固氮粪产碱菌（Alcaligenes faecalis）胞外多糖突变株的筛选及特性

应用Ｔｎ５转座子对粪产碱菌野生型菌株Ａ１５０１进行诱变处理，用荧光增白剂及ＴＴＣ为标记筛选，分别获得胞外多糖缺陷型和丰富型突变株。激光共聚焦观察证实，ＥＰＳ突变株确实具有与野生型不同的表面特性，贴根试验表明，突变株贴根菌数均少于野生型，胞外多糖生成过多或过少均影响菌体与根表的有效结合。     

盐胁迫下粪产碱菌在水稻根表的定殖(英文)

粪产碱菌A1 5 0 1能在氯化钠浓度高达 4%的固体和液体介质中良好生长 ,合成IAA和固定空气中的氮素。当氯化钠浓度超过 3 %时 ,A1 5 0 1丧失运动和趋化能力。一定盐浓度胁迫下A1 5 0 1能定殖在水稻根表和根毛区和侧根伸出部位 ,表现出较高的联合固氮活性。0 5 %的氯化钠能促进A1 5 0 1在水稻根表的定殖能力。A1 5 0 1能通过侧根伸出部位侵入根内。     

固氮粪产碱菌（Alcaligenes faecalis）表面蛋白变构作用的研究

采用电子自旋共振技术（ＥＳＲ），以马来酰亚胺自旋标记粪产碱菌野生型（Ａ１５０１）、胞外多糖突变株（ｅｘｏ＾－和ｅｘｏ＾＋＋）及工程菌（ｎｉｆ某Ａ和ｎｔｒＣ－ｎｉｆＡ重组子），监测由粪产碱菌表面多肽及些膜蛋白构象变化引起的ＥＳＲ波谱的变化。结果表明，粪产碱菌野生型菌株细胞表面特性显著不同于胞外多糖突变株及工程菌，根系粘质及ＮＨ４＾＋都能引起粪产碱菌表面蛋白的构象变化，标记后菌体ＥＳＲ参数τｃ的变化与     

联合固氮遗传工程菌的田间释放与风险评价

应用遗传工程技术分别将含有ｎｉｆＡ＾ｃＤＮＡ片段的质粒及含有Ｔｎ５：：ｎｉｆＡ的广泛宿主自杀性质粒ｐＳＺ３６转入了三个联合固氮菌，即粪产碱菌，阴沟肠杆菌及催娩克氏菌中；把Ｔｎ５：：ｎｉｆＡ－ｎｔｒＣ转入了粪产碱菌野生型菌株Ａ１５０１。这些转化结合子在高铵下表现出固氮活性，并能集聚在水稻根表，而野生型菌在相同条件下远离水稻根表。这些菌株的田间释放实验在严格控制条件下进行，应用＾１５Ｎ稀释技术测定其固     

水稻根际固氮粪产碱菌的研究（综述）

粪产碱菌分离自我国南方水稻田中，根据系统发育分类法属变形细菌中β亚类。粪产碱菌不仅能附着于水稻根表，部分细菌还能进入稻根细胞内，有较高的固氮能力，在高铵下它仍能合成固氮酶，并具有明显的ＰＧＰＲ作用。本文系统地论述了粪产碱菌的生物学特性及其联合固氮研究的进展。     

异源nifL在粪产碱菌的表达及功能研究

本实验采用两亲结合方法，将棕色固氮菌（Ａｚｏｔｏｂａｃｔｅｒ ｖｉｎｅｌａｎｄｉｉ）的ｎｉｆＬ－ｌａｃＺ融合基因转入固氮粪产碱菌（Ａｌｃａｌｉｇｅｎｅｎｓ ｆａｅｃａｌｉｓ）Ａ１５０１中获得了携带异源ｎｉＬ－ｌａｃＺ融合基因的结合子Ａ１５Ｌ１。对野生型和结合子的固氮活性，β－半乳糖苷酶活性及根表定殖能力的测定结果表明：（１）异源ｎｉｆＬ在粪产碱菌中的表达受Ｏ２和ＮＨ＾＋４的抑制，好氧条件下基因表达     

