醉马草内生真菌与宿主种带真菌、根际微生物的互作及其进化研究

发布时间：2018-06-08 17:24

  本文选题：醉马草 + 内生真菌　； 参考：《兰州大学》2017年博士论文

【摘要】：禾草内生真菌(Epichlo?spp.)常与早熟禾亚科(Pooideae)冷季型禾草形成一类较为特殊的禾草-内生真菌互惠共生体。醉马草-内生真菌共生体(Achnatherum inebrians-Epichlo?sp.)是国际上研究较多的三大类禾草-内生真菌共生体之一,其广泛分布于我国西部和北部的大部天然草地。已知的侵染醉马草的禾草内生真菌主要有Epichlo?gansuensis和E.inebrins,这两种禾草内生真菌属于Epichlo?内生真菌的无性态,其常通过宿主醉马草的种子进行垂直传播,同时共生体种子也是一些种传真菌进行传播的重要载体,其中包括部分引起种子苗期病害的重要病原真菌。前人报道称醉马草-内生真菌共生体对于退化草地物种丰富度的维持和恢复具有重要的生态意义,但是在醉马草-内生真菌共生体种子传播和植株生长过程中与种传真菌及根际土壤微生物群落的相关性尚缺乏研究。对于禾草内生真菌的分类学、生理生化、生态学等的研究中发现其在生态系统中扮演着重要的角色,因此有必要基于其基因组采用其他方法对其进化关系进行进一步的明确。本研究以带菌(E+)和不带菌(E-)醉马草种子及其种传真菌为研究对象,分别探讨内生真菌和种传真菌之间的相关性、内生真菌侵染对醉马草根际土壤微生物群落结构的影响及其机理,并结合Epichlo?模式菌Epichlo?festucae基因组对比分析醉马草内生真菌E.gansuensis基因组的进化途径,以期为醉马草内生真菌的开发利用及其生态意义的深入研究提供较为重要的科学依据。研究得到的主要结果如下:1.以E+和E-醉马草种子为研究材料,对比种子表面消毒处理发现,表面消毒能显著(P0.05)降低醉马草种子的霉变率并提高其发芽率、发芽指数和活力指数;相对于未携带内生真菌的醉马草种子,未消毒处理下,内生真菌的侵染能显著(P0.05)降低醉马草种子的霉变率并提高其发芽率、发芽指数和活力指数;干旱与消毒处理条件下,内生真菌的侵染能明显缓解干旱和种传真菌给醉马草种子的萌发带来的抑制作用,低度(-0.3MPa)干旱胁迫条件下内生真菌能显著(P0.05)降低醉马草种子的霉变率并提高其发芽率、发芽指数和活力指数;渗透势为-0.3MPa的PEG模拟干旱处理下,萌发期野大麦、麦宾草和中华羊茅种子的霉变率均显著低于(P0.05)对照处理且各萌发指标明显提高;贮藏期醉马草种子的带菌率和发芽率、发芽指数及活力指数随着种子含水量的上升表现出不同程度的下降趋势,E+醉马草种子萌发活力较强,这可能与内生真菌诱导宿主醉马草种子抗氧化酶系统快速反应有关。2.以分离自6个不同种群的醉马草(A.inebrians)、中华羊茅(Festuca sinensis)和野大麦(Hordeum brevisublatum)的12个(每种禾草选择两个种群,每个种群选择一个菌株)内生真菌菌株为研究材料,分析其与根腐离孺孢(Bipolaris sorokiniana)、燕麦镰孢(Fusarium avenaceum)、细交链孢(Alternaria alternata)、德氏霉(Drechslera erythrospila)、新月弯孢(Curvularia lunata)5种病原真菌之间的互作关系,平板拮抗试验表明12个内生真菌菌株相对于对照均能显著(P0.05)抑制5种病原真菌菌落的生长,其中分离自醉马草的2个菌株对所有病原真菌生长的抑制作用较为明显,不同地理种群内生菌株之间的抑菌活性表现出一定的差异性;相对于对照,12株内生真菌培养液均能显著(P0.05)抑制5种病原真菌菌株菌落的生长,其中5种病原真菌菌落的生长对于分离自醉马草的2个内生真菌菌株较为敏感,不同地理种群内生真菌菌株之间的抑菌活性具有一定的差异性;分析显示内生真菌对病原真菌的作用机理可能与其对病原真菌分生孢子较高的萌发抑制率具有一定的相关性。3.以分离自醉马草的内生真菌菌株Eg(E.gansuensis)、分离自野大麦的内生真菌菌株Eb(E.bromicola)和分离自麦宾草的内生真菌菌株Et(Epichlo?sp.)为研究材料,探讨其与分离自老芒麦(Elymus sibiricus)的4种种传病原真菌细交链孢(A.alternaria)、根腐离孺孢(B.sorokinianum)、燕麦镰孢(F.avenaceum)和镰刀菌(Fusarium sp.)之间互作对老芒麦种子萌发和幼苗生长的影响,结果显示相对于空白对照,不同稀释倍数内生真菌培养液的浸种处理均能显著(P0.05)提高4种种传真菌胁迫下老芒麦种子的萌发及幼苗的生长,不同内生真菌菌株之间作用结果具有一定的差异,并且同一菌株的不同稀释倍数之间也有较大的差异,不同菌株的最佳作用的稀释倍数不同;分别选用Eb03、Et03和Eg02三个稀释倍数的内生真菌菌液进行浸种预处理之后,在温室栽培条件下观测老芒麦的株高、分蘖和生物量发现均显著(P0.05)高于空白对照,其作用机理可能与不同内生真菌菌株产生的生物碱类次生代谢产物浓度有关。4.以E+、E-醉马草植株及其根际土壤为研究材料,分析内生真菌侵染对宿主醉马草的生长和根际土壤微生物的影响,结果显示内生真菌的侵染能显著(P0.05)提高宿主醉马草的株高、分蘖、地上生物量、地下生物量及根冠比;E+、E-醉马草根际土壤中除土壤容重、碳氮比(C:N)比之间差异显著(P0.05)之外,其余各土壤物理和营养状况指标均不显著;基于高通量测序结果显示,E+醉马草根际土壤中真菌和细菌群落丰富度均明显高于E-醉马草根际土壤,内生真菌的侵染提高了宿主醉马草对根际土壤微生物多样性的庇护;结合土壤因子和营养差异分析显示,醉马草根际土壤中C:N的改变可能是引起真菌和细菌群落变化的重要因素之一,同时尽管土壤pH值差异并不显著(P0.05),但是它也是引起微生物多样性差异的重要因素之一。5.以醉马草内生真菌E.gansuensis基因组和E.festucae基因组为研究材料,基于两个基因组密码子的使用模式和偏好性分析其进化因子,中性绘图分析显示中性进化对这两个基因组的影响较小,而自然选择在其中扮演着主导作用,其对二者密码子偏好性的影响分别达到了82.71%和95.14%;通过有效密码字数(ENC)和第3位同义密码子(GC3s)之间的相关性分析显示,两个基因组分别仅有2.08%和2.76%的基因表现出较高的密码子偏好性,这就意味着供试内生真菌基因组并没有受到强烈的密码子偏好性,其基因组密码子的使用应该是通过随机选择完成的;对密码子使用的各项指标的相关性分析显示,两个内生真菌基因组密码子使用的偏好性除受有效密码字数的影响之外,同样受到核苷酸的组成和基因的表达水平的影响;两个内生真菌基因组中分别鉴定出了26个和27个以G或C结尾的最优密码子,这些最优密码子与转录水平显著相关,有助于利用这一特点进行简并引物的设计,进一步追溯内生真菌的进化起源。
