KMS XINJIANG INSTITUTE OF ECOLOGY AND GEOGRAPHY，CAS
| 新疆三种外来入侵植物内生菌及活性代谢产物的分离鉴定 | |
| Alternative Title | ENDOPHYTIC MICROBES AND THEIR BIOACTIVITIES ISOLATED FROM 3 INVASIVE PLANTS GROWING IN XINJIANG |
| NIGORA KUCHKAROVA | |
| Subtype | 博士 |
| Thesis Advisor | 邵华 ; 张道远 |
| 2020-05-30 | |
| Degree Grantor | 中国科学院大学 |
| Place of Conferral | 北京 |
| Degree Discipline | 理学博士 |
| Keyword | 内生菌 意大利苍耳 刺苍耳 黄花刺茄 植物生长调节活性 Endophytic microbes Xanthium italicum Xanthium spinosum Solanum rostratum plant growth regulatory activity |
| Abstract | 随着全球经济一体化进程的加快，外来生物入侵已成为世界性的社会经济和环境问题。外来入侵物种在不同国家、不同生态区域间进行不断的迁移和转移，加剧了外来物种的传入频率和扩散风险。入侵植物不仅造成了巨大的经济损失，也导致了全球生物多样性的快速丧失，这使得对其入侵机理的研究更具有现实意义与紧迫性。越来越多的研究表明，入侵植物与微生物之间的相互作用关系深刻影响着入侵植物的适应性和竞争力，并在其成功入侵的过程中发挥着重要的作用。其中，入侵植物对病原微生物的响应直接影响到外来植物在入侵地的生存状态与成功入侵进程，土壤微生物为外来植物的成功入侵提供了多样的异质性栖息环境，而植物内生菌在外来植物成功入侵进程中也发挥着抗逆、促生等重要作用。植物内生菌能够通过产生具有生物活性的化合物，来促进寄主植物生长，增强其抵抗不利环境的能力，促进外来物种成功入侵。 外来植物意大利苍耳、刺苍耳和黄花刺茄能够在新疆恶劣的环境条件下强势入侵且迅速繁衍，其内生菌可能扮演着重要角色。 对其内生菌多样性的研究，不仅有助于未来对其入侵机理的深度解析，更是为开发利用内生菌次生代谢产物中具有植物生长调节活性的化合物为对环境友好的植物生长调节剂或除草剂提供了新的可能。本研究采用纯培养手段，从野外自然入侵地采集三种入侵植物材料， 对其内生菌进行分离、纯化，通过 ITS rDNA 和 16S rRNA 基因基因片段进行测序，对内生菌的种属进行初步的分子和形态鉴定。 此外，选择部分潜在新种， 对其次生代谢产物的植物生长调节活性进行检测，从中选择三个菌株进行了大规模摇床培养和次生代谢产物的提取，完成了第一步的硅胶柱层析分离工作，并对其中 SR84 菌株以活性跟踪方法，结合反复硅胶柱层析，制备 HPLC 等手段分离鉴定活性化合物。 主要研究结果包括：(1) 从意大利苍耳种子中共分离到 316 个内生细菌菌株，其中上位种子 78 株，下位种子 107 株，果皮 131 株，初步归类为 58 种，其中果皮分离 51 种，上位种子 30 种，下位种子 48 种。 经统计发现果实各部位分离的菌株数与其重量具有显著相关性，重量大的下位种子内生菌多余上位种子。 内生细菌的丰富度及多样性按如下顺序排列: 下位种子>果皮>上位种子；其中 XIXJ104 (Bacilluszhangzhouensis) 、 XIXJ116 (Bacillus safensis) 、 XIXJ053 (Bacillusparalicheniformis) 、 XIXJ160 (Bacillus mojavensis) 和 XIXJ226 (Staphylococcusepidermidis)为优势菌株，分别占总分离数的 9.5%， 5.38%， 4.11%， 4.11%和4.43%，以 Bacillus 属的细菌占绝对优势地位。意大利苍耳果实中， 下位种子重量大，易于萌发， 是否与其内生细菌具有较高的多样性及丰富度有关值得研究；(2) 从刺苍耳植株中共分离到 102 个内生真菌菌株，其中叶片 22 株，茎 28株，根 52 株。 这些菌株归类为 54 种，其中根 31 种，茎 21 种，叶 16 种；Aspergillus 和 Penicillium 是刺苍耳内生真菌群落的优势属，其中菌株 X2(Aspergillus flavus), X32 (Penicillium oxalicum) 和 X96 (Alternaria alternate) 的分离频率最高， 合计占总分离数的 5.88%，而菌株 X37 与比对菌株 Penicilliumcrustosum (HF680261.1) 最大相似性只有 77%， 提示该菌株可能为潜在新种；；(3) 从黄花刺茄植株中共分离到 176 个内生真菌菌株，其中叶片 57 株，茎57 株，根 62 株，分属于 45 种。 其中， 菌株 SR66 (Penicillium chrysogenum、SR15 (Penicilliu oxalicum) 和 SR46 (Aspergillus tubingensis) 分别占总分离数的29%、 11%和 8%，属于优势菌株， 与刺苍耳类似， Aspergillus 和 Penicillium 也是黄花刺茄内生真菌群落的优势属，它们在寄主的成功入侵中有无作用尚有待进一步的研究；(4) 经 BLAST 比较分析， 发现有 11 个菌株相似度低于 97%，有可能为潜在新种。 其中黄花刺茄 5 种： SR59 Aspergillus niger, SR65 Doratomyces stemonitis,SR84 Purpureocillium lilacinum , SR152 Mucor circinelloides, SR192 Penicilliumchrysogenum；刺苍耳 6 种： X1 Dichomitus albidofuscus, X15 Dematiopleosporafusiformis, X37 Penicillium crustosum, X53 Penicillium oxalicum, X60 Lagenariasiceraria , X 120 Fusarium petroliphilum。对这 11 株真菌的次生代谢产物进行了提取及活性测定，发现其中 8 个菌株的代谢产物具有显著的植物生长调节活性。(5) 为了从潜在新种中分离得到新颖的活性化学物质， 选择活性最强的三个真菌菌株 SR192， XSP15 和 SR84 三个菌株以 PDB 培养基在 25℃温度下进行大规模的摇床培养，以乙酸乙酯进行萃取，依次从 SR192 菌株中得到次生代谢产物 2.3g，从 XSP15 中得到 9.77g，在 SR84 中得到 5.73g，经测试均具有显著的植物生长调节活性，对上述三个菌株的提取物均已完成了第一次的硅胶柱层析工作。