發(fā)布時間：2018-06-08 17:24

  本文選題：醉馬草 + 內(nèi)生真菌��； 參考：《蘭州大學(xué)》2017年博士論文

【摘要】：禾草內(nèi)生真菌(Epichlo?spp.)常與早熟禾亞科(Pooideae)冷季型禾草形成一類較為特殊的禾草-內(nèi)生真菌互惠共生體。醉馬草-內(nèi)生真菌共生體(Achnatherum inebrians-Epichlo?sp.)是國際上研究較多的三大類禾草-內(nèi)生真菌共生體之一,其廣泛分布于我國西部和北部的大部天然草地。已知的侵染醉馬草的禾草內(nèi)生真菌主要有Epichlo?gansuensis和E.inebrins,這兩種禾草內(nèi)生真菌屬于Epichlo?內(nèi)生真菌的無性態(tài),其常通過宿主醉馬草的種子進(jìn)行垂直傳播,同時共生體種子也是一些種傳真菌進(jìn)行傳播的重要載體,其中包括部分引起種子苗期病害的重要病原真菌。前人報道稱醉馬草-內(nèi)生真菌共生體對于退化草地物種豐富度的維持和恢復(fù)具有重要的生態(tài)意義,但是在醉馬草-內(nèi)生真菌共生體種子傳播和植株生長過程中與種傳真菌及根際土壤微生物群落的相關(guān)性尚缺乏研究。對于禾草內(nèi)生真菌的分類學(xué)、生理生化、生態(tài)學(xué)等的研究中發(fā)現(xiàn)其在生態(tài)系統(tǒng)中扮演著重要的角色,因此有必要基于其基因組采用其他方法對其進(jìn)化關(guān)系進(jìn)行進(jìn)一步的明確。本研究以帶菌(E+)和不帶菌(E-)醉馬草種子及其種傳真菌為研究對象,分別探討內(nèi)生真菌和種傳真菌之間的相關(guān)性、內(nèi)生真菌侵染對醉馬草根際土壤微生物群落結(jié)構(gòu)的影響及其機(jī)理,并結(jié)合Epichlo?模式菌Epichlo?festucae基因組對比分析醉馬草內(nèi)生真菌E.gansuensis基因組的進(jìn)化途徑,以期為醉馬草內(nèi)生真菌的開發(fā)利用及其生態(tài)意義的深入研究提供較為重要的科學(xué)依據(jù)。研究得到的主要結(jié)果如下:1.以E+和E-醉馬草種子為研究材料,對比種子表面消毒處理發(fā)現(xiàn),表面消毒能顯著(P0.05)降低醉馬草種子的霉變率并提高其發(fā)芽率、發(fā)芽指數(shù)和活力指數(shù);相對于未攜帶內(nèi)生真菌的醉馬草種子,未消毒處理下,內(nèi)生真菌的侵染能顯著(P0.05)降低醉馬草種子的霉變率并提高其發(fā)芽率、發(fā)芽指數(shù)和活力指數(shù);干旱與消毒處理?xiàng)l件下,內(nèi)生真菌的侵染能明顯緩解干旱和種傳真菌給醉馬草種子的萌發(fā)帶來的抑制作用,低度(-0.3MPa)干旱脅迫條件下內(nèi)生真菌能顯著(P0.05)降低醉馬草種子的霉變率并提高其發(fā)芽率、發(fā)芽指數(shù)和活力指數(shù);滲透勢為-0.3MPa的PEG模擬干旱處理下,萌發(fā)期野大麥、麥賓草和中華羊茅種子的霉變率均顯著低于(P0.05)對照處理且各萌發(fā)指標(biāo)明顯提高;貯藏期醉馬草種子的帶菌率和發(fā)芽率、發(fā)芽指數(shù)及活力指數(shù)隨著種子含水量的上升表現(xiàn)出不同程度的下降趨勢,E+醉馬草種子萌發(fā)活力較強(qiáng),這可能與內(nèi)生真菌誘導(dǎo)宿主醉馬草種子抗氧化酶系統(tǒng)快速反應(yīng)有關(guān)。2.以分離自6個不同種群的醉馬草(A.inebrians)、中華羊茅(Festuca sinensis)和野大麥(Hordeum brevisublatum)的12個(每種禾草選擇兩個種群,每個種群選擇一個菌株)內(nèi)生真菌菌株為研究材料,分析其與根腐離孺孢(Bipolaris sorokiniana)、燕麥鐮孢(Fusarium avenaceum)、細(xì)交鏈孢(Alternaria alternata)、德氏霉(Drechslera erythrospila)、新月彎孢(Curvularia lunata)5種病原真菌之間的互作關(guān)系,平板拮抗試驗(yàn)表明12個內(nèi)生真菌菌株相對于對照均能顯著(P0.05)抑制5種病原真菌菌落的生長,其中分離自醉馬草的2個菌株對所有病原真菌生長的抑制作用較為明顯,不同地理種群內(nèi)生菌株之間的抑菌活性表現(xiàn)出一定的差異性;相對于對照,12株內(nèi)生真菌培養(yǎng)液均能顯著(P0.05)抑制5種病原真菌菌株菌落的生長,其中5種病原真菌菌落的生長對于分離自醉馬草的2個內(nèi)生真菌菌株較為敏感,不同地理種群內(nèi)生真菌菌株之間的抑菌活性具有一定的差異性;分析顯示內(nèi)生真菌對病原真菌的作用機(jī)理可能與其對病原真菌分生孢子較高的萌發(fā)抑制率具有一定的相關(guān)性。