本文關(guān)鍵詞：設(shè)施土壤氮素積累條件下番茄枯萎病發(fā)生的微生態(tài)機(jī)制研究　出處：《沈陽農(nóng)業(yè)大學(xué)》2016年博士論文　論文類型：學(xué)位論文

  更多相關(guān)文章： 土壤氮素積累 微生物區(qū)系 番茄發(fā)病率 枯萎菌 拮抗菌

【摘要】：設(shè)施栽培目前已成為人們獲得高產(chǎn)、優(yōu)質(zhì)果蔬的重要途徑,但是設(shè)施栽培土壤中普遍存在過量施肥問題,其中氮肥的過量施用現(xiàn)象尤為嚴(yán)重。隨著種植年限長、土壤氮磷鉀速效養(yǎng)分的不斷積累,引起設(shè)施土壤養(yǎng)分嚴(yán)重不平衡、氮素等積累現(xiàn)象非常明顯,這導(dǎo)致了設(shè)施土壤酸化、鹽漬化現(xiàn)象越來越嚴(yán)重,番茄枯萎病等蔬菜的土傳病害發(fā)生率一直居高不下�，F(xiàn)有的研究主要集中于從植物保護(hù)的角度對土傳病害的防治,而從土壤養(yǎng)分角度出發(fā)研究關(guān)于設(shè)施土壤養(yǎng)分積累條件下番茄枯萎病等土傳病害的發(fā)生機(jī)制方面的研究則相對較少。本研究以番茄枯萎病菌為研究對象,設(shè)置土壤不同速效氮含量,分別從宏觀(土壤微生物生態(tài))角度和微觀(微生物生理生化)角度兩方面來探索設(shè)施土壤氮素積累條件下番茄枯萎病發(fā)生的機(jī)制。通過土壤培養(yǎng)試驗和液體發(fā)酵純培養(yǎng)試驗,綜合分析了氮素含量和氮素形態(tài)對微生物數(shù)量、微生物群落動態(tài)及微生物區(qū)系變化的影響,同時探究了不同速效氮含量土壤接種枯萎菌條件下番茄的發(fā)病率及病情指數(shù)的變化情況。本研究從微生物平衡角度對設(shè)施土壤長期氮積累條件下土傳病害發(fā)生及發(fā)病率升高的機(jī)制提出合理假設(shè)：隨著土壤微生物區(qū)系平衡(主要是土壤微生物比例變化)被打破,當(dāng)超過“閾值”時將導(dǎo)致土傳病害的發(fā)生,微生物比例越偏離“閾值”,發(fā)病率則相應(yīng)越高。同時調(diào)查發(fā)現(xiàn)在實際生產(chǎn)中長期種植作物的土壤和長期大量施氮肥的土壤更容易感染土傳病害,而土壤中硝態(tài)氮的大量累積有利于病原菌生長,不利于病原菌的拮抗微生物生長。定量化研究了氮素積累對土壤微生物及番茄枯萎菌數(shù)量的影響,發(fā)現(xiàn)土壤中各種微生物對氮素形態(tài)及含量有不同的響應(yīng),進(jìn)而發(fā)現(xiàn)氮素積累量過高時細(xì)菌、放線菌的比例嚴(yán)重下降,真菌、枯萎菌的比例有所上升,土壤微生物原有的區(qū)系平衡被打破,土壤微生物多樣性減少,最終導(dǎo)致番茄枯萎病發(fā)病率升高,得出了土壤氮素大量積累是影響番茄枯萎病發(fā)病率升高的重要原因之一。由此可見本試驗的研究結(jié)論與生產(chǎn)實踐相符。本研究成果將為設(shè)施栽培土壤中合理施肥以控制土傳病害提供理論支持。主要研究結(jié)果如下：1.在土壤培養(yǎng)試驗中,不同氮素積累試驗(速效氮含量為95.96、203.56、257.62、279.36、319.59、348.94 mg·kg-1)條件下,隨著培養(yǎng)時間的延長,土壤中可培養(yǎng)細(xì)菌、真菌、放線菌數(shù)量均表現(xiàn)出前期增加,培養(yǎng)末期緩慢降低的趨勢；而土壤中枯萎菌數(shù)量則表現(xiàn)為前期增加,培養(yǎng)后期緩慢增加的趨勢。速效氮含量為279.36 mg·kg-1～319.59mg·kg-1的土壤可培養(yǎng)細(xì)菌數(shù)量達(dá)到最多,而土壤可培養(yǎng)真菌數(shù)量、可培養(yǎng)放線菌數(shù)量、土壤枯萎菌數(shù)量分別是在土壤速效氮含量為279.36mg·kg-1、257.62mg·kg-1、 319.59mg·kg-1的條件下達(dá)到最多。因此,土壤中氮素積累能顯著影響土壤可培養(yǎng)微生物(三大類菌群數(shù)量和枯萎菌數(shù)量)的數(shù)量。2.氮素積累不僅能顯著影響土壤微生物的數(shù)量,同時引起了微生物比例的改變。不同氮素積累條件下,土壤中細(xì)菌與真菌的比值(B/F)、放線菌與真菌的比值(A/F),隨著土壤中氮素積累量的增加均呈先升高后降低的趨勢；土壤速效氮含量為203.56mg·kg-1時土壤中B/F、A/F均達(dá)到最高,說明此時土壤微生物中細(xì)菌、放線菌所占比例最高。隨著土壤中氮素積累量的增多,土壤中細(xì)菌、放線菌不僅數(shù)量減少并且所占比例降低。通過不依賴培養(yǎng)的16S rDNA(V4區(qū))高通量測序分析發(fā)現(xiàn),土壤中氮素積累的增加會減少細(xì)菌和古菌的生物多樣性及細(xì)菌的物種豐度。3.土壤氮素積累能顯著引起土壤pH降低,同時引起土壤No3--N、NH4+-N、堿解氮、有效氮含量的增加,土壤pH與土壤中可培養(yǎng)細(xì)菌、真菌數(shù)量及枯萎菌數(shù)量呈負(fù)相關(guān)關(guān)系,與可培養(yǎng)放線菌數(shù)量呈正相關(guān)關(guān)系；土壤NO3--N、NH4+-N、堿解氮、有效氮含量與土壤中可培養(yǎng)細(xì)菌、真菌數(shù)量及枯萎菌數(shù)量呈正相關(guān)關(guān)系,而與可培養(yǎng)放線菌數(shù)量呈負(fù)相關(guān)關(guān)系。由此可見土壤氮素積累引起的土壤指標(biāo)的變化能顯著影響土壤微生物區(qū)系變化和枯萎菌的數(shù)量變化。4.在土壤培養(yǎng)試驗中,不同氮素積累條件下向土壤中接種枯萎菌后,隨著培養(yǎng)時間的延長土壤中可培養(yǎng)細(xì)菌、真菌、放線菌數(shù)量變化及枯萎菌數(shù)量變化與未接菌土壤變化的趨勢相一致。在接菌土壤中,氮素含量為279.36mg·kg-1的土壤可培養(yǎng)細(xì)菌數(shù)量達(dá)到最多,土壤速效氮含量為319.59mg·kg-1的土壤可培養(yǎng)真菌數(shù)量達(dá)到最多,土壤速效氮含量為257.62mg·kg-1的土壤可培養(yǎng)放線菌數(shù)量達(dá)到最多,土壤速效氮含量為319.59mg·kg-1的土壤枯萎菌數(shù)量達(dá)到最多。人為向土壤中接入枯萎菌后,能顯著改變土壤三大微生物種群的數(shù)量,顯著提高了真菌數(shù)量,抑制細(xì)菌和放線菌數(shù)量的增加,從而引起微生物區(qū)系平衡的劇烈變化,進(jìn)而引起番茄枯萎病的發(fā)生。5.在土壤培養(yǎng)試驗中,不同氮素積累條件下向土壤中接種枯萎菌后,土壤不同速效氮含量下番茄均不同程度發(fā)病,當(dāng)土壤中速效氮含量為203.56mg·kg-1時番茄發(fā)病率及病情指數(shù)均達(dá)到最低,發(fā)病率和病情指數(shù)分別為41.82%、16.22%。此時土壤中細(xì)菌、放線菌所占比例最高,真菌所占比例最低。隨著土壤中細(xì)菌、放線菌所占比例的降低,真菌所占比例的升高,番茄枯萎病發(fā)病率和病情指數(shù)均出現(xiàn)升高趨勢。由此可見,氮素積累量加重土壤微生物平衡被打破,細(xì)菌、放線菌所占比例降低,真菌所占比例升高是致使番茄發(fā)病率升高的重要因素。6.在純培養(yǎng)試驗中,NO3--N最為唯一氮源時,枯萎菌的繁殖能力、產(chǎn)酸能力、分泌毒素及植物細(xì)胞壁降解酶活的能力均達(dá)到最好；NH4+-N作為唯一氮源時,以解淀粉芽孢桿菌、枯草芽孢桿菌、草酸青霉菌為代表的番茄枯萎菌的拮抗菌的生長能力、產(chǎn)酸能力、分泌水解性酶的能力達(dá)到最高。然而當(dāng)?shù)貪舛冗^高時微生物的生長及代謝均會受到抑制�？菸陨砜梢援a(chǎn)生酸性物質(zhì),并且它們更易于在酸性環(huán)境中生長繁殖。土壤中硝態(tài)氮的大量累積會引起土壤pH值降低,這更有利于枯萎菌的繁殖。