粪产碱菌耐铵工程菌与水稻联合共生固氮作用

粪产碱菌（ Ａａｅｃａｌｉｓｆａｅｃａｌｉｓ） 在ＬＷ 培养基下能产生植物激素ＩＡＡ。盆栽结果表明,接种Ａ．ｆａｅｃａｌｉｓ 显著提高了水稻的生长,接种Ａ１５０１ 和Ａ１５１３ 的水稻稻谷产量分别比未接种提高了８５％ 和１０３ ％ ,用１５ Ｎ 同位素稀释法估测水稻地上部总氮中来源联合固氮的百分率达９００ ％ 和１１５ ％ ,其结果与水稻地上部（ 茎叶及稻谷） 总氮量的增加幅度（８６ ％ 和１１６ ％ ）基本一致。研究还表明Ａ１５１３ 耐铵工程菌的产ＩＡＡ作用以及对水稻的生长和产量、联合固氮作用均优于原始菌株Ａ１５０１ 。     

粪产碱菌(Alcaligenes faecalis)的反硝化及固氮作用

粪产碱菌生长在NH_4~+和NO_3~-两种氮源的介质中时,优先利用NH_4~+,在好气条件下,NH_4~+的存在抑制了粪产碱菌对NO_3~-的同化作用。在厌氧条件下,粪产碱菌能以乳酸为碳源,NH_4~+为唯一氮源进行反硝化作用,NO_3~-作为最终电子受体,接受无氧呼吸链传递的末端电子,经NO_2~-等氮氧化物最终还原为N_2。在好气和厌气条件下,低浓度的NO_2~-对粪产碱菌生长有一定抑制作用,但NH_4~-的存在并不抑制细菌对NO_2~-的利用。粪产碱菌在厌氧条件下还具有需硝酸盐的固氮酶活性。当氧存在或以NO_2~-代替NO_3~-时,固氮活性均受抑制,硝酸盐或亚硝酸盐浓度愈高,抑制愈强。反硝化作用产生的N_2能为微好氧条件下生长的粪产碱菌所重新固定。     

固氮球形芽孢杆菌与巨大芽孢杆菌的混合增效作用

球形芽孢杆菌Bs7与巨大芽孢杆菌Bm1 0 1混合培养时具有固氮增效作用。稀释 40 0倍的巨大芽孢杆菌的无菌上清液与球形芽孢杆菌共培养 ,同样能增强后者的固氮活性。Bs7(pAB5 76 )在纯培养时的nifA表达活性为 6 849 5Miller单位 ,与巨大芽孢杆菌上清培养液进行混合培养时的表达活性为 75 0 0 4Miller单位。上述结果表明 :某些微量非碳源的活性分泌物和某些固氮调节基因可能在固氮增效作用中起重要作用。在与水稻联合培养条件下 ,混合接种球形芽孢杆菌与巨大芽孢杆菌可以提高固氮菌在水稻根表的定殖能力     

根际促生菌防控土传病害的机理与应用进展

根际促生菌作为一类活跃于植物根际的有益微生物,对抑制土传病害的发生,改善和维护土壤生态质量具有重要作用以及广阔的发展前景。本文综述了根际促生菌直接或间接的土传病害防控机理,包括产抗生素、产水解酶、释放挥发性气体、诱导抗性、分泌铁载体、分泌激素和固氮解磷等;亦从信号识别与迁移、定殖规律以及定殖的微生物群落影响等方面对根际促生菌的定殖防病机理予以了总结;此外,笔者还就根际促生菌的应用现状进行了概述。     

Differential colonization of Azospirillum lipoferum on roots of two varieties of rice (Oryza sativa L.)

Summary Seedlings of rice (IR42 and IR50) were aseptically dipped into Azospirillum lipoferum strain 34H suspension under dark, and the presence of bacteria on the differentiating regions of rice roots was observed by scanning electron microscopy. The bacterium did not colonize the root tips of IR42, while it colonized this region in the case of IR50, within 24 h after inoculation. In the early stages, most of the bacteria were embedded in the ruptured mucigel below the root cap cells of IR42. Mucigel was hardly detectable in IR50. While the root hair primordia of IR50 were colonized heavily with the bacterium within 24 h, the root hairs of IR42 were colonized 48 and 72 h after inoculation. This phenomenon in relation to plant varietal differences was discussed.     

