影响根瘤菌竞争结瘤的生态学因素分析

根瘤菌的发现并确证其共生固氮作用已逾100年,根瘤菌剂的制备和应用也已超过半个世纪,实践效果有目共睹。如美国对豌豆根瘤菌、三叶草根瘤菌和大豆根瘤菌的应用以及澳大利亚对三叶草根瘤菌的应用都取得显著成绩。我国在豆科作物和豆科绿肥上应用根瘤菌接种措施已有30余年历史,采用筛选的优良菌     

Number of Rhizobia and delayed inoculation influence nodulation of clovers

The relationship between numbers of rhizobia and nodulation response of legumes is of considerable practical importance. Experiments were done under controlled conditions to determine the influence of numbers of Rhizobium leguminosarum biovar. trifolii on nodulation of arrowleaf clover (Trifolium vesiculosum Savi.) and crimson clover (T. incarnatum L.). Numbers of rhizobia in excess of 1000 per seed did not substantially increase earliness of nodulation or total number of nodules formed on the taproot. Nodules, however, were formed nearer the top of the taproot as numbers of rhizobia increased to 100,000 per seed. Delayed inoculation experiments indicated that nodulation sites for these clovers only remained susceptible to infection for less than 1 day. Delaying inoculation for 4 days resulted in only a 1 to 2 day delay in nodulation for arrowleaf and crimson clovers respectively and no delay for subterranean clover (T. subterraneum L.). Apparently, larger seedlings nodulated faster.     

草木樨属根瘤菌的16S rDNA-RFLP分析

采用16S rDNA-RFLP分析方法对草木樨属根瘤菌进行了遗传多样性及系统分类研究。结果表明,3种限制性内切酶(HaeⅢ、HinfⅠ、MspⅠ)对所有供试菌株的酶切图谱类型组合只有2种。类型I的代表菌株CC-NWSX0003-1与豌豆根瘤菌(R.leguminosarum)的模式菌株USDA2370的序列相似性达到99.8%,在分类地位上属于根瘤菌属(Rhizobium)分支;类型Ⅱ的代表菌株CCNWGS0006与草木樨中华根瘤菌(Sinorhizobium meliloti)的16S rDNA相似性为100%,属于中华根瘤菌属(Sinorhizobium)分支。     

外源质粒（基因）导入花生根瘤菌的行为分析

利用二亲本或三亲本杂交的方法,将携带有共生固氮基因的外源重组质粒或外源载体质粒导入慢生型花生根瘤菌［Ｂｒａｄｙｒｈｚｏｂｉｕｍｓｐ．（Ａｒａｃｈｉｓ）］１４７－３和快生型花生根瘤菌［Ｒｈｉｚｏｂｉｕｍｓｐ．（Ａｒａｃｈｉｓ）］８５－７中。探讨了转移接合子中外源质粒在人工培养条件下和共生条件下的稳定性,发现外源质粒在花生根瘤菌中的稳定性与质粒的类型、受体菌的特性和环境条件有关。同时还探讨了外源质粒上的共生基因对受体菌１４７－３共生固氮效率的影响。结果表明,外源共生基因对共生固氮能力的影响是复杂的,既可以产生正效应,也可以产生负效应。     

Effects of Co-Inoculation with Arbuscular Mycorrhizal Fungi and Rhizobia on Fungal Occupancy in Chickpea Root and Nodule Determined by Real-Time PCR

Legume roots in nature are usually colonized with rhizobia and different arbuscular mycorrhizal fungi (AMF) species. Light microscopy that visualizes the presence of AMF in roots is not able to differentiate the ratio of each AMF species in the root and nodule tissues in mixed fungal inoculation. The purpose of this study was to characterize the dominant species of mycorrhiza in roots and nodules of plants co-inoculated with mycorrhizal fungi and rhizobial strains. Glomus intraradices (GI), Glomus mosseae (GM), their mix (GI + GM), and six Mesorhizobium ciceri strains were used to inoculate chickpea. Quantitative real-time polymerase chain reaction (qRT-PCR) was used to assess occupancy of these fungal species in roots and nodules. Results showed that GI molecular ratio and relative density were higher than GM in both roots and nodules. These differences in molecular ratio and density between GI and GM in nodules were three folds higher than roots. The results suggested that M. ciceri strains have different effects on nodulation and mycorrhizal colonization pattern. Plants with bacterial S3 and S1 strains produced the highest root nodulation and higher fungal density in both the roots and nodules.     

