本文選題：硅 + 黃瓜��； 參考：《東北農業(yè)大學》2016年碩士論文

【摘要】：硅作為地殼中含量僅次于氧的礦質營養(yǎng)元素,其對增強作物抗病性的的有益作用越來越受到關注和重視。對此,國內外學者針對硅提高作物抗病性的作用機制開展了大量的研究,前人采用水培研究發(fā)現,加硅能通過增加黃瓜(Cucumis sativas L.)植株體內抗病相關酶的活性降低枯萎病的發(fā)病率,但是在土壤條件下,硅是否能通過改變土壤微生物群落提高黃瓜對枯萎病的抗性,目前尚不清楚。本實驗通過設置無苗土壤施硅、有苗土壤施硅和葉面噴施硅等處理,分別研究了不同施硅方式對黃瓜植株生長、枯萎病發(fā)病率的影響,以及利用PCR-DGGE,qPCR,Mi Seq等分子生物技術研究了其對土壤微生物群落結構、組成和豐度的影響;并且利用植物-土壤反饋的方法研究了不同施硅方式引起的土壤微生物的變化對黃瓜植株枯萎病發(fā)病率的反饋作用;旨在明確土壤微生物群落的改變對黃瓜植株枯萎病抗性的影響;此外我們進一步通過體外試驗研究了硅對尖孢鐮刀菌黃瓜專化型和鏈霉菌DHV3-2孢子數量和生長以及其對尖孢鐮刀菌黃瓜�；彤a鐮刀菌酸的影響,旨在明確硅對尖孢鐮刀菌的直接影響,進一步揭示硅介導的土壤微生物群落的改變在提高黃瓜植株枯萎病抗性的重要作用,為從土壤微生物的角度解釋硅提高作物抗病機制提供理論基礎。所得主要結果如下:(1)加硅處理后第20d,有苗土壤施硅和葉面噴施硅處理顯著增加了黃瓜植株的鮮重和干重;有苗土壤施硅處理顯著降低了黃瓜植株枯萎病的發(fā)病率,而葉面噴施硅對發(fā)病率無顯著影響。(2)PCR-DGGE和qPCR結果表明,無苗土壤施硅處理增加了土壤細菌和真菌條帶數、多樣性指數和群落豐度,有苗土壤加硅提高了細菌多樣性指數和群落豐度,但降低了土壤真菌條帶數、多樣性指數和群落豐度,葉面噴施硅對土壤細菌和真菌條帶數、多樣性指數和群落豐度無影響,說明不同施硅方式對土壤微生物群落結構和豐度的影響存在差異。(3)Mi Seq測序結果發(fā)現,不同施硅方式對土壤細菌群落豐富度指數、多樣性指數和組成無影響。所有17015個OTU分屬于細菌的40個門,其中主要包括變形菌門、放線菌門、酸桿菌門、綠彎菌門、擬桿菌門、芽單胞菌門、浮霉菌門、厚壁菌門等,但改變了土壤真菌群落豐富度指數、多樣性指數和組成。所有4468個OTU分屬于真菌的8個門,主要為子囊菌門、接合菌門、擔子菌門等。無苗土壤加硅處理提高了真菌豐富度指數和多樣性指數,降低了接合菌門、接合菌綱、座囊菌綱、,增加了擔子菌門、銀耳綱、鐮刀菌屬的相對百分豐度;有苗土壤加硅處理降低了真菌豐富度指數、多樣性指數和糞殼菌綱的相對百分豐度,但增加了擔子菌門、盤菌綱、鐮刀菌屬的相對百分豐度;葉面噴施硅處理降低了真菌多樣性指數及接合菌門、擔子菌門、糞殼菌綱的相對百分豐度,但增加了子囊菌門、盤菌綱、座囊菌綱、莖點霉屬的相對百分豐度。(4)主坐標軸(PCoA)和非線性多維標度(NMDS)分析表明,不同施硅方式改變了土壤細菌和真菌群落結構。對于細菌群落結構而言,有苗土壤施硅和葉面噴施硅兩者之間距離較近,但兩者與無苗土壤加硅距離較遠,說明有苗土壤施硅和葉面噴施硅與無苗土壤施硅的細菌群落結構差異較大;對于真菌群落結構而言,有苗土壤施硅、葉面噴施硅和無苗土壤施硅3種不同施硅方式之間距離均較遠,說明3種不同施硅方式真菌群落結構差異較大。(5)不同施硅方式土壤細菌群落和真菌群落ANOSIM相似性分析可知,不同處理間真菌群落結構差異顯著;不同施硅方式在綱水平共有和獨有的OTU結果表明,就細菌群落結構而言,共有的綱為酸桿菌綱、放線菌綱、β-變形菌綱、α-變形菌綱、γ-變形菌,無苗土壤施硅獨有的綱為疣微菌綱、柔膜菌綱、BD1-5_norank,有苗土壤施硅獨有的綱為Caldisericia,葉面噴施硅處理獨有的綱為Actinobacteria_unclassified,VadinHA17,擬桿菌綱;就真菌群落結構而言,共有的綱主要為主要為糞殼菌綱,Zygomcota_incertae_sedis,無苗土壤施硅獨有的綱為囊擔菌綱,有苗土壤施硅處理獨有的綱為Archacorhizomycetes,葉面噴施硅處理獨有的綱為芽枝霉綱。(6)植物-土壤反饋實驗結果表明,接種6%有苗土壤施硅的土壤微生物顯著降低了黃瓜幼苗枯萎病的病情指數,增加了黃瓜植株根的SOD,POD,PAL,GLU酶的活性,接種6%葉面噴施硅土壤和無苗施硅土壤微生物對黃瓜枯萎病的發(fā)病率和抗性相關酶活的影響較小,說明有苗土壤施硅能夠通過改變土壤微生物群落提高黃瓜幼苗對枯萎病的抗性。(7)體外實驗結果表明,加硅未調節(jié)pH條件下降低了尖孢鐮刀菌和鏈霉菌DHV3-2的孢子數量,加硅調節(jié)p H后增加了尖孢鐮刀菌黃瓜�；秃玩溍咕鶧HV3-2的孢子數量,說明了硅體外抑制尖孢鐮刀菌黃瓜�；秃玩溍咕鶧HV3-2孢子數量是由于基質pH的增加造成的;此外,我們利用HPLC檢測發(fā)現,在培養(yǎng)基中加硅抑制了尖孢鐮刀菌的產酸量;土壤試驗結果表明,土壤施硅不能直接抑制尖孢鐮刀菌的生長,但增加了鏈霉菌DHV3-2的數量,說明在土壤中加硅降低黃瓜枯萎病的發(fā)病率,并不是通過直接抑制尖孢鐮刀菌的生長而實現的。綜上所述,PCR-DGGE,qPCR,Mi Seq結果表明,無苗土壤施硅、有苗土壤施硅和葉面噴施硅3種方式對土壤微生物群落結構和豐度的影響不同;有苗土壤施硅是通過改變土壤微生物群落結構增強黃瓜對枯萎病的抗性,進而降低了黃瓜枯萎病的發(fā)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.
【學位授予單位】：東北農業(yè)大學
【學位級別】：碩士
【學位授予年份】：2016
【分類號】：S154.3;S436.421.13
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本文編號：1974870