【作者】 李剑峰；

【导师】 师尚礼；

【作者基本信息】 甘肃农业大学 ， 草业科学， 2011， 博士

【摘要】 磷是植物生长必需的主要营养元素,由于72%到90%的土壤磷会被土壤中的钙、铝、铁离子和有机化合物所固定,使土壤磷缺乏成为限制农产品产量和质量的重要因素。苜蓿和红豆草作为最重要的牧草,栽培面积超过两百万公顷,占全国近四分之三的人工草地。产量集中于西北高原干旱半干旱地区的谷物作物轮作区。土壤全磷含量随磷肥的不断施用而逐渐增加,但作物及牧草依旧由于有效磷过低而产生明显的缺磷症状。解磷微生物能够分解土壤中的难溶性无机磷供植物吸收利用,其中的解磷根瘤菌作为一种同时具备解磷和结瘤固氮作用的植物促生菌,对豆科作物的生长和产量有很好的促进作用,能够减少磷化肥的施用,并降低农业生产的成本。但目前解磷根瘤菌剂的应用受到下列因素的限制:1.菌种的选育效率较低,筛选出的菌种综合促生能力不高。2.菌种的应用范围仅限于其寄主植物。3.国内外根瘤菌剂普遍存在污染率高、功能单一、保质期短和占瘤率低的问题。由于解磷根瘤菌本身会分泌有机酸,因此更容易造成菌剂产品的酸化,引起菌体的死亡和杂菌的侵入;4.目前采用的通用泥炭载体基质为不可再生资源,其采集过程会对产地的生态环境造成破坏,而泥炭菌剂也存在一些质量上的缺陷。针对上述问题,本研究从以下方面进行了探讨:1.优化解磷根瘤菌的筛选方法和菌种的诱变增效;2.将能够解磷并有分泌生长素能力的根瘤菌作为根际解磷固氮促生菌在非寄主植物上应用,拓宽其应用范围;3.利用抑菌剂降低根瘤菌剂的污染率,提高根瘤菌活菌数和目标根瘤菌的占瘤率;4.以青秸秆粉浸提液作为解磷根瘤发酵培养基,降低菌剂生产成本。并以黄绵土和秸秆粉代替泥炭载体作为固体菌剂载体基质,降低根瘤菌菌剂制备的经济成本和环境成本。根据这样的指导思想和目标,本研究在逐级分离、定向选育的方法筛选出有利用潜力的解磷分泌生长素根瘤菌Rhizobium. Meliloti L-5和Rhizobium sp.RS-1菌株的基础上,通过微波诱变和验证选育得到两株原始菌株的增效耐药突变株Rhizobium. meliloti LW107和Rhizobium sp.RSW96,使菌株的解磷和分泌生长素能力得到增强,并获得对两种抗生素的抗药标记特性。经过遗传稳定性验证后,在以难溶性无机磷为唯一磷素来源的栽培条件下考察诱变选育得到的解磷根瘤菌突变株LW107和RSW96对寄主及非寄主植物的促生性能。在抗污染菌剂的试制过程中,利用突变株的耐药特性,以其耐受的氨苄青霉素作为抑菌剂,进行不同浓度氨苄青霉素对菌株和寄主植物幼苗的毒性测验,优化出液体菌剂的最适抑菌剂浓度和含药菌剂施用时的最佳稀释度。为了最大程度的降低菌剂成本并提高其质量,本研究优化出最适于解磷根瘤菌发酵培养的碳源种类,讨论了调整成份后的青秸秆粉浸提液作为解磷根瘤菌液体发酵培养基和固体培养基的可行性。随后以灭菌的玉米青秸秆粉、黄绵土以及常用的泥炭和蛭石进行载体基质的筛选,试制出含抑菌剂的解磷根瘤菌液体菌剂和固体菌剂,经不同贮存时期进行菌剂性质的测定和验证,得到性质稳定、造价低廉、污染率低、有效期长的解磷根瘤菌剂及其制备工艺的关键技术。研究的主要结果如下:1.以固氮菌-解磷固氮菌-解磷根瘤菌的逐级筛选模式可以从豆科植物根瘤内分离得到具有良好促生性能的解磷根瘤菌。以该方法筛选出的苜蓿根瘤菌Rhizobium meliloti L-5和红豆草根瘤菌Rhizobium sp. RS-1均为快生型根瘤菌,具有结瘤固氮能力较强的特点,兼备解磷和产生长素的特性,耐盐和耐酸碱能力强,可以作为高效解磷根瘤菌进一步选育的基础材料。2.根瘤中可溶解无机磷的解磷固氮菌株以土壤杆菌、放线菌、芽孢杆菌、氮单胞杆菌和其他固氮菌属中的菌株为主,经回接鉴定的根瘤菌仅有5株,仅占总固氮菌株数的1.78%。表明根瘤内有大量无结瘤能力的解磷固氮菌存在。3.采用菌株诱变的方法,可利用已有菌种资源,提高菌种选育效率,弥补野生菌种解磷能力弱的缺陷。作为一种简便、安全、清洁、廉价的辐射源,微波辐照诱变的条件更易于掌控和变更。本研究中随着诱变目的和根瘤菌株的不同,具体的最佳微波辐照参数有较大差异,但最高正突变率均出现于致死率为87%-90%之间的辐照处理。经诱变得到的部分高效突变菌株存在遗传基因不稳定、出现回复突变或产量下降的现象,需验证至少6代菌体的遗传稳定性。4.在仅提供难溶性无机磷的栽培条件下,所有植物都表现出明显的生长受抑,叶片生长迟缓、颜色变深,根系变细。具备产生长素能力的解磷根瘤菌对寄主和非寄主植物有良好促生效果,能有效解除寄主和非寄主植物的缺磷胁迫,促进植株生长量和生物量的增长,提高氮素和磷素的吸收。解磷根瘤菌通过溶解难溶性无机磷解除寄主或非寄主植物遭受的缺磷胁迫,使之进入相对均衡的营养环境,同时侵染寄主植株产生根瘤固氮。对非寄主植物,解磷根瘤菌具有根际解磷菌相似的特性,但接种于寄主植物时,植株的叶面积、地上及地下生物量干重、根瘤个数、根瘤直径等均优于接种的非寄主植物。5.在低磷条件下,普通根瘤菌依然具备侵染寄主植株并结瘤固氮的能力,但根瘤直径、鲜重和固氮酶活性明显下降。缺磷胁迫下接种普通根瘤菌12531的植株生长状况优于未接种根瘤菌也未施用可溶性磷素的处理CK1,但单叶面积仅分别为接种LW107、RSW96菌株和施用1/4量可溶性磷素处理(CK2)的65.9%、79%和67.4%。接种普通根瘤菌12531的植株生物量干重较CK1处理增加35.93%,但仅为接种解磷根瘤菌LW107和RSW96处理的73.1%和85.29%,也低于施用化学磷肥的处理17.92%。6.氨苄青霉素作为具有低毒性的抑菌剂,低剂量时刺激植物生长而高剂量时产生毒害效应。200mg/L以上剂量氨苄青霉素能够抑制根瘤菌诱导植株结瘤使根瘤数量减少,根瘤剖面颜色变浅,个体变小,固氮酶活性降低至其正常根瘤的1/5以下。低浓度的氨苄青霉素对结瘤和固氮酶活性无抑制作用,还能提高植株结瘤的数目。由于物种间存在差异,苜蓿和红豆草对于同一氨苄青霉素浓度的反应并不相同。7.保存120d、以泥炭、蛭石、秸秆粉和黄绵土为载体基质的固体菌剂中,活菌数随菌剂载体吸附菌液量的增多而升高。由于青秸杆粉疏松多孔,可吸附达自重2.7倍的菌液,并含有大量适于微生物生长的氮源、核酸、可溶性糖和几乎菌种所需的全部矿质元素。菌种进入载体后继续大量繁殖,保存120d时的最大有效菌含量达到泥炭菌剂的155.48%和138.44%,用于菌剂载体有很大潜力。但贮存1a后,由于解磷根瘤菌酸性代谢物的过量积累,使pH值显著降低,杂菌数量上升而根瘤菌数急剧下降。比较发现,黄绵土载体制备的菌剂中LW107和RSW96菌株的有效菌含量显著高于其他载体基质(P<0.05),泥炭载体次之。黄绵土和泥炭基质菌剂的主要性状都达到微生物菌剂国家标准,可以作为菌剂载体基质利用。黄绵土基质由于价格低廉,原料易得,贮存后活菌数量高而杂菌率低,性质稳定,具有良好的应用潜力。8.秸秆粉浸提液培养基(CSE)中的碳源和磷素营养都能被菌体很好的利用。补充葡萄糖并调整pH后的秸秆粉浸提液培养基(CSE-2,青秸秆粉与H2O的比例为1:50)适用于LW107菌株的液体发酵,在降低菌剂成本的同时加快菌液的发酵速度,优于通用的YEM培养基;CSE-2中1:100的青秸秆粉浸提液(CSE-3)加入葡萄糖和琼脂后制成的固体培养基对根瘤菌的平板培养效果等同或优于标准YMA培养基,可以作为根瘤菌平板培养的高效廉价培养基。9.降低温度和添加抑菌剂均能提高根瘤菌剂中的有效菌数,降低杂菌率并维持菌剂内pH值的稳定。温度对长期保藏菌剂(1a)的活菌数和污染率影响最大,抑菌剂次之,4oC低温下保存抑菌剂可以有效提高活菌数,并使杂菌的数量得到有效控制;室温下菌剂中的菌体代谢活跃,有机酸积累量高,pH值偏低,而低温保存或添加抗生素的菌剂则pH值相对稳定。解磷根瘤菌菌剂贮藏1a后的pH值大小顺序为:低温贮藏的含抗生素菌剂>常温贮藏的含抗生素菌剂>低温贮藏的普通菌剂>常温贮藏的普通菌剂。10.以对300mg/L氨苄青霉素和80mg/L卡那霉素的双抗药性和解无机磷特性作为解磷根瘤菌高效突变株的标记特征,获得的菌株可以通过观测解磷透明圈、含药平板甄别的方式进行示踪和检测,使菌剂中目标菌和杂菌的测定过程得以简化,也使含抑菌剂的抗污染剂型制备和目标菌的占瘤率测定得以实现。11.以乙炔还原法测定固氮酶活性时发现,由于缺乏厌氧环境或低氧环境对固氮酶的保护,根瘤菌纯培养物的固氮酶活性远低于其诱导植物形成的根瘤,但源于同一原始菌株的突变株纯培养物固氮酶活性和其同一寄主的根瘤固氮酶活性间,存在正相关,且苜蓿根瘤菌L-5和红豆草根瘤菌RS-1的突变株系都得到一致的结果。这一现象值得进一步研究和发掘,可对高效固氮根瘤菌突变株的快速选育提供借鉴。更多还原

