【作者】 廖敏；

【导师】 张小平；

【作者基本信息】 四川农业大学 ， 微生物学， 2016， 博士

【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。更多还原

【Abstract】 Plants and endophytes have passed through a long procedure of evolution. They had been fit for each other already. Plants provide nutrients for endophytes, endophytes can absorb nutrient from the host plants for survival, which assist them in adapting to a variety of environmental stresses. The endophytes can promote the growth of the host plants and also can induce plant’s tolerance to both abiotic and biotic stress through a synergistic mechanism involving synthesis of active substances such as antibiotics、 hormone and inductor, competition for nutrition, induced systemic resistance, etc. Aba region in Sichuan in China with unique climate and ecosystem is a habitat of a great variety of medicinal plants. We can isolate a lot of endophytes from these medicinal plants.In this study,737 endophytic actinomycetes were isolated from 36 medicinal plant species in Aba region by using ten isolation mediums. The results indicated that the Raffinose-histidine medium was the optimal one and the Glucose-asparagine medium was the worst one to isolate endophytic actinomycetes from medicinal plants. The types and quantities of endophytic actinomycetes in different tissues of medicinal plants have been analyzed, the results showed that the types and quantities in roots were the largest, and then were the leaves and stems, the minimum number of endophytic actinomycetes were in the fruits and flowers.Of these, endophytic actinomycetes isolated with high inhibitory activity against Magnaporthe grisea were selected by stepwise screening using the plate confrontation and mycelium growth rate measurement. At the same time, plant growth promoting activity of endophytic actinomycetes were also analyzed. Finally strains 3-17 4-21&4-70 with high inhibitory activity against Magnaporthe grisea and plant-growth promotion were selected for follow-up study.Strain 3-17 was identified as Streptomyces exfoliates, strain 4-21 was identified as Streptomyces albidoflavus and strain 4-70 was identified as Streptomyces albus on the basis of the morphological features, cultutal, physiological & biochemical characteristics and phylogenetic analysis of 16S rDNA.Sequence Guided Genetic screening was performed to evaluate the potential ability of synthesizing antibiotic substances.55 strains with high inhibitory activity against Magnaporthe grisea were amplified the type Ⅰ& type Ⅱ polyketide synthase gene and non-ribosomal peptide synthetases (NRPS) gene by PCR. The results showed,31 strains contain PKS Ⅰ gene,15 strains contain PKS II gene and 18 strains contain NRPS gene, respectively. Type I polyketide synthase (PKSI) is successfully amplified out from strain 4-70, which indicates that strain 4-70 has the potential ability of generating polyketide.The antagonisms of strains 3-17,4-21 and 4-70 to Magnaporthe grisea were combined with antifungal substance, hydrolase, spatial and nutritional competition. Inhibitory activities of antifungal substance were significantly promoted by inhibiting conidiospore germination and germ tube growth of Magnaporthe grisea and arousing hyphal aberrance of Magnaporthe grisea.The rice seedlings were treated with strains 3-17,4-21&4-70 by root-irrigating, then inoculate with Magnaporthe grisea. Studying the change of several key defensive enzymes activities in rice seedlings leaves, we found that PAL、POD、LOX activities were higher than control which treated with strains 3-17,4-21&4-70. The PPO activities were higher than control which treated with strains3-17,4-21; but strain 4-70 treatment can not enhance PPO activity. Strain 3-17 treatment AOS activity was higher than control, but strains 4-21&4-70 treaments AOS activities were lower than control.Strains 3-17,4-21&4-70 could enhance the disease-resistance and defense function against Magnaporthe grisea and cause the induced systemic resistance ISR response by root-irrigating treatments. All 3-17,4-21&4-70 strains-treated rice seedlings, resistance was likely to induce SA signal transduction pathway.Strains 3-17,4-21&4-70 could colonize in rice seedlings, and promoted the growth of seedlings. Treatments with strains 3-17,4-21&4-70 significantly improved the length and weight of roots, chlorophyll content, root activity, but reduced the length and weight of stems and leaves. Treatments’with strains 3-17,4-21&4-70 and Magnaporthe grisea significantly improved the length and weight of roots and stems and leaves, chlorophyll content, root activity.In conclusion, this study indicated the plant growth-promoting effects of strains 3-17, 4-21&4-70 and enhancement of disease resistance on rice seedlings.更多还原