【作者】 乔康；

【导师】 王开运；

【作者基本信息】 山东农业大学 ， 农药学， 2013， 博士

【摘要】 我国是重要的设施蔬菜生产国，因长期连作种植导致以根结线虫为代表的土传病虫害发生严重，制约了设施蔬菜的发展。实践证明，使用甲基溴熏蒸处理土壤是防治土传病虫害最有效的方法。但因其破坏臭氧层，亟须寻找新的替代药剂。近年来，国内外都在大力研究甲基溴的替代品，并发现1,3-D（1,3-二氯丙烯）不仅具有良好的杀线虫活力，而且对某些土传病原菌和杂草也具有一定的防效，同时对蔬菜还有一定的增产作用，是一种很有开发潜力的甲基溴替代品。目前1,3-D已在国外推广应用，但该化合物作为农药在我国尚未获得登记。针对我国农业的特点、经济水平、农药的使用和管理等要求，1,3-D可否作为甲基溴替代品在国内获得登记并应用，仍需进行系统的研究。本研究拟通过室内毒力测定、温室盆栽试验和大田验证试验，评价1,3-D对南方根结线虫、土传病原菌和杂草的生物活性，明确其在我国保护地蔬菜上实际应用的效果和价值。同时，通过传统分析方法研究1,3-D对可培养微生物数量及土壤酶活的影响，结合分子生物学技术（T-RFLP）和最新的高通量测序技术（Roche454GS FLX+）探讨1,3-D对细菌微生物多样性的影响，为支撑其在我国作为甲基溴替代药剂获得农药登记，并为其在设施蔬菜上的安全合理应用提供科学依据，也为克服土壤连作障碍和保障设施蔬菜可持续生产奠定理论基础。主要研究结果如下：1.采用触杀法测定了1,3-D对南方根结线虫的毒力，表明1,3-D对南方根结线虫的LC50和LC90分别为1.20和3.74mg/L。采用美国农业部杂草种子处理方法研究了1,3-D对多种杂草种子的剂量-响应关系，发现杂草种子对1,3-D敏感性由大到小顺序为：马唐＞牛筋＞稗草＞反枝苋，其LC90在14.23-73.59mg/kg之间。采用十字交叉法测定了1,3-D对辣椒疫霉病菌、草莓枯萎病菌、棉花立枯病菌、烟草黑胫病菌和番茄灰霉病菌的毒力，明确1,3-D对辣椒疫霉病菌和草莓枯萎病菌的LC50分别为0.24和1.55g/m2，辣椒疫霉病菌对1,3-D最为敏感，草莓枯萎病菌对1,3-D最不敏感，其他种类病原菌对1,3-D则表现出中等程度的敏感性。2.通过分别在温室大棚番茄和黄瓜上进行田间药效试验，证明1,3-D（90、120和180L/ha）对南方根结线虫、杂草和土传病害病原菌均有较高的防治效果。1,3-D施用后能够有效抑制根结线虫侵染和种群数量，降低根结指数，减少土传病害发生率，还有显著地促进作物生长、增强植株活力和增加作物产量的作用，并且1,3-D中、高剂量熏蒸处理小区在除杂草防治以外的其他各种防治指标上均达到甚至超过甲基溴处理小区，尤其在作物产量上1,3-D处理与甲基溴处理之间无显著性差异。3.采用传统方法研究1,3-D对土壤可培养微生物数量和土壤酶的影响。结果表明，1,3-D熏蒸土壤后，各处理浓度对土壤可培养真菌、细菌和放线菌的影响表现为抑制-激活再逐渐恢复作用，最后各处理与对照无显著性差异。1,3-D熏蒸土壤后，各浓度处理对土壤脲酶、蔗糖酶、过氧化氢酶、脱氢酶、酸性磷酸酶与FDA水解酶等6种主要土壤酶的影响，在处理开始时均表现为激活或抑制作用，然后逐渐恢复，最后各处理与对照无显著性差异。4. T-RFLP分析结果表明，1,3-D熏蒸土壤后，在不同采样时期存在23个主要的长度片段，1,3-D施用之后不同时间对不同细菌种群影响存在显著差异。从3个不同取样时期可以看出，一开始5WAT，1,3-D对各种细菌种群主要呈现抑制作用；随着时间推移，在10WAT各种细菌种群开始慢慢恢复，并且对有些种群刺激作用比较明显；而在15WAT，处理组菌群与对照组菌群之间并无显著性差异。T-RFLP结果证明了1,3-D与其他甲基溴替代药剂相比，其对细菌微生物群落的影响相对比较短暂。5.本研究首次将第二代高通量测序技术应用于熏蒸土壤的研究，明确了1,3-D熏蒸处理设施蔬菜连作土壤后对土壤细菌微生物多样性的影响，全面揭示了熏蒸土样中的微生物组成及丰度。高通量测序结果发现变形菌门、拟杆菌门、放线菌门、酸杆菌门和厚壁菌门是样品中主要门类。细菌种群多样性在施药前期受到一定抑制，然而在熏蒸中后期各处理逐渐恢复至对照水平，甚至还表现出刺激生长的趋势，熏蒸土样的微生物多样性及丰度均高于对照土样，这表明1,3-D熏蒸土壤对细菌微生物多样性只有短暂影响，并且1,3-D熏蒸处理能够刺激某些细菌种群的增殖。更多还原

