硅对黄瓜枯萎病及土壤微生物的影响

发布时间：2018-06-04 00:11

  本文选题：硅 + 黄瓜　； 参考：《东北农业大学》2016年硕士论文

【摘要】：硅作为地壳中含量仅次于氧的矿质营养元素,其对增强作物抗病性的的有益作用越来越受到关注和重视。对此,国内外学者针对硅提高作物抗病性的作用机制开展了大量的研究,前人采用水培研究发现,加硅能通过增加黄瓜(Cucumis sativas L.)植株体内抗病相关酶的活性降低枯萎病的发病率,但是在土壤条件下,硅是否能通过改变土壤微生物群落提高黄瓜对枯萎病的抗性,目前尚不清楚。本实验通过设置无苗土壤施硅、有苗土壤施硅和叶面喷施硅等处理,分别研究了不同施硅方式对黄瓜植株生长、枯萎病发病率的影响,以及利用PCR-DGGE,qPCR,Mi Seq等分子生物技术研究了其对土壤微生物群落结构、组成和丰度的影响;并且利用植物-土壤反馈的方法研究了不同施硅方式引起的土壤微生物的变化对黄瓜植株枯萎病发病率的反馈作用;旨在明确土壤微生物群落的改变对黄瓜植株枯萎病抗性的影响;此外我们进一步通过体外试验研究了硅对尖孢镰刀菌黄瓜专化型和链霉菌DHV3-2孢子数量和生长以及其对尖孢镰刀菌黄瓜专化型产镰刀菌酸的影响,旨在明确硅对尖孢镰刀菌的直接影响,进一步揭示硅介导的土壤微生物群落的改变在提高黄瓜植株枯萎病抗性的重要作用,为从土壤微生物的角度解释硅提高作物抗病机制提供理论基础。所得主要结果如下:(1)加硅处理后第20d,有苗土壤施硅和叶面喷施硅处理显著增加了黄瓜植株的鲜重和干重;有苗土壤施硅处理显著降低了黄瓜植株枯萎病的发病率,而叶面喷施硅对发病率无显著影响。(2)PCR-DGGE和qPCR结果表明,无苗土壤施硅处理增加了土壤细菌和真菌条带数、多样性指数和群落丰度,有苗土壤加硅提高了细菌多样性指数和群落丰度,但降低了土壤真菌条带数、多样性指数和群落丰度,叶面喷施硅对土壤细菌和真菌条带数、多样性指数和群落丰度无影响,说明不同施硅方式对土壤微生物群落结构和丰度的影响存在差异。(3)Mi Seq测序结果发现,不同施硅方式对土壤细菌群落丰富度指数、多样性指数和组成无影响。所有17015个OTU分属于细菌的40个门,其中主要包括变形菌门、放线菌门、酸杆菌门、绿弯菌门、拟杆菌门、芽单胞菌门、浮霉菌门、厚壁菌门等,但改变了土壤真菌群落丰富度指数、多样性指数和组成。所有4468个OTU分属于真菌的8个门,主要为子囊菌门、接合菌门、担子菌门等。无苗土壤加硅处理提高了真菌丰富度指数和多样性指数,降低了接合菌门、接合菌纲、座囊菌纲、,增加了担子菌门、银耳纲、镰刀菌属的相对百分丰度;有苗土壤加硅处理降低了真菌丰富度指数、多样性指数和粪壳菌纲的相对百分丰度,但增加了担子菌门、盘菌纲、镰刀菌属的相对百分丰度;叶面喷施硅处理降低了真菌多样性指数及接合菌门、担子菌门、粪壳菌纲的相对百分丰度,但增加了子囊菌门、盘菌纲、座囊菌纲、茎点霉属的相对百分丰度。(4)主坐标轴(PCoA)和非线性多维标度(NMDS)分析表明,不同施硅方式改变了土壤细菌和真菌群落结构。对于细菌群落结构而言,有苗土壤施硅和叶面喷施硅两者之间距离较近,但两者与无苗土壤加硅距离较远,说明有苗土壤施硅和叶面喷施硅与无苗土壤施硅的细菌群落结构差异较大;对于真菌群落结构而言,有苗土壤施硅、叶面喷施硅和无苗土壤施硅3种不同施硅方式之间距离均较远,说明3种不同施硅方式真菌群落结构差异较大。(5)不同施硅方式土壤细菌群落和真菌群落ANOSIM相似性分析可知,不同处理间真菌群落结构差异显著;不同施硅方式在纲水平共有和独有的OTU结果表明,就细菌群落结构而言,共有的纲为酸杆菌纲、放线菌纲、β-变形菌纲、α-变形菌纲、γ-变形菌,无苗土壤施硅独有的纲为疣微菌纲、柔膜菌纲、BD1-5_norank,有苗土壤施硅独有的纲为Caldisericia,叶面喷施硅处理独有的纲为Actinobacteria_unclassified,VadinHA17,拟杆菌纲;就真菌群落结构而言,共有的纲主要为主要为粪壳菌纲,Zygomcota_incertae_sedis,无苗土壤施硅独有的纲为囊担菌纲,有苗土壤施硅处理独有的纲为Archacorhizomycetes,叶面喷施硅处理独有的纲为芽枝霉纲。(6)植物-土壤反馈实验结果表明,接种6%有苗土壤施硅的土壤微生物显著降低了黄瓜幼苗枯萎病的病情指数,增加了黄瓜植株根的SOD,POD,PAL,GLU酶的活性,接种6%叶面喷施硅土壤和无苗施硅土壤微生物对黄瓜枯萎病的发病率和抗性相关酶活的影响较小,说明有苗土壤施硅能够通过改变土壤微生物群落提高黄瓜幼苗对枯萎病的抗性。(7)体外实验结果表明,加硅未调节pH条件下降低了尖孢镰刀菌和链霉菌DHV3-2的孢子数量,加硅调节p H后增加了尖孢镰刀菌黄瓜专化型和链霉菌DHV3-2的孢子数量,说明了硅体外抑制尖孢镰刀菌黄瓜专化型和链霉菌DHV3-2孢子数量是由于基质pH的增加造成的;此外,我们利用HPLC检测发现,在培养基中加硅抑制了尖孢镰刀菌的产酸量;土壤试验结果表明,土壤施硅不能直接抑制尖孢镰刀菌的生长,但增加了链霉菌DHV3-2的数量,说明在土壤中加硅降低黄瓜枯萎病的发病率,并不是通过直接抑制尖孢镰刀菌的生长而实现的。综上所述,PCR-DGGE,qPCR,Mi Seq结果表明,无苗土壤施硅、有苗土壤施硅和叶面喷施硅3种方式对土壤微生物群落结构和丰度的影响不同;有苗土壤施硅是通过改变土壤微生物群落结构增强黄瓜对枯萎病的抗性,进而降低了黄瓜枯萎病的发病率。
[Abstract]:As a mineral nutrient element in the earth's crust only inferior to oxygen, silicon has attracted more and more attention and attention to enhance the disease resistance of crops. A lot of researches have been carried out by scholars at home and abroad on the mechanism of silicon to improve the disease resistance of crops. S L.) the activity of resistance related enzymes in the plant reduces the incidence of Fusarium wilt, but it is not clear whether silicon can improve the resistance of cucumber to Fusarium wilt by changing soil microbial community in soil conditions. The effects of silicon application on the growth of cucumber plant, the incidence of Fusarium wilt, and the effects of PCR-DGGE, qPCR and Mi Seq on the structure, composition and abundance of soil microbial community were studied, and the changes of soil microorganism caused by different silicon application methods on cucumber plants were studied by the method of plant soil feedback. The feedback effect of the incidence of Fusarium wilt; to determine the effect of soil microbial community changes on the resistance of cucumber plant Fusarium wilt. In addition, we further studied the number and growth of the spores