3个harziane二萜(2–4)、10个没药烷倍半萜(5–14)、1个杜松烷倍半萜(15)和3个环橙花烷倍半萜(16–18)。化合物4是首次报道的A环开环的harziane二萜,化合物16为首次发现的含有环戊烯环的环橙花烷倍半萜,化合物18是首次发现的A环降解的环橙花烷倍半萜。 对类棘孢木霉RR-dl-6-11粗提物和部分单体化合物进行了抑微藻和抗菌活性测试。抑微藻活性结果表明,类棘孢木霉RR-dl-6-11的粗提物及倍半萜类化合物(5–18)能显著抑制赤潮异弯藻(Heterosigma akashiwo)、海洋卡盾藻(Chattonella marina)、东海原甲藻(Prorocentrum donghaiense)和强壮前沟藻(Amphidinium carterae),IC50值为0.54–13 μg/mL,受试倍半萜对四种微藻的抑制活性优于harziane二萜类化合物。机理研究表明粗提物及化合物5、15、20和23使微藻细胞内的超氧歧化酶、过氧化物酶、可溶性蛋白、丙二醛、叶绿素a和类胡萝卜素的含量发生变化,影响藻细胞内氧化–抗氧化的平衡,造成细胞膜脂质过氧化,破坏光合作用系统,进而影响藻细胞的细胞稳态,最终导致藻细胞死亡。类棘孢木霉RR-dl-6-11的粗提物对围小丛壳(Colletotrichum cingulate)和香蕉枯萎病菌(Fusarium oxysporum f. sp. cubense)具有良好的抑制活性,MIC值分别为6.25和12.5 μg/mL。同时,类棘孢木霉RR-dl-6-11粗提物及部分单体化合物对病原细菌也具有一定的抑制作用,包括鳗弧菌(Vibrio anguillarum)、灿烂弧菌(V. splendidus)、哈维氏弧菌(V. harveyi)、副溶血弧菌(V. parahemolyticus)和柠檬假交替单胞菌(Pseudoalteromonas citrea)。 类棘孢木霉RR-dl-6-11与其它海藻真菌及植物致病真菌的平板对峙实验表明类棘孢木霉RR-dl-6-11可有效抑制受试真菌,作用机制包括生长竞争作用、重寄生作用和抗生作用。酶活实验表明,该木霉通过向胞外分泌纤维素酶和几丁质酶分解镰刀菌细胞壁中的纤维素和几丁质,达到抑制镰刀菌生长的作用。鉴于类棘孢木霉RR-dl-6-11的抗菌优势,利用农产品加工副产物(谷壳:麦麸 = 2:1)将该其开发成类棘孢木霉菌剂应用到盐碱地农业中。小麦的平板萌发实验、温室盆栽实验和田间实验的结果均表明类棘孢木霉菌剂有显著的促生作用,能使小麦苗的总根长、根表面积、根尖数、平均根体积和茎长等指标显著增加。对施用菌剂的大田土壤进行了微生物高通量测序分析,结果表明类棘孢木霉菌剂的施用使大田土壤中的微生物丰富度增加,类棘孢木霉菌剂对细菌群落的影响大于对真菌群落的影响。 本文首次对类棘孢木霉RR-dl-6-11的次生代谢产物进行了研究,其结构类型涵盖二萜、倍半萜、聚酮和甾体,并评价它们的抗赤潮微藻、抗农业植物病原真菌和抗水产病原细菌的活性,探索了抗微藻作用机理,丰富了海洋木霉次生代谢产物的结构和活性多样性。结合菌株抗真菌活性的进一步研究,将类棘孢木霉RR-dl-6-11开发为一种农用菌剂,并应用于盐碱地农业中,具有小麦促生和根际菌群调节的双重作用,为海洋木霉在盐碱地农业中的应用提供了重要参考。
Other AbstractTrichoderma spp. are widespread in nature and have important application in agriculture. They can not only control phytopathogens but also colonize on the root surface of plants to promote their growth. With the prevalence of green agriculture, environment-friendly microbial agents are urgently needed. Marine-derived Trichoderma refers to the Trichoderma spp. obtained from marine environments. Due to the unique habitats, the research on marine-derived Trichoderma species and its secondary metabolites may have important significance. Trichoderma asperelloides RR-dl-6-11 was isolated from the inner tissue of the marine alga Rhodomela confervoides. It was subjected to batch fermentation, and its total secondary metabolites were extracted. The monomeric compounds were separated by silica gel chromatography, reversed-phase chromatographic column, Sephadex LH-20 gel chromatography, preparative thin layer chromatography and HPLC. Their structures were further determined by NMR, IR, MS, ECD and X-ray. Effects on T. asperelloides RR-dl-6-11 resulted in the isolation and identification of 44 monomer compounds, with eighteen (1–18) including one rare proharziane diterpene (1), three harziane diterpenes (2–4), ten bisabolane sesquiterpenens (5–14), one cadinene sesquiterpenen (15) and three cyclonerane sesquiterpenens (16–18). Compound 4 features an opened ring A that has not been reported previously. Compound 16 represents the first occurrence of cyclopentenyl-bearing cycloneranes, and 18 seems a cyclopentyl-degrading cyclonerane derivative. The crude extract and some monomeric compounds of T. asperelloides RR-dl-6-11 were evaluated for antimicroalgal and antimicrobial activities. The results showed that crude extract and sesquiterpenoids (5–18) exhibited significant inhibitory activity against Amphidinium carterae, Chattonella marina, Heterosigma akashiwo and Prorocentrum donghaiense, with IC50 values ranging from 0.54 to 13 μg/mL. Sesquiterpenoids were more active to four phytoplankton species than harziane diterpenes. Mechanistic