(6) 在上述菌株中，黄花刺茄内生真菌 SR84 具有最强的植物生长调节活性，同时该菌株是一个潜在新种，因此对其次生代谢产物进行了进一步的研究。以活性跟踪手段，经反复硅胶柱层析、凝胶柱层析、半制备 HPLC 等方法，从SR84 代谢产物中分离鉴定出 3 个具有植物生长活性调节的化合物，分别为：腺苷(adenosine), 啤酒甾醇 (cerevisterol)和 5-甲基脲嘧啶 (thymine)， 三个化合物均表现出较强的植物生长抑制活性，其中 5-甲基脲嘧啶是作用最强的化合物，在最低浓度（4 μg/ml）下即可抑制受试植物反枝苋(Amaranthus retroflexus)根长生长的 63.8%，当浓度增加至 100 和 500 μg/ml 时，反枝苋种子萌发被完全抑制。腺苷显示出中等的抑制活性，在 100 μg/ml 浓度时可使受试植物根长降低为对照的 53.3%，而 500 μg/ml 处理可完全抑制种子的萌发。啤酒甾醇的生物活性最弱，在 100 和 500 μg/mL 浓度下对反枝苋根长生长抑制为对照的 56.6%和 32.1%。三个化合物对反枝苋苗高的作用与根长的作用类似，但程度较轻。所有化合物均是首次从 Purpureocillium 属真菌中分离得到，并且其植物毒活性均为首次报道。综上所述，本研究首次对入侵新疆的外来植物意大利苍耳、刺苍耳、黄花刺茄的内生菌进行了分离纯化，共分离得到 594 株植物内生菌，对其多样性有了初步的了解， 并且发现多种内生菌的次生代谢产物均具有植物生长调节活性，为后期继续研究外来种内生菌是否与入侵相关奠定了基础。从潜在新种黄花刺茄内生真菌 SR84 的次生代谢产物中分离鉴定出具有植物生长调节活性的 3 个化合物， 为首次从 Purpureocillium 属真菌中分离得到，并且三个化合物的活性均为首次报道， 具有开发为生物源除草剂的潜力。 |
| Other Abstract | With the acceleration of global economic integration, invasive plant species areworldwide socio-economic and environmental problem. Invasive species in differentcountries and different ecological regions are constantly migrating and transferring,which aggravates the introduction frequency and diffusion risk of invasive species.More and more studies showed that the interaction between invasive plants andmicroorganisms has a profound impact on the adaptability and competitiveness ofinvasive plants, and plays an important role in the process of their successful invasion.Among them, endophytes might play an essential role in the successful invasion processof alien plants due to their ability of enhancing resistance to adverse environment andpromote growth. Exploring the biodiversity of endophytes of these invasive species laythe foundation for future work on the possible involvement of endophytes in theirinvasion success. Besides, the secondary metabolites produced by endophytes withplant growth regulatory activity have the potential to be explored as environmentfriendly plant growth regulator.In this study, endophytes of three plants were isolated and purified by conventionalculture and the species and morphology of endophytes were identified by ITS rDNAand 16S rRNA sequencing; Bioassay guided fractionation and column chromatographymethod were applied to isolate secondary metabolites with biological activity. The mainwork includes investigation of the biodiversity of endophytes of 3 exotic invasive plantsvia isolation and identification of their endophytes in order to analyze their possibleinvolvement of their invasion process, and isolation and identification of 3 activecompounds from the potential new strain SR84. The main findings include:1. In total 316 bacteria strains were isolated and identified from the upper seeds(78), lower seeds (107) and shells (131) of Xanthium italicum. Among them, from fruitsof Xanthium italicum, 58 endophytic bacteria species were identified, including 