光敏生物素标记总DNA探针对大豆根瘤菌的检测

以光敏生物素标记慢生型大豆根瘤菌(Bradyrhizobium japonicum)USDA110总DNA作为探针,与快生型大豆根瘤菌杂交时,没有杂交斑点形成,而与慢生型大豆根瘤菌中的部分菌株能形成杂交斑点,表明该探针具有种和部分菌株特异性,用该探针与压碎的根瘤汁液进行DNA杂交,检测USDA110在不灭菌的盆栽土壤中的竞争结瘤能力,发现USDA110在大豆不同生育期的占瘤率为70%～90%。     

Isolation of mutants of fast-growing soybean strains that are effective on commercial soybean cultivars

Fast-growing rhizobia that nodulate soybeans ( Glycine max L. cvs Peking and Malayan) have been isolated. Initial inoculation of commercial cultivars of soybean with these strains results in the formation of a few pink nodules. Isoiates from these nodules form fully effective symbiosis with the soybean cultivar from which they were isolated. These results demonstrate that is is possible to isolate effective mutants of fast-growing soybean strains by allowing the plants to select for effective mutants. These mutants are potentially valuable because, if their symbiotic properties can be improved, they could possibly be used as commercial soybean inoculants.     

Evolving ideas of legume evolution and diversity: a taxonomic perspective on the occurrence of nodulation

Legumes evolved about 60 million years ago (Ma), and nodulation 58 Ma. Nonnodulation remains common in Caesalpinioideae, with smaller numbers in Mimosoideae and Papilionoideae. The first type of infection by bacteria may have been at junctions where lateral roots emerged, followed by formation of infection threads to confine bacteria and convey them to some cells in the developing nodule, where they were generally released into symbiosomes. Infection threads were a prerequisite for root-hair infection, a process better controlled by the host, leading to a higher degree of specificity between symbionts. An alternative process, dating from the same time and persisting in about 25% of legumes, did not involve infection threads, bacteria entering a few host cells, surrounded by an undefined matrix. These cells divided repeatedly to give uniform infected tissue, with bacteria released into symbiosomes. Such legumes may have less stringent control of nodulation processes, and are found mainly in tropical and warm temperate areas. In each type of nodule, meristems may or may not be retained, leading to indeterminate or determinate forms. Nodule morphology and structure are host-determined, but the effectiveness of nitrogen fixation is largely controlled by the bacterial symbionts, which vary greatly in genotypic and phenotypic characters.     

Burkholderia species are ancient symbionts of legumes

Burkholderia has only recently been recognized as a potential nitrogen-fixing symbiont of legumes, but we find that the origins of symbiosis in Burkholderia are much deeper than previously suspected. We sampled 143 symbionts from 47 native species of Mimosa across 1800 km in central Brazil and found that 98% were Burkholderia . Gene sequences defined seven distinct and divergent species complexes within the genus Burkholderia . The symbiosis-related genes formed deep Burkholderia -specific clades, each specific to a species complex, implying that these genes diverged over a long period within Burkholderia without substantial horizontal gene transfer between species complexes.     