The flavonoid naringenin enhances intercellular colonization of rice roots by<Emphasis Type="Italic"> Azorhizobium caulinodans</Emphasis>

The intercellular colonization of rice roots by Azorhizobium caulinodans and other diazotrophic bacteria has been studied using strains marked with the lacZ reporter gene. A. caulinodans were able to enter the roots of rice at emerging lateral roots (lateral root cracks) by crack entry and this was observed by light microscopy. After colonization of lateral roots, bacteria moved into intercellular space within the cortical cell layer of roots. Naringenin at 1×10^-5 and 5×10^-5 M concentration significantly enhanced root colonization. The role of nodABC and regulatory nodD genes was also studied; lateral root crack (LRC) colonization of rice was shown to be Nod factor and NodD independent. Lateral root crack colonization of rice was also observed with similar frequency following inoculation with Azospirillum brasilense and the colonization by A. brasilense was stimulated by naringenin and other flavonoid molecules.     

田菁茎瘤固氮根瘤菌在小麦体内的定殖及营养元素相关miRNA的表达

【目的】随着化肥过度使用引起的环境问题的出现,无公害农业的推广,以及新型肥料研究领域不断拓宽,微生物肥料,尤其是植物根际促生菌的研究成为近年来的热点。然而微生物肥料的增产机理还基本停留在作物农学性状的表观调查上,没有从分子水平进行研究。因此,本文用田菁茎瘤固氮根瘤菌（Azorhizobium caulinodans ORS571）侵染小麦,探索GFP-A.caulinodans在小麦幼苗组织中的分布与定殖规律以及营养元素相关miRNAs在小麦与A.caulinodans互作中的作用机制。【方法】使用A.caulinodans侵染小麦种子（品种为小偃22）,将接菌6 d后的小麦幼苗的根和接菌12 d后的叶制作玻片,利用激光共聚焦显微镜对样品进行逐层扫描,检测GFP-A.caulinodans在幼苗不同组织中的分布与定殖情况。同时,采集接菌后0 h、 6 h、 12 h、 24 h、 48 h、 72 h、 96 h的小麦幼根取样,用Trizol法提取总RNA。利用试剂盒进行加尾和反转录反应,将样品总RNA中的miRNA合成为cDNA,使用-tubulin作为内参基因,进行实时定量PCR反应,使用2-CT方法计算相对表达量。利用psRNATarget 在线软件,采用默认参数,对miRNA的靶基因进行预测。【结果】 1）激光共聚焦结果显示,GFP-A.caulinodans可定殖于根的表皮细胞、 细胞间隙、 根尖破损处和根毛,在根维管组织和叶片气孔部位,也发现有GFP-A.caulinodans存在。2）实时定量PCR分析结果表明,6条与营养元素代谢有关的miRNA表达发生变化,其中miR164、 miR167和miR827相对表达量呈现出先上调后下调的趋势,miR169 和miR398相对表达量也基本呈现出这一趋势。miR164、 miR167、 miR169和miR398的相对表达量在接菌12 h时上调至最高点,分别为对照的4.13、 2.84、 2.46和3.99倍; miR827相对表达量在接菌24 h时达到最高点,为对照的2.17倍。miR399相对表达量呈现出先下调后上调的趋势,在接菌24 h时降至最低点,为对照的0~21倍。3）通过靶基因预测,6条miRNA的靶基因分别编码了NAC1转录因子、 生长素响应因子、 HAP转录因子、 Cu/Zn 超氧化物歧化酶、 蛋白缀合酶PHO2和SPX-MFS亚家族蛋白。【结论】GFP-A.caulinodans能够从根毛和根尖破损处等部位进入小麦幼苗根部定殖,并可通过向上迁移到达叶片,在气孔处定殖。接种A.caulinodans可不同程度增加小麦根中响应氮素、 磷素、 微量元素的miRNAs相对表达量,增强小麦幼苗对营养元素的吸收和利用,促进小麦根的形态建成。     