[Abstract]:Protected cultivation has become an important way for people to obtain high yield, high-quality fruits and vegetables, but the ubiquitous problem of excessive fertilization soil cultivation, the phenomenon of excessive application of nitrogen fertilizer is particularly serious. Along with the planting years long, continuous accumulation of nitrogen phosphorus and potassium in soil available nutrient, soil nutrient imbalance caused by facilities, such as nitrogen accumulation phenomenon is very obvious this leads to the facilities, soil acidification, salinization phenomenon is more and more serious, such as vegetable Fusarium Wilt of tomato soil borne disease incidence rate has been high. The prevention and control of existing research focused on plant protection from the perspective of soil borne diseases, and starting from the angle of soil nutrient research studies on the pathogenesis of the disease from tomato wilt disease and other soil conditions of soil nutrient accumulation facilities is relatively small. In this study, Fusarium oxysporum as the research object, not the same set of soil available nitrogen The content, from the macro (soil microbial ecology) and micro angle (microbial physiology and Biochemistry) two aspects to explore the soil nitrogen accumulation mechanism of tomato Fusarium wilt occurrence conditions. Through soil culture experiment and liquid fermentation of pure culture test, a comprehensive analysis of the content of nitrogen and nitrogen forms on microorganisms, effects of microbial community dynamics and microbial flora, and explore the changes of disease incidence and disease index of different soil available nitrogen content in tomato Fusarium wilt pathogen inoculation conditions. This study proposes reasonable assumptions from under the condition of soil borne diseases occurrence and mechanism of increased incidence angle of microbial balance to soil nitrogen accumulation: with the long-term balance of soil microbial flora (changes of soil microbial proportion) is broken, when more than the "threshold" will cause the occurrence of soil borne diseases, the proportion of microorganisms Deviate from the "threshold", the incidence rate is higher and higher. At the same time, the corresponding survey found that long-term crop planting in the actual production of the soil and a large number of long-term fertilization soil more susceptible to soil borne disease, and nitrate nitrogen in soil is conducive to the accumulation of a large number of pathogenic bacteria growth, is not conducive to the growth of antagonistic microorganism pathogens. Quantitative study on the nitrogen