四川阿壩地區(qū)藥用植物內生拮抗放線菌的篩選及其防病促生機制研究
發(fā)布時間：2018-07-31 20:17
【摘要】：植物和內生菌經過漫長的進化,彼此之間早已形成和諧的共生關系。植物為內生菌提供營養(yǎng)物質；內生菌通過不同方式促進宿主植物生長發(fā)育,增強宿主植物抗脅迫能力。四川阿壩州具有獨特的氣候和生態(tài)環(huán)境,生長著多種多樣的藥用植物,為發(fā)掘植物內生放線菌提供了豐富的資源。本研究選擇10種分離培養(yǎng)基,從36種阿壩地區(qū)野生藥用植物中分離得到737株內生放線菌。結果表明,選用的10種分離培養(yǎng)基中,棉子糖組氨酸培養(yǎng)基分離效果最好,葡萄糖天冬酰胺培養(yǎng)基分離效果最差。從藥用植物的不同部位分離的放線菌數量有較大的差異,其中根部分布最多,占總數的37%,其次為葉和莖,分別占總數的29%和27%,其余部位如花和果實等,占總數不足10%。從這737株內生放線菌中,通過平板對峙法和生長速率法逐級篩選,得到對稻瘟病菌有較強抑制作用的菌株。同時對這些菌株進行產鐵載體、產纖維素酶、產吲哚乙酸和溶磷等促生指標篩選,最終篩選出3株具有抑制稻瘟病菌活性和促進植物生長雙重功能的內生放線菌3-17、4-21、4-70。通過形態(tài)和培養(yǎng)特征觀察、生理生化指標檢測和16SrDNA序列分析,將菌株3-17鑒定為Streptomyces exfoliates;菌株4-21鑒定為Streptomyces albidoflavus:菌株4-70鑒定為Streptomyces albus。選取55株初篩具有抑制稻瘟病菌活性的內生放線菌,包括菌株3-17、4-21、4-70在內,進行Ⅰ型聚酮合酶PKS Ⅰ、Ⅱ型聚酮合酶PKS Ⅱ和非核糖體肽合成酶NRPS功能基因篩選,發(fā)現55株供試菌株中有31株菌含有PKS功能基因片段;有15株菌含有PKS Ⅱ功能基因片段；有18株菌含有NRPS功能基因片段,且?guī)缀跞繉儆阪溍咕鷮�。結果表明,能抑制稻瘟病菌的內生放線菌具有合成聚酮類化合物及非核糖體肽類抑菌物質的潛力。菌株4-70與Streptomyces sp.KE1未知代謝產物的PKS I氨基酸序列同源性為100%,說明該菌株具有產生聚酮類化合物的能力,可能通過非芳香環(huán)聚酮化合物產生抑制稻瘟病菌的效果。通過顯微鏡觀察和水解酶活力檢測,發(fā)現內生放線菌3-17、4-21、4-70通過分泌胞外水解酶和生長競爭相互協同的機制來抑制稻瘟病菌。內生放線菌3-17、4-21、4-70無細胞發(fā)酵液不僅對稻瘟病菌有很好的抑制作用,且對稻瘟病菌孢子萌發(fā)及芽管伸長也有顯著的抑制作用。根據結果推測菌株3-17、4-21也可能產生抑菌物質,但是抑菌物質的種類和合成途徑還有待進一步研究探索。采用內生放線菌3-17、4-21、4-70孢子混懸液灌根預處理水稻后,再接種稻瘟病菌,觀測了離體水稻葉片防治效果；結果表明,內生放線菌灌根處理后,葉片病斑個數及直徑大小均較對照組有明顯的降低,能顯著抑制水稻稻瘟病的發(fā)生。測定水稻葉片防御酶活性,發(fā)現菌株3-17、4-21、4-70處理組PAL、POD、LOX活性均高于對照組；菌株3-17、4-21處理組PPO活性高于對照組,但菌株4-70處理并不能顯著增加水稻PPO活性；菌株3-17處理組AOS活性高于對照組,但菌株4-21、4-70處理組AOS活性與對照組差異不大甚至低于對照組。采用內生放線菌3-17、4-21、4-70孢子混懸液灌根預處理水稻后,再接種稻瘟病菌,運用熒光定量PCR技術觀測了水楊酸以及茉莉酮酸/烯信號轉導途徑中關鍵防御基因的表達情況,結果發(fā)現內生放線菌3-17、4-21、4-70主要都是通過水楊酸信號轉導途徑誘導水稻抗病性增強,從而達到防病促生的效果。在盆栽溫室培養(yǎng)條件下,內生放線菌3-17、4-21、4-70灌根預處理水稻苗均對稻瘟病表現出一定的防治效果。在盆栽促生研究中發(fā)現,無論接種稻瘟與否,內生放線菌3-17、4-21、4-70灌根預處理都能增強水稻幼苗根系活力。但是內生放線菌3-17、4-21、4-70單獨灌根預處理,導致水稻幼苗植株地上部分變矮,根系變長而發(fā)達,其中菌株3-17和4-70增加葉綠素含量從而促進植株光合作用。當有稻瘟病菌存在時,內生放線菌3-17、4-21、4-70促進水稻幼苗地上部分增高,根系發(fā)達生長,其中菌株4-21增加葉綠素含量促進植株光合作用。內生放線菌3-17、4-21、4-70灌根預處理能促進水稻生長。綜上所述,本研究從阿壩藥用植物中篩選到的內生放線菌3-17、4-21、4-70通過產生不同抑菌物質；分泌胞外水解酶和生長競爭相互協同；誘導宿主植物水楊酸信號轉導途徑；增強宿主植株光合作用及根系活力進而促進植株生長這四方面機制,達到防治水稻稻瘟病和促進植株生長的效果,并且能在水稻植株內有效定殖,具備開發(fā)成生防菌劑的潛力。
[Abstract]:After long evolution, plants and endophytes have formed a harmonious symbiotic relationship with each other. Plants provide nutrients for endophytes; endophytes promote the growth and development of host plants in different ways and enhance the resistance to stress of host plants. Sichuan Aba state has a unique climate and ecological environment, and grows a variety of medicinal herbs. Plants have provided abundant resources for the exploration of endophytic actinomycetes. In this study, 10 kinds of isolation medium were selected to separate 737 Endophytic Actinomycetes from 36 wild medicinal plants in Aba region. The results showed that in the 10 separate medium, the isolation effect of histidine culture medium was the best, and the culture of glucosamine asparagine was the best medium. The number of actinomycetes isolated from different parts of the medicinal plants is different, the root distribution is the most, 37% of the total, followed by leaves and stems, accounting for 29% and 27% of the total, the rest of the total, such as flowers and fruits, accounting for less than 10%. from the 737 endogenic Actinomycetes, through flat confrontation and growth rate method. In addition, 3 strains of endophytic actinomycetes, which could inhibit the activity of blast fungus and promote the dual power of plant growth, were screened out, and 3 strains of endophytic actinomycetes, which could inhibit the activity of blast fungus and promote the dual power of plant growth, were screened. The strain 3-17 was identified as Streptomyces exfoliates, the strain 3-17 was identified as Streptomyces exfoliates, and the strain 4-21 was identified as the endophytic