设施土壤氮素积累条件下番茄枯萎病发生的微生态机制研究

发布时间：2018-01-18 03:29

本文关键词：设施土壤氮素积累条件下番茄枯萎病发生的微生态机制研究　出处：《沈阳农业大学》2016年博士论文　论文类型：学位论文

【摘要】：设施栽培目前已成为人们获得高产、优质果蔬的重要途径,但是设施栽培土壤中普遍存在过量施肥问题,其中氮肥的过量施用现象尤为严重。随着种植年限长、土壤氮磷钾速效养分的不断积累,引起设施土壤养分严重不平衡、氮素等积累现象非常明显,这导致了设施土壤酸化、盐渍化现象越来越严重,番茄枯萎病等蔬菜的土传病害发生率一直居高不下。现有的研究主要集中于从植物保护的角度对土传病害的防治,而从土壤养分角度出发研究关于设施土壤养分积累条件下番茄枯萎病等土传病害的发生机制方面的研究则相对较少。本研究以番茄枯萎病菌为研究对象,设置土壤不同速效氮含量,分别从宏观(土壤微生物生态)角度和微观(微生物生理生化)角度两方面来探索设施土壤氮素积累条件下番茄枯萎病发生的机制。通过土壤培养试验和液体发酵纯培养试验,综合分析了氮素含量和氮素形态对微生物数量、微生物群落动态及微生物区系变化的影响,同时探究了不同速效氮含量土壤接种枯萎菌条件下番茄的发病率及病情指数的变化情况。本研究从微生物平衡角度对设施土壤长期氮积累条件下土传病害发生及发病率升高的机制提出合理假设：随着土壤微生物区系平衡(主要是土壤微生物比例变化)被打破,当超过“阈值”时将导致土传病害的发生,微生物比例越偏离“阈值”,发病率则相应越高。同时调查发现在实际生产中长期种植作物的土壤和长期大量施氮肥的土壤更容易感染土传病害,而土壤中硝态氮的大量累积有利于病原菌生长,不利于病原菌的拮抗微生物生长。定量化研究了氮素积累对土壤微生物及番茄枯萎菌数量的影响,发现土壤中各种微生物对氮素形态及含量有不同的响应,进而发现氮素积累量过高时细菌、放线菌的比例严重下降,真菌、枯萎菌的比例有所上升,土壤微生物原有的区系平衡被打破,土壤微生物多样性减少,最终导致番茄枯萎病发病率升高,得出了土壤氮素大量积累是影响番茄枯萎病发病率升高的重要原因之一。由此可见本试验的研究结论与生产实践相符。本研究成果将为设施栽培土壤中合理施肥以控制土传病害提供理论支持。主要研究结果如下：1.在土壤培养试验中,不同氮素积累试验(速效氮含量为95.96、203.56、257.62、279.36、319.59、348.94 mg·kg-1)条件下,随着培养时间的延长,土壤中可培养细菌、真菌、放线菌数量均表现出前期增加,培养末期缓慢降低的趋势；而土壤中枯萎菌数量则表现为前期增加,培养后期缓慢增加的趋势。速效氮含量为279.36 mg·kg-1～319.59mg·kg-1的土壤可培养细菌数量达到最多,而土壤可培养真菌数量、可培养放线菌数量、土壤枯萎菌数量分别是在土壤速效氮含量为279.36mg·kg-1、257.62mg·kg-1、 319.59mg·kg-1的条件下达到最多。因此,土壤中氮素积累能显著影响土壤可培养微生物(三大类菌群数量和枯萎菌数量)的数量。2.氮素积累不仅能显著影响土壤微生物的数量,同时引起了微生物比例的改变。不同氮素积累条件下,土壤中细菌与真菌的比值(B/F)、放线菌与真菌的比值(A/F),随着土壤中氮素积累量的增加均呈先升高后降低的趋势；土壤速效氮含量为203.56mg·kg-1时土壤中B/F、A/F均达到最高,说明此时土壤微生物中细菌、放线菌所占比例最高。随着土壤中氮素积累量的增多,土壤中细菌、放线菌不仅数量减少并且所占比例降低。通过不依赖培养的16S rDNA(V4区)高通量测序分析发现,土壤中氮素积累的增加会减少细菌和古菌的生物多样性及细菌的物种丰度。3.土壤氮素积累能显著引起土壤pH降低,同时引起土壤No3--N、NH4+-N、碱解氮、有效氮含量的增加,土壤pH与土壤中可培养细菌、真菌数量及枯萎菌数量呈负相关关系,与可培养放线菌数量呈正相关关系；土壤NO3--N、NH4+-N、碱解氮、有效氮含量与土壤中可培养细菌、真菌数量及枯萎菌数量呈正相关关系,而与可培养放线菌数量呈负相关关系。由此可见土壤氮素积累引起的土壤指标的变化能显著影响土壤微生物区系变化和枯萎菌的数量变化。4.在土壤培养试验中,不同氮素积累条件下向土壤中接种枯萎菌后,随着培养时间的延长土壤中可培养细菌、真菌、放线菌数量变化及枯萎菌数量变化与未接菌土壤变化的趋势相一致。在接菌土壤中,氮素含量为279.36mg·kg-1的土壤可培养细菌数量达到最多,土壤速效氮含量为319.59mg·kg-1的土壤可培养真菌数量达到最多,土壤速效氮含量为257.62mg·kg-1的土壤可培养放线菌数量达到最多,土壤速效氮含量为319.59mg·kg-1的土壤枯萎菌数量达到最多。人为向土壤中接入枯萎菌后,能显著改变土壤三大微生物种群的数量,显著提高了真菌数量,抑制细菌和放线菌数量的增加,从而引起微生物区系平衡的剧烈变化,进而引起番茄枯萎病的发生。5.在土壤培养试验中,不同氮素积累条件下向土壤中接种枯萎菌后,土壤不同速效氮含量下番茄均不同程度发病,当土壤中速效氮含量为203.56mg·kg-1时番茄发病率及病情指数均达到最低,发病率和病情指数分别为41.82%、16.22%。此时土壤中细菌、放线菌所占比例最高,真菌所占比例最低。随着土壤中细菌、放线菌所占比例的降低,真菌所占比例的升高,番茄枯萎病发病率和病情指数均出现升高趋势。由此可见,氮素积累量加重土壤微生物平衡被打破,细菌、放线菌所占比例降低,真菌所占比例升高是致使番茄发病率升高的重要因素。6.在纯培养试验中,NO3--N最为唯一氮源时,枯萎菌的繁殖能力、产酸能力、分泌毒素及植物细胞壁降解酶活的能力均达到最好；NH4+-N作为唯一氮源时,以解淀粉芽孢杆菌、枯草芽孢杆菌、草酸青霉菌为代表的番茄枯萎菌的拮抗菌的生长能力、产酸能力、分泌水解性酶的能力达到最高。然而当氮素浓度过高时微生物的生长及代谢均会受到抑制。枯萎菌自身可以产生酸性物质,并且它们更易于在酸性环境中生长繁殖。土壤中硝态氮的大量累积会引起土壤pH值降低,这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.

【学位授予单位】：沈阳农业大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：S436.412.1

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