51 fromthe shells, 30 from the upper seeds and 48 from the lower seeds. Statistically, the number of strains isolated from each part of the fruit was significantly correlated withits weight; bacterial richness and diversity were at the following order: lower seeds>shells> upper seeds; among them, XIXJ104 (Bacillus zhangzhouensis), XIXJ116(Bacillus safensis), XIXJ053 (Bacillus paralicheniformis), XIXJ160 (Bacillusmojavensis), and XIXJ226 (Staphylococcus epidermidis) were dominant strains whichaccounted for 9.5%, 5.38%, 4.11%, 4.11%, and 4.43% of the total isolated strains,respectively; the genus Bacillus was the most dominate genus;2. In total 102 endophytic fungal isolates were collected from leaves (22), stems(28), and roots (52) of 15 Xanthium spinosium, which were classified into 54 species,including 31 from roots, 21 from stems and 16 from leaves. Fifty-four endophytic fungiwere isolated from Xanthium spinosum, including 31 from roots, 21 from stems and 16from leaves. The separation frequencies of strains X2 (Aspergillus flavus), X32(Penicillium oxalicum) and X96 (Alternaria alternate) were the highest, accounting for5.88% of the total isolates; the maximum similarity between strain X37 and the relativestrain Penicillium crustosum (HF680261.1) was only 77%, therefore it was suspectedto be a potential new species; Aspergillus and Penicillium were the dominant genus ofthe endophytic fungal community;3. In total 176 endophytic fungal isolates were collected from leaves 57, stems 57,and roots 62 of Solanum rostratum, which were classified into 45 species accordingly.SR66 (Penicillium chrysogenum), SR15 (Penicillium oxalicum) and SR46 (Aspergillustubingensis) were dominant, which accounted for 29%, 11% and 8% of the totalisolates, respectively. The maximum similarity between the strain SR84 and thealignment strains Purpureocillium lilacinum (KJ938575.1) and SR152 and the strainSR65 Doratomyces stemonitis (KX302062.1) was 95% and 85%, respectively,indicating they might be potential new species. Aspergillus and Penicillium were thedominant genus of the endophytic fungal community. SR84 was chosen for furtherstudy of its secondary metabolites due to its high plant growth regulatory activity andthe fact that it is a potential new species.4. According to the phylogenetic analysis, 11 potentially new fungal strains (SR59 Aspergillus niger, SR65 Doratomyces stemonitis, SR84 Purpureocillium lilacinum,SR152 Mucor circinelloides, SR192 Penicillium chrysogenum) from Solanumrostratum and (X1 Dichomitus albidofuscus, X15 Dematiopleospora fusiformis, X37Penicillium crustosum, X53 Penicillium oxalicum, X60 Lagenaria siceraria, X120Fusarium petroliphilum) Xanthium spinosium plants were isolated. Secondarymetabolites of these 11 fungi strains were tested for their plant growth regulatoryactivity, and 8 of these strains showed significant inhibiting activity at the concentration500 