[Abstract]:The endophytic fungi (Epichlo? Spp.) often form a special kind of symbiotic symbiotic symbiotic with the cold season grasses of precocious grasses (Pooideae). The drunkah endophyte symbiotic (Achnatherum inebrians-Epichlo? Sp.) is one of the three major grasses endophytic symbionts which are widely studied in the world. It is widely distributed in me. The large natural meadows of Western and northern countries. The known endophytic fungi of the grass infected grass are mainly Epichlo? GANSUENSIS and E.inebrins. These two kinds of endophytic fungi belong to the asexual state of the Epichlo? Endophytic fungi, which are often transmitted vertically through the seeds of the host drunkweed, and the symbiotic seeds are also some species of fungi. Important carriers of transmission, including some important pathogenic fungi that cause seed seedling disease, have been reported to have important ecological significance for the maintenance and recovery of species richness in degraded grassland, but in the process of seed propagation and plant growth during the process of seed propagation and plant growth of drunken endophyte symbiosis The correlation between bacteria and rhizosphere soil microbial communities is still lack of research. It has been found in the study of taxonomy, physiology, biochemistry and ecology of endophytic fungi that it plays an important role in the ecosystem. Therefore, it is necessary to use other methods to further clarify the relationship between the endophytic fungi and their genomes. (E+) and non fungus (E-) Drunkie seed and its seed borne fungi as the research object, the correlation between endophyte and seed borne fungi, the effect of endophyte infection on the microbial community structure in the rhizosphere soil of drunken drunkweed and its mechanism, and the comparison and analysis of the endophytic fungus E.g with the Epichlo? Festucae genome of Epichlo? The evolutionary pathway of the ansuensis genome provides an important scientific basis for the development and utilization of endophytic fungi and the in-depth study of its ecological significance. The main results are as follows: 1. the seeds of E+ and E- were used as the research materials. Compared with the seed surface disinfection treatment, the surface disinfection could be significantly lower than that of P0.05 (P0.05). The mildew rate and the germination index and vigor index of the seeds of the horse grass were improved. Compared with the unsterilized seeds of the drunkweed without endophytic fungi, the infestation of endophytic fungi could significantly (P0.05) reduce the mildew rate of drunkah seeds, increase the germination rate, germination index and vigor index, and endophyte under the conditions of drought and disinfection. Fungal infection could significantly alleviate the inhibitory effects of drought and seed borne fungi on the germination of drunkah seeds. Endophytic fungi with low degree (-0.3MPa) drought stress can significantly (P0.05) reduce the mildew rate of drunkah seeds and increase their germination rate, germination index and vigor index, and PEG simulated by the osmotic potential of -0.3MPa under