【Abstract】 China is one of the most important producting country of protected vegetablein the world.Meantime, protected vegetable is the most dynamic industry in agriculture of China, playing akey role in the development of modern agriculture. However, many greenhouse crops sufferfrom attack by nematodes, soil-borne pathogens as well as to competition from weeds,especially in monocropping systems. The risk of nematodes and soil-borne epidemic whencrops are grown under such intensive regimes is high, and often results inreduction ofquantity and quality of vegetables. At present, the standard treatment for the management ofnematodes, soil-borne pathogens and weeds in many high-value crop production systems ispreplant soil fumigation with methyl bromide (MB). However, developing countries arecommitted to totally phasing out MB production and use by2015due to its detrimental effectson stratospheric ozone. The absence of MB from market will impact agricultural, silvicultural,and horticultural production unless safe and efficacious alternatives are found. Based on ourprevious study,1,3-D (1,3-dichloropropene), is one of the potential candidates of MB as a soildisinfectant. However, due to the limitation by the current level of economic development,many advanced fumigant application methods and much of the equipment cannot be used inChina. As one of the most promising short-term alternatives to MB,1,3-D is going to beregistered as preplant fumigant in China. Therefore, in this study,a series of laboratory test,greenhouse pot experiment and field trials were conducted to determine thebioactivities of1,3-D for its potential to control nematode, soil-borne pathogens andweeds.Meanwhile,traditional physiological and biochemical methods, associate with T-RFLPandhigh-throughput DNA sequencing (Roche454GS FLX+) were used to determine theeffects of1,3-D on soil culturable microorganismspopulation, enzyme activities and microbialdiversity to obtain microorganism flora and species richness at different levels. The results ofthis studywill evaluate the bioactivities and ecological safetyof1,3-D,offering a theoreticalfoundation to the use of1,3-D on greenhouse vegetable industry. The results were as follows:1. Laboratory toxicity results showed that the LC50and LC90of1,3-D to second-stagejuveniles (J2)of Meloidogyne incognitawere1.20and3.74mg/L, respectively. Dose-responsestudies found that the seeds of Digitaria chinensis were the most sensitive to soil fumigationwith1,3-D, followed by Eleusina indica, Echinochloa crusgalli and Amaranthus retroflexuswith the LC90values between14.23and73.59mg/kg soil. Among the pathogens,Phytophthora capsici was the most sensitive and Fusarium oxysporum was the least sensitiveto1,3-D fumigation with the LC50values were0.24and1.55g/m2. Rhizoctonia solani, Phytophthora nicotianae and Botrytis cinerea exhibited intermediate susceptibility.2. Field trials in tomato and cucumber were conducteddetermine the efficacy of1,3-D(90,120and180L/ha) for its potential to control nematode, soil-borne pathogens and weeds.The results revealed that1,3-D exhibited excellent control effects to M. incognita while withcertain yield increase. In both trials,1,3-D at the doses of120and180L/ha were as effectiveas MB in increasing plant height, vigor, tomato yield and in reducing the incidence ofsoil-borne disease, especially in maintaining excellent nematode control efficiency, but itproviding relatively poor control over weeds.3.Traditional physiological and biochemical methods were used to determine the effectsof1,3-D on population of culturable microorganisms and soil enzyme activities. The resultsshowed that all doses of1,3-D exhibited a inhibition initially and then recovery. There wereno signifant effects between fumigation treatments and the control. The activities of theurease,invertase,hydrogen peroxidase,dehydrogenase, acid phosphataseand FDA hydrolyticactivity were also investigated. The results showed that all doses of1,3-D exhibited ainhibition initially and then recovery. There were no signifant effects between fumigationtreatments and the control in the end.4. T-RFLPresults showed that there were23main TRFs in different sampling time. At5WAT,1,3-D exhibited a inhibition effects to soil bacteria. While, at10WAT, bacteriarecovered and stimulation effects developed. At15WAT,there were no signifant effectsbetween fumigation treatments and the control.5. To the best of our knowledge, this study is the first application of PCR-based454pyrosequencing to characterize fumigating soil samples.454sequencing results revealed thatProteobacteria, Bacteroidetes, Actinobacteria, Acidobacteria and Firmicutes werepredominant phylum in soils. Bacterial diversity was affected initially, while recovered in thelater treatments and soils from1,3-D treatment plots had a more bacterial diversity.1,3-D hadonly a short-term and transitory impact on the indigenous soil microbial community, andapplication of1,3-D fumigation would stimulate the proliferationof some microbialpopulation.更多还原