of the cucumber specific and Streptomyces DHV3-2 by silicon to Fusarium oxysporum and the Fusarium oxysporum of Fusarium oxysporum. The effect was aimed at determining the direct effect of silicon on Fusarium oxysporum, and further revealing the important role of silicon mediated soil microbial community change in improving the resistance of cucumber plant Fusarium wilt, providing a theoretical basis for the interpretation of silicon to improve the mechanism of crop resistance from the angle of soil microorganism. The main results are as follows: (1) 20d after adding silicon, Silicon treatment with silicon in seedling soil and spraying silicon on leaf surface significantly increased fresh weight and dry weight of cucumber plants, and silicon treatment in seedlings significantly reduced the incidence of Cucumber Fusarium wilt, while foliar spraying silicon had no significant influence on the incidence of plant disease. (2) PCR-DGGE and qPCR results showed that the application of silicon treatment in seedling free soil increased soil bacterial and fungal bands. The number, the diversity index and the abundance of the community increased the bacterial diversity index and the community abundance, but decreased the number, the diversity index and the community abundance of the soil fungi. The foliar spraying silicon has no effect on the number, the diversity index and the community abundance of the soil bacteria and fungi and the diversity index and the community abundance. The effects of falling structure and abundance were different. (3) Mi Seq sequencing results showed that different silicon application methods had no influence on soil bacterial community richness index, diversity index and composition. All 17015 OTU fractions belonged to 40 doors of bacteria, including deformable bacteria doors, actinomycetes, acid bacilli doors, greenben doors, Pseudomonas, and butoomonas There are 4468 OTU points that belong to 8 doors of fungi, mainly ASCO bacteria gate, conjugant gate, basidiomycetes and so on. The class, cysts, increased the relative percent abundance of basidiomycetes, tremella, and Fusarium; the seedling soil and silicon treatment reduced the fungal richness index, the diversity index and the relative percent abundance of the faeciomycetes, but increased the relative percent abundance of the basidiomycetes, the fungi, the Fusarium, and the foliar spraying silicon treatment reduced the fungi. The relative percentage abundances of the phylum, basidiomycetes, and fecal bacteria, but increased the relative percent abundance of the ascysta, the phylum, the cystomycetes, and the Rhizopus. (4) the main coordinate axis (PCoA) and the nonlinear multidimensional scaling (NMDS) analysis showed that different methods of silicon application changed the soil bacterial and fungal community structure. In terms of the structure, the distance between the silicon and the foliar spraying silicon in the seedling soil is close, but the distance between the two and the seedlings is far away. It shows that the structure of the bacterial community structure with silicon and the foliage spraying silicon and the seedlings without seedling soil is larger. For the community structure of the fungi, the silicon in the seedling soil and the foliage spraying silicon and the seedling free soil are applied to the fungi community structure. The distance between 3 different silicon application methods showed that there were great differences in the structure of Fungi Community in 3 different