accumulation effect on soil microorganism and Fusarium wilt, found all kinds of microorganisms in soil have different responses to nitrogen form and content, and then found the nitrogen accumulation of excessive bacteria, actinomycetes decreased the proportion of serious fungi, Fusarium oxysporum, a rise in the proportion of the original soil microbial flora balance break, soil microbial diversity decreased, resulting in increased incidence of Fusarium Wilt of tomato, the soil nitrogen accumulation effect of Fusarium Wilt of tomato increased incidence of important reasons A study on the test. Thus the conclusion and production practice. The results of this study will provide reasonable fertilization in greenhouse soil to control soil borne diseases provide theoretical support. The main results are as follows: 1. in the soil culture experiment, experiment of different nitrogen accumulation (available nitrogen content for 95.96203.56257.62279.36319.59348.94 Mg - kg-1) conditions, with the culture the extension of time, soil bacteria, fungi, actinomycetes showed increased in early stage, the end of the incubation decreased slowly; while the number of Fusarium oxysporum in soil showed early increase, later increased slowly in culture. The content of available N was 279.36 Mg - kg-1 - kg-1 ~ 319.59mg soil the number of bacteria reached the maximum, while the soil culturable fungi, culturable actinomycetes and the number of soil bacteria wilt respectively in soil available nitrogen content is 279.36mg Kg-1257.62mg kg-1, 319.59mg kg-1 the most. Therefore, soil nitrogen accumulation can significantly affect soil microbial (three kinds of bacteria and the number of the number of Fusarium oxysporum).2. nitrogen accumulation can significantly affect the quantity of soil microorganism, also caused a change in the proportion of microbial accumulation under different conditions. N, the ratio of bacteria and fungi in the soil (B/F), the ratio of Actinomyces and fungi (A/F), with the increase of the accumulation of nitrogen in the soil were increased and then decreased; soil available nitrogen content was 203.56mg kg-1 B/F in soil, A/F reached the highest, indicating that soil microbial in bacteria, actinomycetes accounted for the highest proportion. With the increasing accumulation of nitrogen in soil, soil bacteria, actinomycetes not only reduce the number and the proportion decreased. By culture independent 16S rDNA (V4 Gao Tongliang) Sequencing analysis showed that the increase will reduce the abundance of bacteria and archaea and bacteria biodiversity.3. soil nitrogen accumulation can lead to significant reduction of soil pH nitrogen accumulation in soil, and soil caused by No3--N, NH4+-N, nitrogen, effective nitrogen content, soil pH and soil bacteria, negative correlation between number of fungi and Fusarium wilt, and culturable actinomycetes were positively correlated; soil NO3--N, NH4+-N, alkali hydrolyzable nitrogen, available nitrogen and soil bacteria, fungi and Fusarium oxysporum was positively correlated with the number of relationship, and culture