氮肥和水分条件对长喙田菁生长、结瘤和固氮的影响

 盆栽试验研究了长喙田菁(Sesbania rostrata) 茎瘤固氮根瘤菌（Azorhizobium caulinodans）共生体系在不同水分和无机氮肥条件下的生长、结瘤和氮积累。水分处理为: 不浸水、浅浸水(土壤水分为田间持水量)和深浸水, 氮肥处理分别为每千克土施加无机氮肥10 mg、20 mg和40 mg。结果表明,水分条件对共生体系有较大影响, 浸水环境虽然抑制了根瘤的形成, 却促进了茎瘤的结瘤作用和长喙田菁根系的生长, 田间持水量状态下共生体系能获得最大的生物量和氮累积量; 无机氮肥对共生体系的影响受水分供应的影响较大,实验氮肥施用浓度范围内,长喙田菁的结瘤作用对复合态氮无负敏感反应,明显有别于一般根瘤体系的复合态氮反应。     

营养杯对长喙田菁在铅锌尾矿上的生长、固氮和重金属积累的影响

设置移栽时营养杯的有无及其大小作试验,研究长喙田菁在乐昌铅锌矿强酸化尾矿上的生长、固氮和积累重金属情况。结果表明,强酸性(pH<3)是限制植物定植的主要因素,在pH=5-7情况下,长喙田菁能在该尾矿库中定植、生长和固氮,表现出良好的适应性。未带营养杯移栽的长喙田菁在尾矿上生长84d,其株高117cm、茎基部直径1.35cm、单株生物量(干物质)20.2g、单位面积生物量(干物质)2828kghm-2、氮素积累量40kghm-2;带营养杯移栽的上述各指标分别达到140-144cm、1.59-1.68cm、36.6-38.8g、5124-5432kghm-2和77-107kghm-2,均显著高于未带营养杯处理的。长喙田菁根部铅、锌、铜、镉含量均最高,其次为茎,叶中最低;长喙田菁的4种重金属积累量为锌(186-221mgkg-1)>铅(96-145mgkg-1)>铜(17-30mgkg-1)>镉(3-4mgkg-1)。带营养杯移栽能有效提高长喙田菁的产量和氮积累量,且明显降低其体内的重金属含量。试验证明长喙田菁是较理想的铅锌矿尾矿废弃地植被重建的先锋植物。     

茎瘤对长喙田菁在铅锌尾矿环境适应中的意义II 茎瘤对长喙田菁固氮和积累重金属的影响

探讨茎瘤对长喙田菁(Sesbania rostrata)在铅锌矿尾矿、客土和纯土环境中结瘤、固氮和积累重金属的影响。结果表明：保留茎瘤使长喙田菁全株植物氮含量和单株植物氮积累量分别比去除茎瘤处理提高了12.4%～34.0%和43.3%～131.4%,且环境愈恶劣,这种作用愈为显著。同时,保留茎瘤处理显著抑制了长喙田菁根瘤的生长,同样表现出环境愈恶劣,抑制作用愈显著的趋势。因此,在尾矿环境胁迫下,长喙田菁更趋向于选择茎瘤作为固氮途径。由此可见,茎瘤对生长在铅锌尾矿环境中的长喙田菁获得氮素及支持其生长有着特别     

茎瘤对长喙田菁在铅锌尾矿环境适应中的意义II 茎瘤对长喙田菁固氮和积累重金属的影响

 探讨茎瘤对长喙田菁(Sesbania rostrata)在铅锌矿尾矿、客土和纯土环境中结瘤、固氮和积累重金属的影响。结果表明：保留茎瘤使长喙田菁全株植物氮含量和单株植物氮积累量分别比去除茎瘤处理提高了12.4%～34.0%和43.3%～131.4%,且环境愈恶劣,这种作用愈为显著。同时,保留茎瘤处理显著抑制了长喙田菁根瘤的生长,同样表现出环境愈恶劣,抑制作用愈显著的趋势。因此,在尾矿环境胁迫下,长喙田菁更趋向于选择茎瘤作为固氮途径。由此可见,茎瘤对生长在铅锌尾矿环境中的长喙田菁获得氮素及支持其生长有着特别     

