四川阿坝地区药用植物内生拮抗放线菌的筛选及其防病促生机制研究

发布时间：2018-07-31 20:17

【摘要】：植物和内生菌经过漫长的进化,彼此之间早已形成和谐的共生关系。植物为内生菌提供营养物质；内生菌通过不同方式促进宿主植物生长发育,增强宿主植物抗胁迫能力。四川阿坝州具有独特的气候和生态环境,生长着多种多样的药用植物,为发掘植物内生放线菌提供了丰富的资源。本研究选择10种分离培养基,从36种阿坝地区野生药用植物中分离得到737株内生放线菌。结果表明,选用的10种分离培养基中,棉子糖组氨酸培养基分离效果最好,葡萄糖天冬酰胺培养基分离效果最差。从药用植物的不同部位分离的放线菌数量有较大的差异,其中根部分布最多,占总数的37%,其次为叶和茎,分别占总数的29%和27%,其余部位如花和果实等,占总数不足10%。从这737株内生放线菌中,通过平板对峙法和生长速率法逐级筛选,得到对稻瘟病菌有较强抑制作用的菌株。同时对这些菌株进行产铁载体、产纤维素酶、产吲哚乙酸和溶磷等促生指标筛选,最终筛选出3株具有抑制稻瘟病菌活性和促进植物生长双重功能的内生放线菌3-17、4-21、4-70。通过形态和培养特征观察、生理生化指标检测和16SrDNA序列分析,将菌株3-17鉴定为Streptomyces exfoliates;菌株4-21鉴定为Streptomyces albidoflavus:菌株4-70鉴定为Streptomyces albus。选取55株初筛具有抑制稻瘟病菌活性的内生放线菌,包括菌株3-17、4-21、4-70在内,进行Ⅰ型聚酮合酶PKS Ⅰ、Ⅱ型聚酮合酶PKS Ⅱ和非核糖体肽合成酶NRPS功能基因筛选,发现55株供试菌株中有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,且几乎全部属于链霉菌属。结果表明,能抑制稻瘟病菌的内生放线菌具有合成聚酮类化合物及非核糖体肽类抑菌物质的潜力。菌株4-70与Streptomyces sp.KE1未知代谢产物的PKS I氨基酸序列同源性为100%,说明该菌株具有产生聚酮类化合物的能力,可能通过非芳香环聚酮化合物产生抑制稻瘟病菌的效果。通过显微镜观察和水解酶活力检测,发现内生放线菌3-17、4-21、4-70通过分泌胞外水解酶和生长竞争相互协同的机制来抑制稻瘟病菌。内生放线菌3-17、4-21、4-70无细胞发酵液不仅对稻瘟病菌有很好的抑制作用,且对稻瘟病菌孢子萌发及芽管伸长也有显著的抑制作用。根据结果推测菌株3-17、4-21也可能产生抑菌物质,但是抑菌物质的种类和合成途径还有待进一步研究探索。采用内生放线菌3-17、4-21、4-70孢子混悬液灌根预处理水稻后,再接种稻瘟病菌,观测了离体水稻叶片防治效果；结果表明,内生放线菌灌根处理后,叶片病斑个数及直径大小均较对照组有明显的降低,能显著抑制水稻稻瘟病的发生。测定水稻叶片防御酶活性,发现菌株3-17、4-21、4-70处理组PAL、POD、LOX活性均高于对照组；菌株3-17、4-21处理组PPO活性高于对照组,但菌株4-70处理并不能显著增加水稻PPO活性；菌株3-17处理组AOS活性高于对照组,但菌株4-21、4-70处理组AOS活性与对照组差异不大甚至低于对照组。采用内生放线菌3-17、4-21、4-70孢子混悬液灌根预处理水稻后,再接种稻瘟病菌,运用荧光定量PCR技术观测了水杨酸以及茉莉酮酸/烯信号转导途径中关键防御基因的表达情况,结果发现内生放线菌3-17、4-21、4-70主要都是通过水杨酸信号转导途径诱导水稻抗病性增强,从而达到防病促生的效果。在盆栽温室培养条件下,内生放线菌3-17、4-21、4-70灌根预处理水稻苗均对稻瘟病表现出一定的防治效果。在盆栽促生研究中发现,无论接种稻瘟与否,内生放线菌3-17、4-21、4-70灌根预处理都能增强水稻幼苗根系活力。但是内生放线菌3-17、4-21、4-70单独灌根预处理,导致水稻幼苗植株地上部分变矮,根系变长而发达,其中菌株3-17和4-70增加叶绿素含量从而促进植株光合作用。当有稻瘟病菌存在时,内生放线菌3-17、4-21、4-70促进水稻幼苗地上部分增高,根系发达生长,其中菌株4-21增加叶绿素含量促进植株光合作用。内生放线菌3-17、4-21、4-70灌根预处理能促进水稻生长。综上所述,本研究从阿坝药用植物中筛选到的内生放线菌3-17、4-21、4-70通过产生不同抑菌物质；分泌胞外水解酶和生长竞争相互协同；诱导宿主植物水杨酸信号转导途径；增强宿主植株光合作用及根系活力进而促进植株生长这四方面机制,达到防治水稻稻瘟病和促进植株生长的效果,并且能在水稻植株内有效定殖,具备开发成生防菌剂的潜力。
[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.
【学位授予单位】：四川农业大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：S476

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