磷饥饿诱导水稻根表铁膜形成机理初探

采用溶液培养的方法,初步探索了磷饥饿诱导水稻根表铁膜形成的机理。磷饥饿24h后水稻的根表出现了明显的红棕色物质的沉积,扫描电镜的能谱分析结果显示,红棕色物质是铁的氧化物。针对这一现象,首先研究了没有水稻生长的正常磷营养液和缺磷营养液的变化,结果表明二者之间全波长的扫描图谱没有出现差异。采用酸碱混合指示剂的琼脂染色方法,观察了水稻根系表面及根际pH值的变化情况,并分别测定了正常磷营养（P）和缺磷（P0）2种条件下水稻的根系活力。结果看出,缺磷时水稻根系活力高于磷营养正常的处理,尤其是基因型Jin23A,其P和P0处理间根系活力差异极显著。水稻根表三价铁的浓度高于二价铁,并且缺磷根系表面三价铁和二价铁浓度均明显高于供磷处理;缺磷处理水稻根质外体沉积的铁浓度也明显高于供磷处理。因此,初步确定磷饥饿诱导水稻根表铁膜形成是生物学基础上的化学反应过程。     

Phenotypic variation in Azospirillum brasilense Sp7 does not influence plant growth promotion effects

The Azospirillum genus comprises free-living, plant growth-promoting, nitrogen-fixing bacteria found in the rhizosphere of plant roots. Azospirilla are able to promote plant growth mainly through improvement of root development. Bacterial surface components, such as extracellular polysaccharides and proteins, are involved in root colonization. Phase variation – or phenotypic variation – is one of the mechanisms by which microorganisms adapt to environmental changes. This phenomenon is characterized by the presence of a sub-population of the bacteria presenting a different phenotype relative to the major population. In this study we characterized phenotypic variation of Azospirillum brasilense Sp7. When plated on solid media, some A. brasilense colonies were shown to possess a much more mucoid morphology, producing 7.5–8 times more exopolysaccharide with different monosaccharide composition than the parental strain Sp7. The rate of appearance of this kind of variant colonies was 1 in 5000, in agreement with the accepted rate for the phase/phenotypic variation phenomenon. The variants were significantly more resistant to heat and UV-exposure than the parental strain and displayed genomic changes as seen by the different band patterns following ERIC-PCR, BOX-PCR and RAPD analyses. In plant inoculation experiments under greenhouse conditions, with maize, wheat, soybean and peanuts, the EPS overproducing variants performed as similar as the parental strain. Therefore, EPS overproduction did not confer an apparent advantage to A. brasilense in terms of induction of plant growth promotion.     

Isolation,partial identification and application of diazotrophic rhizobacteria from traditional Indian rice cultivars

A diversity of N₂-fixing (diazotrophic) bacteria was isolated from two traditional rice cultivars, Sataria and Kartiki, from the rice growing area of Mithila region of North Bihar, India, where low levels of nitrogen fertilizers are applied. Nitrogen-free semisolid media NFb, JMV and LGI with different carbon sources and pH-values were used for enrichment and isolation of root-associated diazotrophs. The colonization density of roots by diazotrophs, as estimated from positive pellicle formation at highest dilution in nitrogen-free enrichment media, was 10⁶–10⁸ diazotrophic bacteria per g fresh root weight. Roots of the cultivar Kartiki were found to be more densely colonized endophytically by diazotrophs as detected after chloramine T (1%) surface disinfection. To ascertain the phylogenetic affiliation of the isolates, phylogenetic oligonucleotide probes and the Fluorescent in situ Hybridization (FISH) technique were applied. Using group-specific rRNA directed oligonucleotide probes, the majority of the isolates could be identified as alpha-, beta-, or gamma-proteobacteria. Using 16S and 23S rRNA-directed genus- or species-specific probes, Herbaspirillum seropedicae, Azospirillum amazonense, Burkholderia cepacia/vietnamiensis, Rhizobia and Pseudomonas spp. were found to be the most prominent root associated culturable diazotrophs. Diazotrophic Gluconacetobacter spp. were also demonstrated as colonizers of rice roots. Burkholderia cenocepacia, Pseudomonas sp. and three diazotrophic PGPR reference strains were used for the inoculation of axenically grown rice seedlings to determine the plant growth promoting potential. Significant increases in the shoot length (up to 60%), shoot dry weight (up to 33%) and the grain yield (up to 26%) per plant were observed in non-axenic pot and field trials. Using semisolid enrichment media after surface sterilization of field grown inoculated rice roots and oligonucleotide probing of the diazotrophic enrichment cultures, a sustainable colonization with the inoculated bacteria could be demonstrated.     