drought treatment. The mildew rate of wild barley, wheat binggrass and Chinese fescue seeds were significantly lower than that of (P0.05) control treatment and the germination indexes were obviously improved. The germination rate and germination rate of drunkia seeds at storage period, germination index and vigor index showed a decrease trend with the increase of seed water content, and the germination vigor of E+ drunkworm seeds was more than that of the seeds. Strong, this may be associated with the rapid reaction of endophytic fungi inducing the host inelgrass seed antioxidant enzyme system to isolate.2. from 6 different populations of Drunkie (A.inebrians), 12 of Chinese fescue (Festuca sinensis) and wild barley (Hordeum brevisublatum) (two species selected for each grass, and one strain for each species) endophytic fungi As a study material, the interaction between 5 pathogenic fungi, including root rot (Bipolaris sorokiniana), Fusarium oxysporum (Fusarium avenaceum), Alternaria alternata, Deshi mold (Drechslera erythrospila), and crescent crescent (Curvularia lunata), was analyzed. The plate antagonistic test showed that 12 endophytic fungi were relative to the fungus. The control could significantly (P0.05) inhibit the growth of 5 pathogenic fungi colonies, of which 2 strains isolated from drunkworm had obvious inhibitory effects on the growth of all pathogenic fungi, and the bacteriostasis activity of the endophytic strains in different geographical populations showed a certain difference. Compared with the control, 12 endophytic fungi culture fluid could be significantly (P0.05) inhibited. The growth of the colony of 5 pathogenic fungi strains, of which the growth of 5 pathogenic fungi colonies is sensitive to 2 endophytic fungi isolated from drunkah, and the antimicrobial activity of endophytic fungi in different geographical populations is different. The higher germination inhibition rate of fungal conidia has a certain correlation.3. to isolate endophytic fungus strain Eg (E.gansuensis) from drunkia, Eb (E.bromicola) isolated from wild barley and Et (Epichlo? Sp.), isolated from malt grass, Et (Epichlo? Sp.), and explore its isolation from old manm (Elymus sibiri). CUS) the effects of interaction between 4 pathogenic fungi, A.alternaria, root rot, B.sorokinianum, Fusarium oxysporum (F.avenaceum) and Fusarium oxysporum (Fusarium sp.) on the seed germination and seedling growth of old manm, the results showed that the seed soaking treatment of different dilution multiple endophytic fungi could be shown to be significant relative to the blank contrast. (P0.05) to increase the germination of the seeds and the growth of the seedlings under the stress of 4 facsimile strains, the results of the different endophytic fungi were different, and the different dilution times of the same strain were different. The best Dilution Times of the different strains were different; Eb03, Et03 and Eg02 were selected respectively. The plant height of old manm was observed under the greenhouse cultivation condition after