actinomycetes, including the strain 3-17,4-21,4-70, including the strain 3-17,4-21,4-70, which was identified as Streptomyces albidoflavus: strain 4-70, which was selected as Streptomyces albus.. The screening of the functional gene of type I polyketone synthase PKS I, type II polyketone synthase PKS II and non ribosomal peptide synthetase NRPS found that 31 of the 55 strain strains contained PKS functional gene fragments; 15 strains contained PKS II functional gene fragments; 18 strains contained NRPS functional segments, and almost all belonged to Streptomyces. The endophytic actinomycetes, which can inhibit the pathogen of rice blast, have the potential to synthesize polyketones and non ribosomal peptides. The homology of the PKS I amino acid sequence of the unknown metabolites of strain 4-70 and Streptomyces sp.KE1 is 100%, indicating that the strain has the ability to produce polyketo compounds and may be combined with non aromatic cyclic polyketones. By microscopic observation and hydrolase activity detection, it was found that endophytic actinomycetes 3-17,4-21,4-70 inhibited rice blast by secreting the synergistic mechanism of extracellular hydrolase and growth competition. The actinomycete 3-17,4-21,4-70 acellular fermentation broth of Endophytic Actinomycetes not only had a good inhibitory effect on the rice blast fungus, but also the inhibitory effect of the endophytic actinomycetes not only on the rice blast fungus. The spore germination and sprout elongation of rice blast also had a significant inhibitory effect. According to the results, we speculated that the strain 3-17,4-21 could also produce bacteriostasis, but the species and synthetic ways of the bacteriostat were still to be further studied. After using the endophytic actinomycete 3-17,4-21,4-70 spore suspension to pretreat rice, then inoculate the rice blast. The results showed that the number and diameter of leaf spot and diameter decreased significantly after the treatment of endophytic actinomycetes, and could significantly inhibit the occurrence of rice blast. The activity of defense enzyme in rice leaves was measured, and the activity of PAL, POD and LOX in the 3-17,4-21,4-70 treatment group was higher than that of the control group. The activity of PPO in the strain 3-17,4-21 treatment group was higher than that of the control group, but the strain 4-70 treatment did not significantly increase the activity of PPO in rice; the activity of AOS in the strain 3-17 treatment group was higher than that of the control group, but the activity of AOS in the strain 4-21,4-70 treatment group was not much even lower than that of the control group. The endogenous actinomycetes 3-17,4-21,4-70 spores were used to fill the root preconditioning group. After rice treatment, rice blast fungus was inoculated, and the expression of key defense genes in salicylic acid and jasmonic acid / allene signal transduction pathway was observed by fluorescence quantitative PCR technique. The results showed that endophytic actinomycetes 3-17,4-21,4-70 were mainly induced by salicylic acid signal transduction pathway to enhance resistance to rice, thus preventing disease and promoting growth. Under the greenhouse cultivation conditions, the endophytic actinomycetes 3-17,4-21,4-70 root pretreated rice seedlings all showed a certain control effect on the rice blast. In the pot cultivation study, it was found that the endophytic actinomycetes could strengthen the root vigor of rice seedlings regardless of the inoculation of rice blast or not, but the endophytic actinomycetes were 3, but the endophytic actinomycetes were 3. -17,4-21,4-70 was pretreated separately, which resulted in the growth of the upper part of the rice seedlings and the growth of the root system. Among the strains 3-17 and 4-70, the chlorophyll