is negatively related to changes in the number of actinomycetes. The accumulation of soil index caused by soil nitrogen thus can significantly affect soil microbial flora and Fusarium wilt pathogen quantity changes of.4. in the soil culture experiment, accumulation conditions to soil nitrogen bacteria inoculated Fusarium Later, with the prolongation of the culture time of soil bacteria, fungi, actinomycete quantity changes consistent with changes in the number of soil bacteria and bacteria wilt and missed trends. On the bacteria in the soil, the nitrogen content of 279.36mg kg-1 soil reached the maximum amount of bacteria, soil available nitrogen content is 319.59mg. Kg-1 soil culturable fungi reached maximum, soil available nitrogen content in 257.62mg kg-1 soil culturable actinomycetes quantity reached the maximum, the content of soil available nitrogen as the number of soil bacteria 319.59mg, kg-1 wilt is at a maximum. Human access to Fusarium oxysporum in soil, can significantly change the quantity of soil microbial population three the increased number of fungi. The inhibition increased the number of bacteria and actinomycetes, causing dramatic changes in the balance of microbial flora, causing.5. wilt of tomato breeding experiment in soil fertility In different nitrogen accumulation conditions to the inoculation of Fusarium oxysporum in soil, soil available nitrogen content in Tomato under varying degrees of incidence, when the soil available nitrogen content was 203.56mg kg-1 tomato disease incidence and disease index reached the lowest incidence and disease index were 41.82%, the bacterial 16.22%. in soil, put the highest proportion of actinomycetes, fungi. With the lowest proportion of bacteria in the soil, reduce the proportion of Actinomyces, increased the proportion of fungi, tomato wilt disease incidence and disease index were increased. Thus, the amount of nitrogen accumulation increased soil microbial balance is broken, bacteria, actinomycetes the proportion of fungi decreased, increased the proportion of.6. is the important factor of tomato increased incidence in pure culture experiments, the only nitrogen source was NO3--N, reproduction of Fusarium wilt pathogen, acid producing ability, secretion of toxin The ability of plant cell wall degrading enzyme activity were the best; NH4+-N as the sole nitrogen source, with Bacillus amyloliquefaciens, Bacillus subtilis, growth ability of tomato Fusarium wilt pathogen Penicillium oxalicum represented by antagonistic bacteria, acid producing ability, ability to secrete hydrolysis enzyme reached the highest. However, when growth and metabolism when the concentration of microbial nitrogen was inhibited. Fusarium wilt can produce acidic substances, and they are more easy to reproduce in the acidic environment. A large number of accumulation of nitrate in the soil will cause the soil pH decreased, which is more conducive to the breeding of Fusarium oxysporum.

【學(xué)位授予單位】：沈陽農(nóng)業(yè)大學(xué)
【學(xué)位級別】：博士
【學(xué)位授予年份】：2016
【分類號】：S436.412.1

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