不同接种浓度对野油菜黄单胞菌野油菜致病变种胞外多糖突变体致病性的影响

利用转座子 Tn5gus A5诱变和标记置换方法 ,构建了野油菜黄单胞菌野油菜致病变种( Xanthomonas campestris pv.campestris)系列标记置换突变体 ,其中 6个突变体的胞外多糖产量显著降低。致病试验结果表明 ,胞外多糖产量对 Xcc的致病性有明显的影响 ,不同接种浓度对 Xcc胞外多糖突变体致病性亦有明显影响。     

On the extent to which root properties and transport through the soil limit nitrogen uptake by lowland rice

Measurements of N uptake by rice plants growing in a puddled flooded soil and the corresponding changes in soil solution NH: concentrations and the soil NH: diffusion coefficient are used to calculate how far root uptake properties and transport to the roots limit acquisition of N by rice. With root uptake parameters assigned values such that influx was maximal within realistic ranges, the minimum root length densities required to explain uptake were similar to measured root length densities in both N-fertilized and unfertilized soil. This suggests that most if not all of the root length was active in uptake and that uptake per unit root length was near maximal. A sensitivity analysis showed that the necessary minimum root length is very sensitive to root uptake properties within appropriate ranges. Transport to the roots was mainly by diffusion. Rates of diffusion will generally not limit uptake in well-puddled soils, but they may greatly limit uptake in puddled soils that have been drained and re-flooded and in unpuddled flooded soils. Uptake of fertilizer N broadcast into rice field floodwater and absorbed by roots in the floodwater or soil near the floodwater is not likely to be limited by root uptake properties or transport.     

硒(Ⅳ)预处理下根表铁膜对水稻幼苗吸收和转运汞的影响

采用水培试验的方法研究硒(Se,Ⅳ)预处理下,根表铁膜对水稻幼苗吸收和转运汞(Hg)的影响。将水稻幼苗置于Se0和Se0.5(mg L-1)培养液中培养2周,再用4种不同浓度的Fe2+溶液(0、25、50和100 mg L-1即Fe0、Fe25、Fe50、Fe100)诱导水稻根表形成不同数量的铁膜,随后置于0.3 mg L-1的Hg Cl2培养液中继续培养72 h。结果表明,根表铁膜对水稻幼苗生长无显著影响,但硒可以增加其生物量。碳酸氢钠―柠檬酸三钠―连二亚硫酸钠(DCB)提取液(即根表铁膜)中含铁比例(57.3%~96.2%)显著高于水稻幼苗地上部(1.1%~17.5%)和根部(2.7%~25.9%),水稻幼苗的大部分铁被积累至DCB提取液中。随着根表铁膜数量的增加,根和地上部汞含量均显著降低。在Fe50和Fe100处理中,硒的加入显著减少了地上部和根部的汞含量,也显著降低了汞的分配系数,Se(Ⅳ)预处理能明显提高铁膜固持汞的量。综上所述,Se(Ⅳ)预处理和根表铁膜均能阻碍水稻幼苗对汞的吸收和向地上部的转运,减轻水稻汞胁迫,从而起到保护水稻避免汞毒害的作用。本研究对于提高汞污染区稻米质量和保证粮食安全具有一定的现实意义。