silicon application methods. (5) the similarity analysis of soil bacterial community and fungal community in different silicon fertilization methods showed that the differences of fungi community structure between different treatments were significant; different silicon application methods were common in the class level and unique OTU result table. (5) the ANOSIM similarity analysis between different treatments of soil bacteria community and fungal community In terms of bacterial community structure, the common classes are acidobacteria, actinomycetes, beta deformiae, alpha deformiae, gamma deformable bacteria, and seedling soil silicon, which are unique to verruca Micrococcus, soft membrane bacteria, BD1-5_norank, unique to Caldisericia for silicon application in seedling soil, and a unique outline of Actinobacteria_unclassified for foliar application of silicon. VadinHA17, bacteriobacteria; in terms of the community structure of fungi, the main class is mainly the fecal crustaceans, and Zygomcota_incertae_sedis. The unique outline of silicon application in the seedling free soil is the class cyoscopia. The unique outline of the silicon treatment in the seedling soil is Archacorhizomycetes, and the unique outline of the foliar spraying silicon treatment is the class bud mycoplasma. (6) plant soil feedback real The results showed that the soil microorganism inoculated with 6% seeded Soil Silicon significantly reduced the disease index of cucumber seedling blight, increased the activity of SOD, POD, PAL, GLU enzyme in cucumber root, and had less influence on the incidence of Cucumber Fusarium Wilt and resistance related enzyme activity by spraying silicon soil and seedless silicon soil microorganism on 6% leaf surface. The application of silicon in seedling soil can improve the resistance of cucumber seedlings to Fusarium wilt by changing soil microbial community. (7) in vitro experiment results showed that the number of spores of Fusarium oxysporum and Streptomyces DHV3-2 were reduced under the condition of unregulated pH, and the number of spores of Fusarium oxysporum cucumber and Streptomyces DHV3-2 were increased after adding silicon to regulate P H In addition, we found that silicon inhibited the acidity of Fusarium oxysporum in the culture medium by adding silicon to the increase of the number of DHV3-2 spores of cucumber and Streptomyces of Fusarium oxysporum in vitro. In addition, we found that silicon inhibited the acidity of Fusarium oxysporum in the medium. Soil tests showed that soil soil silicon could not directly inhibit the growth of Fusarium oxysporum, but the soil test results showed that the growth of Fusarium oxysporum was not directly inhibited by soil test. The increase of the number of Streptomyces DHV3-2 indicates that the reduction of Cucumber Fusarium wilt by adding silicon in soil is not achieved by directly inhibiting the growth of Fusarium oxysporum. In summary, the results of PCR-DGGE, qPCR, and Mi Seq show that the soil microorganism community structure of the seedling soil silicon application and the foliar spraying silicon in the seedling soil is 3 methods. The effect is different from the abundance, and the application of silicon in seedling soil is to enhance the resistance of cucumber to Fusarium wilt by changing the soil microbial community structure, and then reduce the incidence of Cucumber Fusarium wilt.
【学位授予单位】：东北农业大学
【学位级别】：硕士
【学位授予年份】：2016
【分类号】：S154.3;S436.421.13
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本文编号：1974870