【Abstract】 Phosphorus is the main nutrient element for plant growth, whereas 72% to 90% phosphate in soil is fixated by calcium, aluminum, iron and organic compounds, which makes phosphate deficiency the main limiting factor that influences the yield and quality of agricultural products. As main forage grasses that ameliorate soil situation and increase animal products, the cultivate area of alfalfa and sainfoin has exceeded 2 million hm2, which is equivalent to 3 quarters of the artificial grassland in the country, and concentrates mainly on the grain rotation area in arid and semi-arid area of northwest plateau, China. Total P content in soil increased as the application of fertilizer, but phosphate deficiency has still been performed for crops and forage grasses due to the low content of available P. P solubilizing microorganisms are capable of solving the mentioned problem, and P solubilizing rhizobium, which is also capable of fixating N and inoculation, is found excellent in promoting growth and yield of legume forage grass, which decreases the cost of agricultural production and the side effect of P application to the environment. Currently, the phosphate-dissolving Rhizobium inoculants has not been widely used in agriculture, the main reasons are as follow: 1: the low Breeding efficiency of strains, and the low comprehensive growth promoting capability of the screened strains; 2: the application of strains was limited only to host plant; 3: the serious contamination of rhizobium inoculants and onefold effect, as well as relatively short shelf life and deficient in the ability to compete for nodulation. Because of the secreting of P dissolving organic acid, the acidification of the products was found more easily, which therefore makes the death of rhizobia and the infection of the undesired bacteria; 4: the currently widely used medium peat is a kind of nonrenewable resource, the collection of which will do harm to the local environment, and the peat itself has some qualitative defects. All the above factors make the application and development of P solubilizing rhizobium inoculants been limited.According to the above problems, the study is going to focus on the following aspects: 1: optimizing for screening method of phosphate-dissolving Rhizobium and the increasing effect of mutant by a rapid intermittent microwave irradiation; 2: to apply the P dissolving and IAA secreting rhizobia as RGPR strains of rhizobacteria is on to other plants but host plants and widen its application range; 3: decrease the contamination and increase the number of viable cells as well as the nodulation competitiveness of the target rhizobium with bacteriostat 4: to use the leach liquor of green stalk powder as the liquid medium of P solubilizing Rhizobium to reduce the cost of production, and use loess soil and stalk powder to instead peat to be used as the carrier of solid inoculants, to lower down the economic and environmental cost of inoculants production. Based on the above goals, the study firstly screened out Rhizobium meliloti. L-5 and Rhizobium sp. RS-1 with P solubilizing and IAA secreting ability by isolation and breeding, and then to obtain the mutated antibiotic resistant muatnts