【作者】 王媛媛；

【导师】 段玉玺；

【作者基本信息】 沈阳农业大学 ， 植物病理学， 2007， 博士

【摘要】 本文以重要的大豆胞囊线虫和大豆根腐病菌为靶标，从大豆根瘤中分离筛选具有对两种病原具有拮抗生物活性的根瘤菌和芽孢杆菌，通过对二者原生质体制备、再生和融合条件的研究，利用细胞融合技术将根瘤菌和芽孢杆菌进行融合，筛选出集固氮促生和拮抗性能为一体的根瘤菌工程菌株；对工程菌株的培养条件、抗菌杀虫机制和诱导抗病性进行了相关研究，为大豆根病的生物防治提供新的方法和思路。结果如下：1．具有拮抗活性的根瘤内生菌株的筛选：从我国20余个省份的138份土样种植大豆共获得786株细菌，其中芽孢杆菌351株，根瘤菌435株。以尖孢镰刀菌（Fusarium oxysporum）、茄腐镰刀菌（Fusarium solani）、立枯丝核菌（Rhizoctonia solani）和腐霉菌（Pythium sp.）4种大豆根腐病病原菌为靶标筛选出具有拮抗作用菌株79株，其中产芽孢杆菌64株，占分离产芽孢杆菌菌株的81.01％，根瘤菌15株，18.98％；在对大豆胞囊线虫有致死作用的菌株中，筛选出根瘤菌L396和产芽孢杆菌Snb2，菌悬液处理96h两株菌对二龄幼虫的致死率分别为21.6％和66.7％；摇瓶复筛结果为，菌株Snb2和L396发酵滤液处理线虫在72h致死率达到69.05％和39.13％；两株菌对供试的12种病原真菌均有拮抗作用；同时菌悬液浸种，两株菌对大豆幼苗也表现明显的促生作用。2．目标菌株的鉴定：采用传统分类学对两株细菌进行鉴定，包括形态观察和18项生理生化反应，将L396和Snb2分别定为中华根瘤菌属（Sinorhizobium sp.）和芽孢杆菌属（Bacillus sp.）；测定细菌16S rDNA序列并进行序列比对，有41个费氏中华根瘤菌（S.fredii）序列与L396的相似性达99％以上，将L396鉴定为费氏中华根瘤菌，有114个枯草芽孢杆菌（B.subtilis）与Snb2相匹配的核苷酸序列，因此将Snb2鉴定为枯草芽孢杆菌。3．目标菌株的原生质体融合：以青霉素（40μg／mE）和热灭活（50℃，120min）作为出发菌株Snb2和L396融合后筛选融合子的有效标记，明确了L396和Snb2原生质体制备、再生和融合的最佳条件。在最适条件下，L396和Snb2原生质体形成率分别为57.17％和43.53％；在最佳再生培养基HCNB上，L396的原生质体再生率达到92.5％，Snb2的再生率也达到66.0％；以pH7、40％的PEG作促融剂、以夹层培养法涂布在再生培养基上，在35℃条件下融合60min，融合率为9.39×10^-4。最终获得398个融合子，在含青霉素40μg／mL的培养基上转代培养6代后，仅有49个融合子能够稳定生长，占所获得融合子的12.31％。4．融合子的生物学特性研究：对获得的遗传稳定性较好的融合子进行了菌落形态、扩展方式、菌体大小、革兰氏染色反应和对病原微生物的活性测定，同时通过SDS-PAGE凝胶电泳分析检测了出发菌株与各个融合子间的可溶性蛋白差异，筛选出优良的融合子菌株RH5。融合子RH5是9株能够在大豆根部形成根瘤、且抗菌杀虫作用优于其他菌株的融合子之一。RH5的发酵液对大豆胞囊线虫二龄幼虫的致死率达到44％。5．融合子RH5的摇瓶发酵条件研究：以拮抗真菌效果、毒杀线虫能力和菌体生长量为指标，研究了融合子RH5的摇瓶发酵条件。通过单因子试验结果，乳糖和尿素为RH5的最佳碳氮源；通过碳氮正交和无机盐正交试验对最终确定RH5摇瓶发酵的最佳培养基配方为：乳糖1.0％，尿素0.35％，NH₄NO₃ 0.35％，MgCl₂ 0.35％，K₂SO₄ 0.8％，K₂HPO₄ 0.3％，FeSO₄ 0.002％；培养液的装液量为60mL、培养液起始pH为6—7和5％的接种量是RH5的最适合培养条件。6．融合子RH5的拮抗机制研究：测定了RH5发酵液对孢子萌发及菌丝形态建成的影响，发现发酵液可以抑制孢子的萌发，发酵原液处理的孢子萌发率仅达到对照的2.30％，也使孢子萌发形成的芽管等结构发生畸变；经发酵液处理的菌丝出现原生质体凝聚、菌丝膨大，并逐渐溶解的现象；RH5还可以产生具有杀线虫活性的有毒物质：HCN、H₂S，并具有淀粉酶氧化酚的能力，对线虫体内的总糖和蛋白含量有一定影响。7．RH5菌悬液接种大豆，对大豆幼苗的生长促进作用明显，还可以使大豆根部防御酶系活性的增加，接种浓度越大，酶活增加的幅度越大，最终研究结果表明，以10¹⁰cfu／mL为最佳接种浓度。更多还原