MtMOT1.2 is responsible for molybdate supply to Medicago truncatula nodules

Symbiotic nitrogen fixation in legume root nodules requires a steady supply of molybdenum for synthesis of the iron‐molybdenum cofactor of nitrogenase. This nutrient has to be provided by the host plant from the soil, crossing several symplastically disconnected compartments through molybdate transporters, including members of the MOT1 family. Medicago truncatula Molybdate Transporter (MtMOT) 1.2 is a Medicago truncatula MOT1 family member located in the endodermal cells in roots and nodules. Immunolocalization of a tagged MtMOT1.2 indicates that it is associated to the plasma membrane and to intracellular membrane systems, where it would be transporting molybdate towards the cytosol, as indicated in yeast transport assays. Loss‐of‐function mot1.2‐1 mutant showed reduced growth compared with wild‐type plants when nitrogen fixation was required but not when nitrogen was provided as nitrate. While no effect on molybdenum‐dependent nitrate reductase activity was observed, nitrogenase activity was severely affected, explaining the observed difference of growth depending on nitrogen source. This phenotype was the result of molybdate not reaching the nitrogen‐fixing nodules, since genetic complementation with a wild‐type MtMOT1.2 gene or molybdate‐fortification of the nutrient solution, both restored wild‐type levels of growth and nitrogenase activity. These results support a model in which MtMOT1.2 would mediate molybdate delivery by the vasculature into the nodules.     

Effects of Oxygen Concentration and Leghemoglobin on Organic Acid Degradation by Isolated Soybean Nodule Bacteriods

Bacteroids retaining high acetylene reduction activity (nitrogenase activity) were prepared anaerobically from soybean nodules. Addition of succinate (or of both leghemoglobin and succinate) to the acetylene reduction assay system greatly increased the activity of the isolated bacteroids.

When various organic acids were incubated with the bacteroids at 2% oxygen concentration, an optimum condition for bacteroid acetylene reduction, the organic acid degradation by bacteroids was very slow, and both lactate and acetate were accumulated in the incubation system, suggesting the operation of fermentative pathway in bacteroids under such low oxygen conditions.

With 20% oxygen, the added organic acids were degraded rapidly by bacteroids without addition of leghemoglobin to the incubation system.

With leghemoglobin in the incubation system, the organic acid degradation by bacteroids was accelerated extensively even at 2% oxygen, and the formation of lactate and acetate were negligible. No significant difference in the organic acid degradation rate was observed between the 2% and 20% oxygen concentrations when the leghemoglobin was present in the incubation system. Addition of acetylene to the assay system slightly inhibited the organic acid degradation.

This data suggests that bacteroids are unable to oxidize organic acid in low oxygen concentration and that the leghemoglobin allows the rapid organic acid dagradation by bacteroids even in such low oxygen concentrations.     

Nodule phosphoenolpyruvate carboxylase: a review

Recent data concerning the fixation of CO₂ and the functioning of phosphoenolpyruvate (PEP) carboxylase (EC 4.1.1.31) in legume, nodules are reviewed. The activites of N₂ fixation (acctylene reduction) and PEP carboxylase are correlated Activities of PEP carboxylase are always higher in nodules than in roots. PEP carboxylase is located in the cytosol of the plant part of the nodules. When nodules are fed with ¹⁴CO₂, radioactivity appears predominantly in malate and aspartate. The resolution of isoenzymes of PEP carboxylase shows one more band in nodules than in related roots. The role of PEP carboxylase in nodule metabolism is discussed.     