Rhizobium meliloti LW107 and Rhizobium sp. RSW96 by microwave mutagenesis, to get greater P solubilizing and IAA secreting capability, and to get the antibiotic resistant characteristics of the 2 strains. After the verifying of genetic stability, the growth promoting capability of LW107 and RSN96 to cultivated host and other plants were observed with Ca3(PO4)2 as the only P source. During the process of anti-contamination inoculants, ampicillin was used as bacteriostat to observe the toxic performance of strains and seedlings to ampicillin with different concentrations to select the optimum concentration of bacteriostat, as well as the optimum diluted concentration in application. The optimum carbon sources were selected out for the culture of P solubilizing rhizobia to decrease cost product and increase the quality, and the feasibility of green stalk powder leach liquor with adjusted components as fermentated and plate media was also observed. Afterwards, the sterilized stalk powder, loess soil, peat and vermiculite were used as media to be selected, and finally the liquid and solid rhizobia inoculants with antibiotics were obtained. According to the test of inoculants in different periods, the stable, cheap P solubilizing inoculants with lower contamination and longer effective time was obtained, together with the important techniques in inoculants production.The main findings are listed as fellow:1. The gradual selection model of acid producing N-fixing and P solubilizing Rhizobium is capable of screening P solubilizing rhizobia with good growth promoting capability from nodules with less procedure. The selected Rhizobium meliloti L-5 and Rhizobium sp. RS-1 were found as fast growing rhizobia, and were found with greater nodulation and N fixing capability, together with P solubilizing and IAA secreting capability, and stronger saline and alkaline resistant capability, and can be used as breeding material of highly efficient P solubilizing rhizobia.2. From the single colony generated by acid secreting N-fixing microbes that isolated from nodules, the inorganic P solubilizing microbes were mainly found as Agrobacteria, Actinomycetes, Bacilli, Pseudomonas denitrificans and other N-fixing strains, but only 5 strains identified as Rhizobium strains through the inoculation test, which take up 1.78% of all N-fixing microbes, indicating lots of P solubilizing and N-fixing microbes without nodulation capability exists in plant nodules.3. To increase breeding effectiveness of the strains and to make up the weak P solubilizing deficiency of the wild species, according to the strains mutagenesis method with the existing strains resources. As a safe, clean and cheap radiation source, the conditions of microwave radiation can be controlled and changed more easily. Because of the difference of mutagenesis goals and strains, the concrete microwave radiation parameters varied from each other, while all the highest positive mutagenesis occurred at the lethality rate between 87% and 90%. Part of the obtained highly efficient strains were found genetically unstable, and some were found back mutation or a decrease in yield, at least 6 generations of genetic stability test is required.4. On condition of only inorganic P be offered, the growth of all plants were found be restrained obviously, the leaves grew retardedly, the leaf colour got darkened, and the root became thinner. The shoot height, root length and leaf area