【作者】 唐勇军；

【导师】 卢向阳；

【作者基本信息】 湖南农业大学 ， 生物化学与分子生物学， 2003， 硕士

【摘要】 【研究背景和意义】水稻是世界上最重要的农作物之一，养育着世界近50％的人口。中国是世界最大的水稻生产和消费国，全国60％以上的人口以大米为主食，每年的总产量达2000亿公斤左右。虽然我国水稻生产已取得举世瞩目的成就，但也面临着新的挑战，粮食生产成本高、效益低、国际竞争力弱的情况依然存在。在生态环境中，影响水稻生长的因子多种多样，并且处于动态的变化中，这些环境因子一旦超出水稻忍耐的范围就成为了逆境胁迫因子，对水稻产生危害，导致了巨大的产量和经济损失。水稻病害是其主要的逆境胁迫因子之一，也是影响水稻产量和质量的重要因素之一。特别是真菌病害，具有传染快、危害大等特点。对水稻危害最大的三种病害为稻瘟病、水稻纹枯病、水稻白叶枯病，其中有两种是真菌病害，稻瘟病在流行年份可减产40％～50％，有的田块甚至颗粒无收。近几十年来，防治水稻病害的措施主要是化学防治，但是化学防治对土壤、空气及水体污染严重，破坏了生态环境的平衡。同时导致农药残留在农副产品中，严重影响了人类健康。为了达到既能有效的防治农作物病虫害，又能减少化学农药用量的目的，生物农药以及一些无害防治途径的研究与开发越来越引起广泛重视。 几丁质酶产生菌是一类能够利用几丁质作为碳源而生存、繁殖的特殊微生物种群，其生理生化方面的共同特征就是能够分泌几丁质酶，并通过这些酶的作用将几丁质降解为几丁质寡糖、几丁质二糖直至几丁质单糖。而几丁质酶和几丁寡糖在植物抗病上的应用已经被广泛的报道，而且有研究证实几丁寡糖还能促进植物的生长发育。几丁质酶产生菌是一类很有前途的抗真菌的微生物，因此，在这方面作更广泛更深入的研究是很有意义的工作。 【目的】获得对病原真菌有较好抑制效果的几丁质酶产生菌及其应用技术，初步探索拮抗菌的作用机理。 【内容】分离纯化对病原真菌有较好抑制作用的几丁质酶产生菌，优化产酶条件，研究拮抗菌株对一些病原真菌的抑菌效果，探讨拮抗菌发酵液对水稻幼苗保护酶活性的影响。 【方法】菌种筛选方法：初步分离：采用平板透明圈方法筛选几丁质酶产生菌菌株， 中文摘要产生透明圈的培养基选用分离培养基。将土样（棉用土．稻田土、桔园土、池塘底泥、水产养殖场的泥土等）或植物器官（水稻、棉花、玉米、烟叶及一些蔬菜的根、茎、叶）用无菌水稀释（梯度稀释法人在稀释倍数为 10叶、10刁、10、时分别取 0．sml涂于分离培养基中，30aC培养3～5天，观察透明圈是否产生。二次分离：继续培养得到的菌株，同样用梯度稀释法在分离培养基中培养菌株单个菌落，观察其在分离培养基中生长情况。根据生长情况和产生透明圈的大小选出较好的菌株。三次分离：在含稻瘟病病原菌细胞壁成分的培养基的平板上培养，在二次分离的基础上，观察筛选菌株的生长情况，并检测菌株的几丁质酶话性。微生物几丁质酶活性测定方法：参照 Ac e介绍的方法。抑菌实验的方法：参照擅建新和李力介绍的方法。过氧化氢酶活性测定：按照《植物生理生化实验》中介绍的方法进行。过氧化物酶活性测定方法：按照《生化实验技术方法和技术》中介绍的方法进行。苯丙氨酸解氨酶活性和植物几丁质酶活性测定方法：按照《现代植物生理学实验指南》中介绍的方法进行。 【结果】1．从自然界中筛选到两株产酶能力较强的几丁质酶产生菌，分别编号为H3和 Hg。初步鉴定结果，H3菌株为苏云金芽抱杆菌，Hg可能为盲霉菌。2．产酶条件：H3菌株以细粉几丁质和酵母粉为 C源和 N源，在 30aC、pH72下培养72小时为最佳产酶条件＊ 菌株以细粉几丁质和酵母粉为 C源和 N源，在 35℃，pH6．9下，培养84小时为最佳产酶条件。3H3和 Hg菌株对几种病原真菌有明显抑制作用，H3对稻瘟病、水稻效枯病、棉花枯萎病、小麦赤霉病、黄瓜枯萎病的抑菌率分别为 73．l％、44．3 ％、62．5 ％、50上％、56．3％：＊9对它们的抑菌率分别为58．2％、忙＊％、56．3％、27．2％、36石％。4．H3菌株发酵液处理水稻种子后，接种稻瘟病，培两优288出苗率、苗高、百苗干物重分别为 31．5％，11．2上1．6cm、1．95土0 089；旱干 127出苗率、苗高、百苗干物重分别为65 ％、12．5土2．Icm、1．67土0．119；新香优80出苗率、苗高、百苗干物重分别为42％、13．2士2，ZCm、2．06土0．149。Hg菌株发酵液处理后，培两优288出苗率、苗高、百苗干物重分别为 33％、8＊h石cm、1．87上0 069。旱干 127出苗率、苗高、百苗干物重分别为 57．5％、11．4士2．Zcm、1．59上0 079；新香优 80出苗率、苗高、百苗干物重分别为35％、11．5土3．ocm、2．10土0．15g。与对照相比均有明显提高。5两株几丁质酶产生菌的产酶发酵液（含几丁质底物）能诱导水稻幼苗保护酶系活性的 改变，使其朝着有利于水稻幼苗生长和抵抗不利因素的方向发展，产酶发酵液作用比更多还原