茎瘤对长喙田菁在铅锌尾矿环境适应中的意义Ⅰ.茎瘤对长喙田菁生长发育的影响

通过设置保留茎瘤和去除茎瘤处理 ,研究长喙田菁 (Sesbaniarostrata)在铅锌尾矿、客土和纯土环境中的生长发育情况。结果表明 :保留茎瘤使长喙田菁的株高、地上部生物量、地下部生物量、全株生物量和叶绿素含量分别比去除茎瘤处理提高了 2 4 %～ 4 8%、2 7 4 %～ 6 7 9%、2 8 5 %～ 99 3%、2 7 6 %～ 72 3%和 17 0 %～ 2 3 4 %,这种作用在纯尾矿处理组最为显著 ,客土处理组次之 ,即环境愈恶劣 ,茎瘤这种作用愈显著 ,证明了茎瘤对长喙田菁适应铅锌尾矿环境有积极的贡献。     

茎瘤对长喙田菁在铅锌尾矿环境适应中的意义I. 茎瘤对长喙田菁生长发育的影响

 通过设置保留茎瘤和去除茎瘤处理,研究长喙田菁(Sesbania rostrata)在铅锌尾矿、客土和纯土环境中的生长发育情况。结果表明:保留茎瘤使长喙田菁的株高、地上部生物量、地下部生物量、全株生物量和叶绿素含量分别比去除茎瘤处理提高了2.4%～4.8%、27.4%～67.9%、28.5%～99.3%、27.6%～72.3%和17.0%～23.4%,这种作用在纯尾矿处理组最为显著,客土处理组次之,即环境愈恶劣,茎瘤这种作用愈显著,证明了茎瘤对长喙田菁适应铅锌尾矿环境有积极的贡献。     

费氏中华根瘤菌共生质粒扩增对结瘤因子组分和共生固氮能力的影响

费氏中华根瘤菌(Sinorhizobium fredii)YC4能在大豆(Glycine max)和野大豆(G．soja)上形成正常固氮的根瘤．人工培养条件下用^14C标记的薄层层析(TLC)法检测根瘤菌产生的结瘤因子(LCOs)的结果表明,与其它4株费氏中华根瘤菌相比,YC4产生的LCOs含有较多的疏水性基团．从YC4菌株中分离到1株共生质粒发生了扩增的自发突变株YSC3,其产生的LCOs中含有较野生型菌株多的1个疏水性组分,28℃培养条件下产生的LCOs量亦较YC4显著增加．结瘤试验结果表明,YSC3菌株只能在大豆和野大豆上形成无效的根瘤．     

植物的共生固氮体系

氮是植物生命活动中不可缺少的重要元素之一。大气中的氮尽管为79％,但这种游离氮只有少数固氮细菌和蓝藻才能吸收利用,绿色植物却不能直接利用。这些细菌、蓝藻把大气中的游离氮固定转化为含氮化合物,成为植物所能吸收的氮,就称为生物固氮作用。固氮生物之所以能催化还原N2成NH3,是由于它含有固氮酶。固氮酶是一种结构复杂、功能特异的酶,由铁蛋白和钼铁蛋白组成。     

铜对大叶相思-根瘤菌共生固氮体系的影响

报道了两种根瘤菌 (大叶相思、美丽胡枝子 )对Cu2 + 的耐受性以及植物 根瘤菌共生固氮体系在Cu2 + 胁迫下结瘤、固氮和生长的变化 ,讨论了大叶相思在矿山尾矿废弃地作为先锋植物结瘤固氮的可能性 .结果表明 ,大叶相思根瘤菌对Cu2 + 离子的耐受性较强 ,可以耐受Cu2 + <0 80mmol的离子浓度 ,Cu2 +对它的半致死浓度为 0 12 9mmol.在无菌砂培无重金属影响条件下 ,其固氮酶活性为 2 7C2 H4 ·μg·g-1·h-1,当Cu2 + >0 12 5mmol会导致大叶相思固氮酶活性急剧下降 ,其有效半抑制浓度 (EC50 )为0 15 1mmol,Cu2 + 为 0 5 0mmol完色抑制大叶相思固氮酶活性 ,不阻碍结瘤 ,但严重抑制植物生长发育 ,引起植物叶片白化、植株矮化 .在外加N源不接菌和不加N源接菌两种处理组间 ,Cu2 + <0 12 5mmol时 ,以不加N接菌处理对大叶相思生长有利 .大叶相思对Cu2 + 吸收积累根部高于地上组织 .     