content was increased and the photosynthesis of the plants was increased. The endophytic actinomycete 3-17,4-21,4-70 promoted the increase of the aboveground part of the rice seedlings and the roots developed, among which the bacteria were developed. Plant 4-21 increased chlorophyll content to promote photosynthesis. Endophytic actinomycetes 3-17,4-21,4-70 root pretreatment could promote the growth of rice. To sum up, the endophytic actinomycete 3-17,4-21,4-70 screened from aba medicinal plants was produced by producing different bacteriostasis; exsecreting extracellular hydrolase and growth competition were synergistic. The signal transduction pathway of salicylic acid in the main plant, enhancing the photosynthesis and root activity of the host plant and promoting the growth of the plant, is the four mechanism to prevent rice blast and promote plant growth, and can be effectively colonized in rice plants, and has the potential to develop biocontrol agents.
【學位授予單位】：四川農業(yè)大學
【學位級別】：博士
【學位授予年份】：2016
【分類號】：S476
本文編號：2156716
[Abstract]:After long evolution, plants and endophytes have formed a harmonious symbiotic relationship with each other. Plants provide nutrients for endophytes; endophytes promote the growth and development of host plants in different ways and enhance the resistance to stress of host plants. Sichuan Aba state has a unique climate and ecological environment, and grows a variety of medicinal herbs. Plants have provided abundant resources for the exploration of endophytic actinomycetes. In this study, 10 kinds of isolation medium were selected to separate 737 Endophytic Actinomycetes from 36 wild medicinal plants in Aba region. The results showed that in the 10 separate medium, the isolation effect of histidine culture medium was the best, and the culture of glucosamine asparagine was the best medium. The number of actinomycetes isolated from different parts of the medicinal plants is different, the root distribution is the most, 37% of the total, followed by leaves and stems, accounting for 29% and 27% of the total, the rest of the total, such as flowers and fruits, accounting for less than 10%. from the 737 endogenic Actinomycetes, through flat confrontation and growth rate method. In addition, 3 strains of endophytic actinomycetes, which could inhibit the activity of blast fungus and promote the dual power of plant growth, were screened out, and 3 strains of endophytic actinomycetes, which could inhibit the activity of blast fungus and promote the dual power of plant growth, were screened. The strain 3-17 was identified as Streptomyces exfoliates, the strain 3-17 was identified as Streptomyces exfoliates, and the strain 4-21 was identified as the endophytic actinomycetes, including the strain 3-17,4-21,4-70, including the strain 3-17,4-21,4-70, which was identified as Streptomyces albidoflavus: strain 4-70, which was selected as Streptomyces albus.. The screening of the functional gene of type I polyketone synthase PKS I, type II polyketone synthase PKS II and non ribosomal peptide synthetase NRPS found that 31 of the 55 strain strains contained PKS functional gene fragments; 15 strains contained PKS II functional gene fragments; 18 strains contained NRPS functional segments, and almost all belonged to Streptomyces. The endophytic actinomycetes, which can inhibit the pathogen of rice blast, have the potential to synthesize polyketones and non ribosomal peptides. The homology of the PKS I amino acid sequence of the unknown metabolites of strain 4-70 and Streptomyces sp.KE1 is 100%, indicating that the strain has the ability to produce polyketo compounds and may be combined with non aromatic cyclic polyketones. By microscopic observation and hydrolase activity detection, it was found that endophytic actinomycetes 3-17,4-21,4-70 