【Abstract】 [ Background ]Rice is one of the most important crops in the world.With nearly 50 % of the world’s population depends on it.China is the biggest rice-producing and rice-cons uming country.More than 60% of the population in our country regard it as the staple f ood,The total output of every year reaches about 200 billion kilograms.Athough our cou ntry have made great achievement of worldwide interests in planting rice,still faces new challenge.The relative high costs,the low efficiency and the weak international competitiv eness still exists.In ecological environment,the factors influencing the growth of rice chan ges.Every time going beyond the range of rice-restrained, these environmental factors bee ome stress factors of adverse circumstances and do harm to rice,they will bring obvious negative effect on rice growth and cause enormous output and economical losses.The di sease of rice is one of the main adverse circumstance and important factors influencing on the output and quality of rice.Especially for fungi disease, it has the characteristics o f fast infection,great harm and so on.As far as the rice is concerned,three kinds of most harmful diseases are rice blast, rice sheath blight, rice bacterial leaf blight, two of whi ch are fungi disease.Among them, rice blast cause 40%-50% decreasing of production in prevailing year,even no harvest at all.In recent decades,the main measures of preventing and controlling the rice diseases is chemical control.However,chemical control gives rise to serious pollution on soil, atmosphere and water, and destroys the balance of the ecol ogical environment.At the same time it leads to the held-up pesticide in the agricultural byproducts and influences on the human’s health seriously. In order to control the grain crop diseases and reduce the quantities of chemical pesticides effectively,the research on biological pesticides and biological controls were paid great attention in the world.Chitinase forming strain is a kind of special microorganisms .This strain can utilize chitin as carbon source to survive and repoduce.And it has the common biochemical ch aracteristics of secreting chitinase.Chitinase can degrade chitin into chitin oligosaccharide, chitin disaccharide,and chitin monosaccharide.The application of chitinase and chitin oligo saccharide on plant resistance are extensively reported.Moreover researches verified that c hitin oligosaccharide can promot the growth of plant.So chitinase froming strain is a kin d of promising fungi-resistant microorgnanism.Therefore,it’s a very meaningful work to d o more extensive and deeper researches in this respect.[Objective] Obstain the chitinase producing strains inhibiting pathogen fungi and the a plicaion technology and explore the mechanism of antifungi strain[Content] Obtained one or several chitinase secreting strains capable with inhibiting pa thogen fungi found out the premium conditions and measuring the effect of inhibiting topathogen fungi.This experiment explore the mechanism of strain’s fermented liquids by examinng the effect of rice defensive enzymes activity.[Method] The screening method of strains: preliminarily isolation: Chitinase producing strains are screened by the method of transparent zones and use selective medium.The s oil samples(cotton field soil,rice field soil,tangerine garden soil,pond bottom mud,aquacult ure mud ,etc.) and plant organs (roots,stems and leaves from rice,cotton,maize,tobacco le aves and some vegetables) are diluted with sterile water(serial dilution method).when thesamples are diluted to 10-4,10-5,10-6 times,o.5ml dilute solution are put into selective medium and are cultived for 3~5 days and observed wheather the transparent zones are produced. The second separation :Strain were obstained by continuing cultivation.single colony wascultivated in separate medium by serial dilution method.,better strain with bisser of tran sparent zones will be choosed.The third separation:Based on the second separation,the colony was cultivated in the pi ate containing blast p更多还原