3.以分離自醉馬草的內(nèi)生真菌菌株Eg(E.gansuensis)、分離自野大麥的內(nèi)生真菌菌株Eb(E.bromicola)和分離自麥賓草的內(nèi)生真菌菌株Et(Epichlo?sp.)為研究材料,探討其與分離自老芒麥(Elymus sibiricus)的4種種傳病原真菌細(xì)交鏈孢(A.alternaria)、根腐離孺孢(B.sorokinianum)、燕麥鐮孢(F.avenaceum)和鐮刀菌(Fusarium sp.)之間互作對老芒麥種子萌發(fā)和幼苗生長的影響,結(jié)果顯示相對于空白對照,不同稀釋倍數(shù)內(nèi)生真菌培養(yǎng)液的浸種處理均能顯著(P0.05)提高4種種傳真菌脅迫下老芒麥種子的萌發(fā)及幼苗的生長,不同內(nèi)生真菌菌株之間作用結(jié)果具有一定的差異,并且同一菌株的不同稀釋倍數(shù)之間也有較大的差異,不同菌株的最佳作用的稀釋倍數(shù)不同;分別選用Eb03、Et03和Eg02三個稀釋倍數(shù)的內(nèi)生真菌菌液進(jìn)行浸種預(yù)處理之后,在溫室栽培條件下觀測老芒麥的株高、分蘗和生物量發(fā)現(xiàn)均顯著(P0.05)高于空白對照,其作用機(jī)理可能與不同內(nèi)生真菌菌株產(chǎn)生的生物堿類次生代謝產(chǎn)物濃度有關(guān)。4.以E+、E-醉馬草植株及其根際土壤為研究材料,分析內(nèi)生真菌侵染對宿主醉馬草的生長和根際土壤微生物的影響,結(jié)果顯示內(nèi)生真菌的侵染能顯著(P0.05)提高宿主醉馬草的株高、分蘗、地上生物量、地下生物量及根冠比;E+、E-醉馬草根際土壤中除土壤容重、碳氮比(C:N)比之間差異顯著(P0.05)之外,其余各土壤物理和營養(yǎng)狀況指標(biāo)均不顯著;基于高通量測序結(jié)果顯示,E+醉馬草根際土壤中真菌和細(xì)菌群落豐富度均明顯高于E-醉馬草根際土壤,內(nèi)生真菌的侵染提高了宿主醉馬草對根際土壤微生物多樣性的庇護(hù);結(jié)合土壤因子和營養(yǎng)差異分析顯示,醉馬草根際土壤中C:N的改變可能是引起真菌和細(xì)菌群落變化的重要因素之一,同時盡管土壤pH值差異并不顯著(P0.05),但是它也是引起微生物多樣性差異的重要因素之一。5.以醉馬草內(nèi)生真菌E.gansuensis基因組和E.festucae基因組為研究材料,基于兩個基因組密碼子的使用模式和偏好性分析其進(jìn)化因子,中性繪圖分析顯示中性進(jìn)化對這兩個基因組的影響較小,而自然選擇在其中扮演著主導(dǎo)作用,其對二者密碼子偏好性的影響分別達(dá)到了82.71%和95.14%;通過有效密碼字?jǐn)?shù)(ENC)和第3位同義密碼子(GC3s)之間的相關(guān)性分析顯示,兩個基因組分別僅有2.08%和2.76%的基因表現(xiàn)出較高的密碼子偏好性,這就意味著供試內(nèi)生真菌基因組并沒有受到強(qiáng)烈的密碼子偏好性,其基因組密碼子的使用應(yīng)該是通過隨機(jī)選擇完成的;對密碼子使用的各項(xiàng)指標(biāo)的相關(guān)性分析顯示,兩個內(nèi)生真菌基因組密碼子使用的偏好性除受有效密碼字?jǐn)?shù)的影響之外,同樣受到核苷酸的組成和基因的表達(dá)水平的影響;兩個內(nèi)生真菌基因組中分別鑒定出了26個和27個以G或C結(jié)尾的最優(yōu)密碼子,這些最優(yōu)密碼子與轉(zhuǎn)錄水平顯著相關(guān),有助于利用這一特點(diǎn)進(jìn)行簡并引物的設(shè)計(jì),進(jìn)一步追溯內(nèi)生真菌的進(jìn)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.
【學(xué)位授予單位】：蘭州大學(xué)
【學(xué)位級別】：博士
【學(xué)位授予年份】：2017
【分類號】：Q948.122.3
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本文編號：1996598