Responses of Azorhizobium caulinodans to cadmium stress

The symbiosis of Azorhizobium caulinodans and an annul legume Sesbania rostrata was recently found to be tolerant to cadmium pollution by an unknown mechanism. In this study, A. caulinodans ORS571 and ZY-20 showed much stronger tolerance to cadmium than a mutant ORS571-X15 and a common Rhizobium sp., with minimum inhibitory concentration values as high as 4 and 5 mM (versus 1 and 0.1 mM) on yeast extract mannitol agar medium, respectively. Although Cd uptake by all three strains of A. caulinodans were mostly from absorption rather than binding (both loosely or tightly) on cell surface, in resistant strains a higher portion of extractable Cd was bound on the cell surface vs. absorbed (about 1:2.5 ratio) compared to the sensitive mutant (about 1:35.1 ratio). These results suggest that certain level of metal exclusion by a permeability barrier was involved in the mechanism of resistance to Cd by A. caulinodans ORS571 and ZY-20. Over the 12-h period of cultivation in yeast extract mannitol agar medium with Cd addition, the Cd concentrations in the outer membrane and periplasm and spheroplast were the highest at the first 3 h, and declined steadily over time. The fact that Cd concentrations in spheroplast of all three strains were many folds higher than those in outer membrane and periplasm, suggests that extracellular sequestration was not the only mechanism of Cd tolerance in A. caulinodans. The decline of Cd concentrations was significantly faster and started earlier in strains ORS571 and ZY-20 than in ORS571-X15. This suggests a second, probably more substantial, mechanism involves active transport of the metal from the cell, e.g., some efflux system for maintaining homeostasis under cadmium stress.     

Growth of Sesbania rostrata (Brem) with stem nodules under controlled conditions

A series of experiments was carried out in an attempt to produce nodulated plants of Sesbania rostrata with qualities more closely resembling those in the wild than has been achieved to date. When groups of five plants were grown in a controlled climate chamber in pipes containing ～12dm³ modified Jensen's medium with 6mol m^?3 nitrate, the daily growth in height reached 5 cm and at 30 d the plants were ～40cm high. At this time, the stems were inoculated with Azorhizobium caulinodans ORS 571 and the medium replaced with Jensen's medium without nitrate. In the subsequent 19-d period ～300 nodules (representing >50% of the potential infection sites) developed on each stem. The nodules increased linearly in size over this time to ～15mg. Specific acetylene reduction activity, ARA ((μmol C₂H₄ mg^?1 h^?1) rose to 45 between days 5 and 10 after inoculation and plateaued; total ARA rose to ～200 μmol C₂H₄ plant^?1 h^?1. Under the conditions described the plants grew vigorously, and reproducibly uniform yields of nodules with high ARA activities were obtained. As outlined, the procedure offers a standard system in which, within a 2-week period after inoculation, individual strains of bacteria can be quantitatively compared in their ability to induce nodulation and N₂-fixation. Physiological and biochemical aspects of the nodulated system can be much more readily approached than with plants producing only root nodules. The inhibitory effects of stem nodules induced by wild type and two mutant strains of Azorhizobium on the development and activity of root nodules are described.     

毛萼田菁茎瘤菌被种子浸提物诱导表达基因的筛选及其功能

摘要：【目的】选择毛萼田菁茎瘤菌ORS571为研究对象,筛选毛萼田菁茎瘤菌在菌植互作早期被宿主植物种子浸提物诱导表达的基因。【方法】采用新颖的基于抗性的活体表达筛选技术,以种子浸提物为诱导物,筛选获得能够稳定被种子浸提物诱导的突变株。通过中间片断融合报告基因的方法研究信号分子对相关基因的诱导情况。【结果】随机引物PCR产物测序及网上BLAST结果表明,被诱导表达的基因为LysE家族的成员。而且,对另外3个属根瘤菌中lysE基因的研究发现,它们都可以被相应的宿主植物种子浸提物诱导表达。此外,这四株根瘤菌的lysE突变株都表现对刀豆氨酸极为敏感。本文首次报道了lysE家族在根瘤菌中的生理功能。【结论】我们推测：LysE家族蛋白可能广泛存在于根瘤菌中,并在根瘤菌与宿主植物的早期相互作用中发挥重要作用。     

毛萼田菁茎瘤豆血红蛋白含量与固氮活性的关系

在个体瘤发育的不同阶段和植株生长的不同时期,以及在不同的光照条件下,分别测定茎瘤的固氨活性和豆血红蛋白含量。结果表明,毛萼田菁茎瘤的固氨活性和豆血红蛋白含量呈正相关,豆血红蛋白在茎瘤的共生固氮中起着重要的作用。     

Effectiveness of Sesbania rostrata and Phaseolus calcaratus as green manure for upland rice grown in acidic soil

