四川阿壩地區(qū)藥用植物內(nèi)生拮抗放線菌的篩選及其防病促生機(jī)制研究
發(fā)布時(shí)間：2018-07-31 20:17
【摘要】：植物和內(nèi)生菌經(jīng)過漫長(zhǎng)的進(jìn)化,彼此之間早已形成和諧的共生關(guān)系。植物為內(nèi)生菌提供營(yíng)養(yǎng)物質(zhì)；內(nèi)生菌通過不同方式促進(jìn)宿主植物生長(zhǎng)發(fā)育,增強(qiáng)宿主植物抗脅迫能力。四川阿壩州具有獨(dú)特的氣候和生態(tài)環(huán)境,生長(zhǎng)著多種多樣的藥用植物,為發(fā)掘植物內(nèi)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(nèi)生放線菌。結(jié)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(shù)量有較大的差異,其中根部分布最多,占總數(shù)的37%,其次為葉和莖,分別占總數(shù)的29%和27%,其余部位如花和果實(shí)等,占總數(shù)不足10%。從這737株內(nèi)生放線菌中,通過平板對(duì)峙法和生長(zhǎng)速率法逐級(jí)篩選,得到對(duì)稻瘟病菌有較強(qiáng)抑制作用的菌株。同時(shí)對(duì)這些菌株進(jìn)行產(chǎn)鐵載體、產(chǎn)纖維素酶、產(chǎn)吲哚乙酸和溶磷等促生指標(biāo)篩選,最終篩選出3株具有抑制稻瘟病菌活性和促進(jìn)植物生長(zhǎng)雙重功能的內(nèi)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(biāo)檢測(cè)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(nèi)生放線菌,包括菌株3-17、4-21、4-70在內(nèi),進(jìn)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(xiàn)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(jié)果表明,能抑制稻瘟病菌的內(nèi)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(zhì)的潛力。菌株4-70與Streptomyces sp.KE1未知代謝產(chǎn)物的PKS I氨基酸序列同源性為100%,說明該菌株具有產(chǎn)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(chǎn)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(cè),發(fā)現(xiàn)內(nèi)生放線菌3-17、4-21、4-70通過分泌胞外水解酶和生長(zhǎng)競(jìng)爭(zhēng)相互協(xié)同的機(jī)制來抑制稻瘟病菌。內(nèi)生放線菌3-17、4-21、4-70無細(xì)胞發(fā)酵液不僅對(duì)稻瘟病菌有很好的抑制作用,且對(duì)稻瘟病菌孢子萌發(fā)及芽管伸長(zhǎng)也有顯著的抑制作用。根據(jù)結(jié)果推測(cè)菌株3-17、4-21也可能產(chǎn)生抑菌物質(zhì),但是抑菌物質(zhì)的種類和合成途徑還有待進(jìn)一步研究探索。采用內(nèi)生放線菌3-17、4-21、4-70孢子混懸液灌根預(yù)處理水稻后,再接種稻瘟病菌,觀測(cè)了離體水稻葉片防治效果；結(jié)果表明,內(nèi)生放線菌灌根處理后,葉片病斑個(gè)數(shù)及直徑大小均較對(duì)照組有明顯的降低,能顯著抑制水稻稻瘟病的發(fā)生。測(cè)定水稻葉片防御酶活性,發(fā)現(xiàn)菌株3-17、4-21、4-70處理組PAL、POD、LOX活性均高于對(duì)照組；菌株3-17、4-21處理組PPO活性高于對(duì)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(duì)照組,但菌株4-21、4-70處理組AOS活性與對(duì)照組差異不大甚至低于對(duì)照組。采用內(nèi)生放線菌3-17、4-21、4-70孢子混懸液灌根預(yù)處理水稻后,再接種稻瘟病菌,運(yùn)用熒光定量PCR技術(shù)觀測(cè)了水楊酸以及茉莉酮酸/烯信號(hào)轉(zhuǎn)導(dǎo)途徑中關(guān)鍵防御基因的表達(dá)情況,結(jié)果發(fā)現(xiàn)內(nèi)生放線菌3-17、4-21、4-70主要都是通過水楊酸信號(hào)轉(zhuǎn)導(dǎo)途徑誘導(dǎo)水稻抗病性增強(qiáng),從而達(dá)到防病促生的效果。在盆栽溫室培養(yǎng)條件下,內(nèi)生放線菌3-17、4-21、4-70灌根預(yù)處理水稻苗均對(duì)稻瘟病表現(xiàn)出一定的防治效果。在盆栽促生研究中發(fā)現(xiàn),無論接種稻瘟與否,內(nèi)生放線菌3-17、4-21、4-70灌根預(yù)處理都能增強(qiáng)水稻幼苗根系活力。但是內(nèi)生放線菌3-17、4-21、4-70單獨(dú)灌根預(yù)處理,導(dǎo)致水稻幼苗植株地上部分變矮,根系變長(zhǎng)而發(fā)達(dá),其中菌株3-17和4-70增加葉綠素含量從而促進(jìn)植株光合作用。當(dāng)有稻瘟病菌存在時(shí),內(nèi)生放線菌3-17、4-21、4-70促進(jìn)水稻幼苗地上部分增高,根系發(fā)達(dá)生長(zhǎng),其中菌株4-21增加葉綠素含量促進(jìn)植株光合作用。內(nèi)生放線菌3-17、4-21、4-70灌根預(yù)處理能促進(jìn)水稻生長(zhǎng)。綜上所述,本研究從阿壩藥用植物中篩選到的內(nèi)生放線菌3-17、4-21、4-70通過產(chǎn)生不同抑菌物質(zhì)；分泌胞外水解酶和生長(zhǎng)競(jìng)爭(zhēng)相互協(xié)同；誘導(dǎo)宿主植物水楊酸信號(hào)轉(zhuǎn)導(dǎo)途徑；增強(qiáng)宿主植株光合作用及根系活力進(jìn)而促進(jìn)植株生長(zhǎng)這四方面機(jī)制,達(dá)到防治水稻稻瘟病和促進(jìn)植株生長(zhǎng)的效果,并且能在水稻植株內(nèi)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(xué)位授予單位】：四川農(nóng)業(yè)大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2016
【分類號(hào)】：S476
本文編號(hào)：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(xué)位授予單位】：四川農(nóng)業(yè)大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2016
【分類號(hào)】：S476
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