inhibited rice blast by secreting the synergistic mechanism of extracellular hydrolase and growth competition. The actinomycete 3-17,4-21,4-70 acellular fermentation broth of Endophytic Actinomycetes not only had a good inhibitory effect on the rice blast fungus, but also the inhibitory effect of the endophytic actinomycetes not only on the rice blast fungus. The spore germination and sprout elongation of rice blast also had a significant inhibitory effect. According to the results, we speculated that the strain 3-17,4-21 could also produce bacteriostasis, but the species and synthetic ways of the bacteriostat were still to be further studied. After using the endophytic actinomycete 3-17,4-21,4-70 spore suspension to pretreat rice, then inoculate the rice blast. The results showed that the number and diameter of leaf spot and diameter decreased significantly after the treatment of endophytic actinomycetes, and could significantly inhibit the occurrence of rice blast. The activity of defense enzyme in rice leaves was measured, and the activity of PAL, POD and LOX in the 3-17,4-21,4-70 treatment group was higher than that of the control group. The activity of PPO in the strain 3-17,4-21 treatment group was higher than that of the control group, but the strain 4-70 treatment did not significantly increase the activity of PPO in rice; the activity of AOS in the strain 3-17 treatment group was higher than that of the control group, but the activity of AOS in the strain 4-21,4-70 treatment group was not much even lower than that of the control group. The endogenous actinomycetes 3-17,4-21,4-70 spores were used to fill the root preconditioning group. After rice treatment, rice blast fungus was inoculated, and the expression of key defense genes in salicylic acid and jasmonic acid / allene signal transduction pathway was observed by fluorescence quantitative PCR technique. The results showed that endophytic actinomycetes 3-17,4-21,4-70 were mainly induced by salicylic acid signal transduction pathway to enhance resistance to rice, thus preventing disease and promoting growth. Under the greenhouse cultivation conditions, the endophytic actinomycetes 3-17,4-21,4-70 root pretreated rice seedlings all showed a certain control effect on the rice blast. In the pot cultivation study, it was found that the endophytic actinomycetes could strengthen the root vigor of rice seedlings regardless of the inoculation of rice blast or not, but the endophytic actinomycetes were 3, but the endophytic actinomycetes were 3. -17,4-21,4-70 was pretreated separately, which resulted in the growth of the upper part of the rice seedlings and the growth of the root system. Among the strains 3-17 and 4-70, the chlorophyll content was increased and the photosynthesis of the plants was increased. The endophytic actinomycete 3-17,4-21,4-70 promoted the increase of the aboveground part of the rice seedlings and the roots developed, among which the bacteria were developed. Plant 4-21 increased chlorophyll content to promote photosynthesis. Endophytic actinomycetes 3-17,4-21,4-70 root pretreatment could promote the growth of rice. To sum up, the endophytic actinomycete 3-17,4-21,4-70 screened from aba medicinal plants was produced by producing different bacteriostasis; exsecreting extracellular hydrolase and growth competition were synergistic. The signal transduction pathway of salicylic acid in the main plant, enhancing the photosynthesis and root activity of the host plant and promoting the growth of the plant, is the four mechanism to prevent rice blast and promote plant growth, and can be effectively colonized in rice plants, and has the potential to develop biocontrol agents.
【學位授予單位】：四川農業(yè)大學
【學位級別】：博士
【學位授予年份】：2016
【分類號】：S476
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