were 61-85.2%, 60.5-81.9% and 32.7-75.6% of the treatments inoculated with P solubilizing rhizobia, indicating that good growth promoting effect were found of P solubilizing rhizobia with IAA secreting capability to both host and other plants, and the P deficiency stress can be solved effectively, the assimilation of N and P, and the growth and biomass of the plants increased. The functional process of P solubilizing rhizobia was: firstly dissolve inorganic P to abate the P deficiency stress of plants, and to offer the plants a relatively balanced nutrient condition, simultaneously, to infect the host plant and generate nodules for N fixation. To other plants, P solubilizing rhizobia were found resemble the characteristics of rhizospheric P solubilizing bacteria, but the growth promoting effect on host plants was found better than on other plants.5. On condition of low P content, normal rhizobia were found capable of infecting host plants and nodulation to fixate nitrogen, but on condition of P deficiency, the diameter, fresh weight and nitrogenase activity of nodules were found decreased conspicuously. On condition of low P content, the growth and the accumulation of biomass of plants inoculated with 12531 rhizobia were found performed better than un-inoculated and non fertilized plants (CK1), but the leaf area was only 65.9%, 79% and 67.4% of the treatments inoculated with LW107. RSW96 and applied with 1/4 P solubilizing rhizobia (CK2), respectively. and also poorer than treatments applied with chemical fertilizer. The dry weight of plants inoculated with 12531 strain was found increased by 35.93%, compared with CK1, and only 73.1% and 85.29% of the treatments inoculated with LW107 and RSW96, and also found 17.92% lower than the treatment applied with chemical fertilizer.6. As bacteriostat with low toxity, ampicillin was found with stimulatory effect of low dose and toxic effect of high dose to plants. 200mg/L ampicillin could cause the decrease of nodule number, and to made the waning and lighten of nodules, simultaneously, the nitrogenase activity of nodules decreased to 1/5 or lower to that of the normal nodules. Ampicillin with low concentration has not depression effect on nodulation and nitrogenase activity, and could even the number of nodulation. Because of the difference between species, the reaction of alfalfa and sainfoin to ampicillin was found different. 7. Among the solid inoculants with different materials as the medium, after 120d of storage, the number of viable cells increased as the increase of the absorption of bacterial liquid. Because of the loose and porous structure, green stalk powder could absorb the bacterial liquid that is 2.7 times of its weight, and the powder also contains mass of carbon source, nucleic acid, soluble sugar and almost all the mineral elements that is required of the growth of the microorganism, which will result in the fast proliferation of bacteria, within the 120d of storage, the effective bacteria content of which were found 155.48% and 138.44% times of the numbers found in peat, which was found of great potential in short time inoculant production. But after 1a storage, because of the accumulation of acidic metabolite generated by P solubilizing rhizobia, the pH value was found decreased significantly, the number of undesired bacteria increased, and the nodule numbers decreased sharply. According to the comparison of inoculants after 1a storage, the