用电脉冲法将外源质粒DNA导入花生根瘤菌的研究

利用基因电转移仪(Gene Pulser^TM|),对快、慢生型花生根瘤菌85-7和1 47-3的电转化条件进行了系统的研究。结果表明:在对数中期(ABC~600|为0.6-0.7)收获的细胞在最高场强(12.5kV/cm)和短脉冲时间(2.5-5.0m sec)时达到最高转化效率(10 E5转化子/μgDNA)。转化子数随DNA终浓度在一定范围(26.24pg-1.5μg/ml)内呈线性增加, 随即表现出“饱和效应”。增加受体菌细胞浓度,能提高电转化效率;而质粒分子量的增加(11.9 -166kb)却使电转化效率降低。来自受体菌自身的同源质粒,因克服了宿主的限制-修饰性,可以极显著地提高电转化效率。电脉冲处理对受体菌自发突变没有影响。 Abstract:A systematic study of transformation conditions of peanut rhizobia 85-7 and 147-3 was conducted by using Bio-Rad Gene Pulser equipment.It was revealed that the highest transformation efficiency(105 transformants/μgDNA) was obtained from cells harvested at mid-log phase of ABC600 on 0.6-0.7 under the highest field strength(12.5kV/cm)and a short pulse length(2.5-5.0 msec).A linear increase of transformants was observed when DNA concentration was increased in the range of 26.24pg-1.5μg/ml and it became saturation afterwards.Transformation efficiency was also increased with the raise of recipient cell concentrations,but decreased with the increase of plasmid sizes from 11.9 to 166kb.A significant increase of transformation efficiency was revealed with the homologous plasmid isolated from fecipient itself since the effects of host restriction and modification were avoided.No significant effect of electroporation on spontaneous mutation was observed.     

Genetic allelism and linkage tests of a soybean gene,Rfg1, controlling nodulation with Rhizobium fredii strain USDA 205

A soybean gene, Rfg1, controlling nodulation with strain USDA 205, the type strain for the fast-growing species Rhizobium fredii, was tested for allelism with the Rj4 gene. The Rj4 gene conditions ineffective nodulation primarily with certain strains of the slow-growing soybean microsymbiont, Bradyrhizobium elkanii. The F2 seeds of the cross of the cultivars Peking, carrying the alleles rfg1, Rj4, i (controlling inhibition of seed coat color) and W1 (controlling flower color), and Kent, carrying the alleles Rfg1, rj4, i-i and w1, were evaluated for nodulation response with strain USDA 205 by planting surface disinfested seeds in sterilized vermiculite in growth trays and inoculating with a stationary phase broth culture of strain USDA 205 at planting. Plants were classified for nodulation response visually after four weeks growth and transplanted to the field for F3 seed production. Flower color, purple (W1) vs white (w1), was determined in the field. The allele present at the i locus was determined by classification of F3 seed coat color. The F3 seeds were planted in growth trays and inoculated with strain USDA 61 of Bradyrhizobium elkanii to determine the genotype for the Rj4 locus. The Rfg1 and Rj4 genes were determined to be located at separate loci. Chi-square analysis for linkage indicated that Rfg1 segregated independently of the Rj4, I and W1 loci.     

利用发光酶基因监测根瘤菌重组质粒的转移性

经三亲本杂交,比较测定了重组大豆根瘤菌HN01DNL和TA11DNL中所含重组质粒pHN307在人工滤膜和灭菌土壤杂交条件下、向华癸中生根瘤菌7653R和荧光假单胞菌Pf.X1-5的转移频率;并初步跟踪了pHN307在根盒-土壤缩影、小区试验和环境释放中向土著细菌的转移性,为考察所构建重组根瘤菌在田间应用时的安全性提供了一定的实验依据。     

Nodule growth and activity may be regulated by a feedback mechanism involving phloem nitrogen*

We present a mechanism of regulation of growth and activity of legume root nodules which is consistent with published experimental observations. The concentration of reduced nitrogen compounds, probably amino acids, flowing into the nodules from the phloem, is sensed by the nodules; growth and activity of the nodules is adjusted accordingly. In many legumes this response may involve changes in the oxygen diffusion resistance of the nodule cortex. A straightforward feedback mechanism in which nodule activity is lowered when reduced N in the phloem is high and increased when it is low is envisaged. Almost all import into nodules is via the phloem sap originating in the lower leaves. As a plant develops, these mature leaves no longer utilize nitrogen delivered in the xylem and so export it in the phloem. In plants with an adequate nitrogen supply (from nodules or combined nitrogen in soil), a high concentration of nitrogen containing compounds in the phloem from the lower leaves may inhibit nodule growth as well as activity. This suggestion is an alternative to the hypotheses of carbohydrate deprivation or nitrate inhibition which are commonly used to explain the effects of combined nitrogen on nodule growth and activity.