Two field experiments on green manuring were conducted under upland acidic soil (pH = 4.35) conditions with the following objectives: (1) to determine the influence of inoculation site, P fertilization, and liming on the biomass production, N content, N accumulation, and N availability of S. rostrata grown in an acidic soil, (2) to compare the effectiveness of S. rostrata, P. calcaratus and urea as N sources for upland rice as affected by liming and N source-sowing time combination, and (3) to assess the effect of liming and N source-sowing time combination on % Ndff (N derived from the fertilizer), % Ndfs (N derived from soil), % FNU (fertilizer N utilization), and FNY or fertilizer N yield (kg N ha^–1) of upland rice grown in acidic soil. At 2 weeks after incorporating S. rostrata (95 days after lime application), liming significantly increased N availability by more than 2-fold suggesting that the decomposition of S. rostrata by soil microflora was stimulated by lime. Liming, phosphorus application, and inoculation site improved significantly the dry biomass production, N content and N accumulation of S. rostrata; thus, enhancing its green manuring potential. Regardless of liming, S. rostrata whether applied at 0 week or 2 weeks before sowing was superior to urea in improving grain and straw yields. P. calcaratus when applied at 2 weeks before sowing also produced higher grain yield than urea. Immediate sowing of upland rice after green manure incorporation did not affect negatively the growth and development of upland rice; hence, farmers could save at least 2 weeks in their cropping calendar. N source-sowing time combination had a highly significant influence on % Ndff, % Ndfs, % FNU, N uptake, and fertilizer N yield of upland rice. However, only N uptake was influenced significantly by liming. The rice plant obtained significantly higher % Ndfs from the soils treated with green manure than those treated with urea regardless of liming. The % FNU and % Ndff from the green manures were 11-37% and 9-25%, respectively. These values are much lower than those obtained under continuously flooded soil conditions possibly because of the differences in the organic matter decomposer populations and N loss mechanisms between sloping upland conditions and continuously flooded conditions.     

An Azorhizobium caulinodans ORS571 locus involved in lipopolysaccharide production and nodule formation on Sesbania rostrata stems and roots.

Azorhizobium caulinodans ORS571 is able to nodulate roots and stems of the tropical legume Sesbania rostrata. An ORS571 Tn5 insertion mutant, strain ORS571-X15, had a rough colony morphology, was nonmotile, and showed clumping behavior on various media. When this pleiotropic mutant was inoculated on roots or stems of the host, no nodules developed (Nod-). Compared with the wild type, strain ORS571-X15 produced lipopolysaccharides (LPS) with an altered ladder pattern on sodium dodecyl sulfate-polyacrylamide gel electrophoresis gels, suggestive of a different O-antigen structure with a lower degree of polymerization. A cosmid clone, pRG20, that fully complemented all phenotypes of ORS571-X15 was isolated. With a 6-kb EcoRI subfragment of pRG20, clumping was relieved and nodulation was almost completely restored, but the strain was still nonmotile. LPS preparations from these complemented strains resembled the wild-type LPS, although minor quantitative and qualitative differences were evident. The sequence of the locus hit by the Tn5 in ORS571-X15 (the oac locus) revealed a striking homology with the rfb locus of Salmonella typhimurium, which is involved in O-antigen biosynthesis. The Tn5 insertion position was mapped to the oac3 gene, homologous to rfbA, encoding dTDP-D-glucose synthase. Biochemical assaying showed that ORS571-X15 is indeed defective in dTDP-D-glucose synthase activity, essential for the production of particular deoxyhexoses. Therefore, it was proposed that the O antigen of the mutant strain is devoid of such sugars.     

Translocation of amino acids from stem nodules of Sesbania rostrata demonstrated by GC-MS in planta 15N isotope dilution

We report a novel use of the ¹⁵N dilution technique to detail the translocation of amino compounds in the legume Sesbania rostrata . The conventional ¹⁵N dilution technique follows the dilution of ¹⁵N within a labelled plant, as ¹⁴N₂ is fixed by symbiotic bacteria. In our experiments, stem-nodulated Sesbania rostrata were enriched by feeding with ¹⁵N ammonium nitrate for 2 weeks, followed by a 1 week period where the only N available to the plants was via nitrogen fixation of atmospheric N₂. We measured the composition, concentration and ¹⁵N enrichment of amino compounds in various plant tissues, both above and below the stem nodules, using GC-MS and isotopic abundance mass spectrometry techniques. Approximately 28% of the total N in the stem nodules was derived from internal plant sources. The ureides allantoic acid and allantoin were not abundant in xylem, leaf or nodule tissues. The amides asparagine and glutamine were the major export products from stem nodules although a wide range of other amino compounds are also synthesized. Amino acids within the nodules had a low level of enrichment, demonstrating that a small fraction (≈ 11%) was derived from outside the nodules, and significant cycling of N (28% of xylem N) through the root system was revealed by measurements of ¹⁵N distribution and amino acid concentrations.