effective bacteria numbers of the two strains in inoculants with loess soil as the medium was found significantly higher than inoculants with other materials as media(P<0.05), followed by peat. Both the 2 media reached the national standard for inoculant, and could be used as inoculant carrier. Because of the low cost and the accessibility, and the high viable cells as well as the low undesired bacteria number, and the stable characteristic, loess soil was found with good potential.8. Both the carbon source and P in medium prepared with corn stalk leach liquor (CSE) could be used by bacteria. When replenished with glucose (with adjusted pH value) (CSE-2, the ratio of corn stalk powder and distilled water was 1:50), the CSE was found good to the fermentation of LW107, the fermentation speed was increased and the cost also decreased, and was found better than YMA medium; to CSE-3 medium (the ratio of corn stalk powder and distilled water was 1:100), when added with glucose and agar, the obtained solid medium was found with good rhizobia culture effect, equivalent to or better than standard YMA medium, and could be used as cheap medium for plate culture of rhizobia.9. The decrease of temperature or the addition of bacteriostat could increase the number of effective bacteria, decrease the number of undesired bacteria and maintain the pH stability in inoculants. Temperature influences most for the number of viable cells and the undesirable microbes in 1a stored inoculants, bacteriostats were secondly. Viable cell number could be maintained and the number of undesired bacteria will be controlled under the condition of addition of bacteriostat and at low temperature simultaneously, the most active metabolism and organic acid accumulation was found at room temperature, and the pH was found relatively lower, while for inoculants stored at low temperature or added with bacteriostat was found relatively stable. The pH order of the inoculants after 1a storage was: inoculants stored at low temperature and with bacteriostat > inoculants stored at room temperature and with bacteriostat > inoculants stored at low temperature > inoculants stored at room temperature.10.The study synthesizes the screening, mutagenesis and the further utilization, and use double antibiotic and inorganic P solubilizing as marked feature of the strains, and the finally obtained strains could be spiked and detected by observing P solubilizing circle and by plates that contain certain chemical compounds, all the above make the test procedure of target and undesired bacteria more concisely, and also make the production of antibiotic inoculation as well as the nodulation competitiveness of target bacteria more achievable.11.According to the determination of nitrogenase activity by ethyne reduction method, because of the lack of oxygen or the protection of low oxygen conditions to nitrogenase, the nitrogenase activity of pure cultured material was found far lower than the induced plant nodules, but positive correlation was found between the nitrogenase activity of the pure culture material and the nitrogenase activity of nodules induced by the same original strain, accordingly, the same result was found in mutant strains derived from Rhizobium meliloti L-5 and sainfoin Rhizobium sp. RS-1. This phenomenon is worth of further study and exploration, and might be a new way for the fast breeding of highly efficient nitrogen fixation mutant strains.更多还原