studies demonstrated that the crude extract and compounds 5, 15, 20 and 23 changed the contents of superoxide dismutase, peroxidase, soluble protein, malondialdehyde, chlorophyll a and carotenoid in microalgae cells. They could disturb the oxidation-antioxidation balance in microalgae cells, cause lipid peroxidation of cell membrane, break down photosynthesis system, affect the cellular homeostasis, and eventually lead to cell death. The crude extract of T. asperelloides RR-dl-6-11 showed excellent inhibitory activity against Colletotrichum cingulate and Fusarium oxysporum f. sp. cubense with MIC values of 6.25 and 12.5 μg/mL, respectively. On the other hand, the inhibition of the marine bacteria Vibrio anguillarum, V. harveyi, V. parahemolyticus, V. splendidus, and Pseudoalteromonas citrea was also assayed, only the crude extract and some monomeric compounds had certain antibacterial activity. Plate confrontation experiments of T. asperelloides RR-dl-6-11 with other algicolous fungi and plant pathogenic fungi indicated that T. asperelloides RR-dl-6-11 could effectively inhibit the tested fungi by growth competition, hyperparasitism and antibiosis. Enzyme activity experiments showed that T. asperelloides could secrete cellulase and chitinase extracellularly to break down the cellulose and chitin in Fusarium cell wall, and achieve the purpose of inhibiting the growth of Fusarium. In view of the significant antifungal activity of T. asperelloides RR-dl-6-11, the T. asperelloides was developed into T. asperelloides agent using by-products of agricultural products processing (chaff: bran = 2: 1) for agriculture in salt-alkaline land. The results of germination experiments on glass plates, pot culture experiments in phytotron and field experiments on wheat showed that the T. asperelloides agent had significant growth-promoting effects, resulting in remarkable increases in total root length, root surface area, root tip number, root projected area, mean root volume and stem length of wheat seedlings. Microbial high-throughput sequencing analysis was carried out on field soils treated with T. asperelloides agent. The results of the analysis revealed that T. asperelloides agent increased the microbial richness in the field soil, and the effects of T. asperelloides agent on bacterial community was greater than that on fungal community. For the first time, the secondary metabolites of T. asperelloides RR-dl-6-11 have been investigated. The structural types cover diterpenes, sesquiterpenes, polyketides and steroids. We also evaluated their activities against red tide microalgae, agricultural plant pathogenic fungi and aquatic pathogenic bacteria, and explored the mechanisms of anti-microalgal action. These enriched the structural and active diversity of the secondary metabolites from marine-derived Trichoderma. Combined with further studies on the antifungal activity of T. asperelloides RR-dl-6-11, RR-dl-6-11 was developed as an agricultural agent and applied in salt-alkaline land. The agent has the dual role of growth-promoting effect on wheat and regulation microflora of rhizosphere, which provides an important reference for the application of marine-derived Trichoderma in saline and alkaline land.
Language中文
Document Type学位论文
Identifierhttp://ir.yic.ac.cn/handle/133337/30521
Collection海岸带生物学与生物资源利用重点实验室
Recommended Citation
GB/T 7714
邹积雪. 一株海洋类棘孢木霉化学成分与生物活性研究[D]. 中国科学院大学,2022.
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