seed soaking with endophytic fungi, and the results were significantly higher than that of the blank control (P0.05). The mechanism may be related to the concentration of the secondary metabolites of alkaloids from different endophytic fungi related to.4., E+, E- drunkworm plant and The rhizosphere soil was used as the research material to analyze the effects of endophytic fungi infection on the growth of the host drunken grass and the rhizosphere soil microorganism. The results showed that the infection of endophyte could significantly increase the plant height, tiller, aboveground biomass, underground biomass and root crown ratio of the host drunkweed (P0.05), and the soil bulk density, carbon and nitrogen in E+, E- drunken rhizosphere soil. In addition to the significant difference (C:N), the other soil physical and nutritional indicators were not significant. High throughput sequencing based on high throughput sequencing showed that the abundance of fungi and bacteria in the rhizosphere soil of E+ drunken horse grass was significantly higher than that of E- drunken rhizosphere soil, and endophytic fungi increased the host drunken grass to the rhizosphere soil microbes. The analysis of soil factors and nutrient differences showed that the change of C:N in the rhizosphere soil of the drunken horse grass may be one of the important factors to cause the change of fungi and bacteria community, while the difference in soil pH value is not significant (P0.05), but it is also one of the important factors causing the diversity of microbial diversity,.5. is endophytic with drunkah. The fungal E.gansuensis genome and the E.festucae genome are the research materials. Based on the use patterns and preferences of two genomic codons, their evolutionary factors are analyzed. Neutral mapping analysis shows that neutral evolution has less influence on the two genomes, while natural selection plays a dominant role in the two codons. The responses were 82.71% and 95.14% respectively; the correlation analysis between the valid cipher numbers (ENC) and the third - bit synonymous codon (GC3s) showed that only 2.08% and 2.76% of the two genomes showed higher codon preference, which meant that the genome of the endogenetic true bacteria was not strongly codon preference, The use of genomic codons should be done by random selection; correlation analysis of the various indicators used in codons shows that the preference of the two endophytic codon used by the genomic codon is influenced by the number of effective ciphers, also influenced by the composition of the nucleotide and the expression level of the gene, and the two endophytes. 26 and 27 optimal codons at the end of G or C were identified in the genome. These best codons were significantly related to the transcriptional level. It was helpful to use this characteristic to design the degenerate primers and further trace the evolutionary origin of endophytic fungi.
【学位授予单位】：兰州大学
【学位级别】：博士
【学位授予年份】：2017
【分类号】：Q948.122.3
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本文编号：1996598