【Abstract】 Rhizobium engineering strain were obtained deriving from rhizobium and bacillus withantagonist to pathogens of soybean root rot and soybean cyst nematode were screened withinsoybean nodule, after study the system of both strains protoplast preparation, regeneration andamalgamation. The characters of this engineering strain were fixing nitrogen, promotinggrowth and antagonist to pathogens. Its fermentation conditions, action mechanism on fungiand plant parasite nematode, resistance induced were studied systematically. Results showedas follows:1. 786 strains were obtained from 138 soil samples collected from more than 20provinces of China. Among them, 351 strain were bacillus and 435 strain were rhizobium. Theresults showed that there were 79 bacteria strains with antagonist to fungi of soybean root rot,in which, the effective bacillus strains were occupied 81.01%, and rhizobium only 18.99%;rhizobium L396 and bacillus Snb2 were screened out from strains with effectively neamticidetoxin to Heterodera glycines. The mortality of H. glycine treated with suspensions of L396and Snb2 were 21.6% and 66.7% respectively. Through re-screening in shaking flasks, affectof L396 and Snb2 bacterial culture filtrates on J2 of H. glycines were 69.05% and 39.13%;meanwhile, strain L396 and Snb2 had high effect against 12 pathogens; After seeking seedwith bacteria suspension, both strains promoted growth the seeding.2. According to traditional taxonomy including morphology and Physio-biochemical,the bio-control bacteria strains L396 and Snb2 were classified as Sinorhizobium sp. andBacillus sp.; The sequences of 16S rDNA gene of L396 and Snb2 were sequenced andanalyzed, the results showed that strain L396 and Snb2 belong to Sinorhizobium fredii,Bacillus subtilis, respectively.3. Take 40μg/mL Penicilium and 50℃, 120min heat treated as fusion marker of parentsstrain Snb2 and L396. The best optimal protoplast preparation, regeneration andamalgamation conditions were researched. Under optimal condition, the ratio of L396 andSnb2 protoplast forming was57.17% and 43.53%, respectively, and protoplast fusion rate was9.39×10^-4. 398 fusants were obtained after amalgamation and only 49 fusants survived transferred six times on medium with 40μg/mL Penicilium.4. RH5 was picked out based on colony morphology, size ofthalli, effect againstpathogens and difference of soluble protein among all fusants. RH5 had distinctive activeagainst pathogens and parasitic nematode among which could nodule on root of soybean. Themortality of J2 treated with Bacterial culture filtrates of RH5 was 44%.5. The fermentation conditions of RH5 were investigated by single factor and designsof orthogonal experiments, results were as follow: the optimum carbon and nitrogen resourcewas lactose and urea, respectively; the liquid medium of fermentation of RH5 constitutes bylactose 1.0%, urea 0.35%, ammonium nitrate0.35%, magnesium chloride 0.35%, PotassiumSulfate0.8%, dipotassium hydrophosphate 0.3%, ferrous sulfate 0.002%; the optimumfermentation condition included the volume of medium of shaking flasks 60mL/250 mL, theinitial pH 6-7, inoculation volume in shaking flasks 3mL.6. The effect of Bacterial culture filtrates of RH5 on spore germination and mycelialconfiguration were tested. The germination of spore were inhibited, the ratio of germation was2.30% treated with culture filtrates without dilution and sprout of germinated spore wasmalformated. Protoplast agglomerate appeared in hypha treated culture filtrates, hypha wereswelled and dissolved gradually. RH5 produced some nematicide: HCN、H2S, had ability ofamylase and oxygenation hydroxybenzene, It had some effect on total sugar content andprotein content of H. glycines.7. The seedling was promoted inoculated with RH5 suspension. The activity defenceenzymes were promoted, the increase range followed the bacteria concentration, the resultindicated the optimum inoculation was 10¹⁰cfu/mL.更多还原