µg/ml.5. In order to find new bioactive substances from isolated potential new species,strains of SR-192， XSP-15 and SR-84 were chosen for large scale cultivation (250Ctemperature with rotation 140 rpm speed using PDB medium). The culture wasextracted with ethyl acetate, which gave the following extracts: SR-192, 2.3g, XSP-15,9.77g and SR-84, 5.73g, all these extracts have completed the first silica columnchromatography procedure.6. Among the above-mentioned strains, SR84 possessed strong plant growthregulatory activity, considering the fact that this fungus was a potential new species,therefore it was chosen for further study. Three compounds, i.e. adenosine, thymine andcerevisterol, were purified and identified from the secondary metabolites produced bySR84 using silica gel column chromatography, Sephadex LH-20 columnchromatography and semi-preparative HPLC which were all found to possesssignificant plant growth regulatory activity. Among these 3 compounds, thymineexhibited the most potent inhibitory activity, which reduced root length of Amaranthusretroflexu to 63.8% of the control at the lowest tested concentration, 4 μg/ml, and seedgermination was completely suppressed when the concentration was increased to 100and 500 μg/ml. Adenosine showed moderate suppressive activity, which reduced rootgrowth to 53.3% of the control at 100 μg/ml, whereas 500 μg/ml adenosine totallyinhibited seed germination. Meanwhile, cerevisterol exhibited the weakest activity,which suppressed root length to 56.6% and 32.1% of the control at 100 and 500 μg/mL,respectively. Shoot length of Amaranthus retroflexus responded similarly to 3compounds but to a lesser extent. All compounds were isolated from Purpureocilliumfungi for the first time, and their phytotoxicity was reported for the first time.Our study was the first to investigate the biodiversity of endophytic microbes ofthese 3 invasive species, which led to the isolation of 594 species as endophytes. Theresults revealed that all 3 invasive species harbor rich endophytic microbes that are ableto produce bioactive secondary metabolites, however, their ecological effect needsfurther investigation. Three compounds with plant growth regulatory activity wereidentified from the secondary metabolites produced by a potential new species SR84isolated as the endophytic fungus of S. rostratum, which have the potential to bedeveloped as environment friendly herbicides. This is the first report on thephytotoxicity of the 3 isolated compounds. |
| Subject Area | 生态学 |
| Language | 英语 |
| Document Type | 学位论文 |
| Identifier | http://ir.xjlas.org/handle/365004/15396 |
| Collection | 中国科学院新疆生态与地理研究所 研究系统 |
| Affiliation | 中国科学院新疆生态与地理研究所 |
| First Author Affilication | 中国科学院新疆生态与地理研究所 |
| Recommended Citation GB/T 7714 | NIGORA KUCHKAROVA. 新疆三种外来入侵植物内生菌及活性代谢产物的分离鉴定[D]. 北京. 中国科学院大学,2020. |
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