厚荚相思根瘤菌对盆栽苗木生长及土壤肥力的影响

从不同立地条件采集到的厚荚相思根瘤分离出9株根瘤菌株,接种于厚荚相思幼茁,6个月后测定苗木生长量及土壤N、P、K含量。试验结果表明：接菌桃株的高生长量、生物量都比对照(未接种根瘤菌)增加,其巾高生长量除R2外,增加幅度为0．89～40．18％,生物量增加52．33～147．67％,接种结瘤率、瘤的生物量也比对照高,结瘤率可达100％,根瘤生物量比对照高163．6～520％。土壤N、P、K元素分析结果表明,接种根瘤菌后土壤的营养元素发生了很大的变化,改善了土壤肥力。通过对接种不同菌株的厚荚相思苗木的茁高、生物量、土壤的全氮、速效磷、速效钾等元素的含量比较分析后筛选出R6和R7菌株为优良菌株。     

优良大豆根瘤菌田间增产效果初报

我所将中国农科院土肥所生物固氮组从佳木斯地区土壤中根瘤分离的快。慢型大豆根瘤菌,从中筛选出的优良菌株２１７８、２１８７及其丸衣、２２３５、混合菌等接种于当地主载大豆品种合丰２５号、３１号、３３号上,表现出较高的田间共生效应,能促进生育、增加根瘤数和固氮量,产量两年比对照增产幅度在９．２—２５．４％,比６１Ａ７６增产４．６—１９．５％。     

果胶菌加根瘤菌诱发油菜结瘤固氮的研究

用果胶细菌(pectin—lytic bacterium)处理油菜幼根,使根瘤菌(Bradyrhizobium spp.Arachis)侵入油菜并结瘤.试验表明,根瘤菌侵入油菜根细胞,且形成含根瘤菌的根瘤,结瘤率为31.25%。从这些根瘤中分离出的菌株,成功地回接到原寄主之上;血清学方法测定油菜根瘤分离物与原接种的根瘤菌起凝集反应。根瘤石蜡包埋切片观察,结果是根瘤细胞内充满了细菌。乙炔还原法测定出低固氮酶活性(0.17—3.43nmC_2H_4/株/小时);酰脲分析结果其相对酰脲丰度为0.1—3.58%。     

大豆血红蛋白基因lba转化根瘤菌工程菌株的构建

以土著大豆根瘤菌接种大豆幼苗45 d后获得的根瘤为材料,提取其总RNA并反转录成cDNA,采用同源序列克隆法扩增大豆血红蛋白基因lba编码区序列。利用DNA重组技术,将lba基因连到lac启动子的下游,利用带有发光酶标记基因luxAB的质粒载体pTR102构建表达载体pTR-Plac-lba。采用三亲本杂交的方式,将表达载体pTR-Plac-lba及作为对照的空载体pTR102分别转化土著大豆根瘤菌,获得根瘤菌工程菌株SFH(pTR-Plac-lba)和SFH(pTR102)。盆栽试验发现,接种SFH(pTR-Plac-lba)的大豆植株各生理指标明显高于接种SFH(pTR102)、土著根瘤菌以及未接菌的大豆植株各生理指标。试验证明,导入大豆血红蛋白基因lba的根瘤菌工程菌株SFH(pTR-Plac-lba)对于提高大豆根瘤的固氮酶活性,增加大豆产量起到显著效果。     

一株能在大豆上结瘤的苜蓿中华根瘤菌

苜蓿中华根瘤菌(Sinorhizobium meliloti)XJ96077分离自新疆的苜蓿根瘤中,其原宿主为紫花苜蓿(Medicago sativa)。交叉结瘤试验发现,它既可在苜蓿上又能在大豆上结瘤固氮。DNA(G C)mol％分析表明,XJ96077的DNA(G C)mol％为61．9％,与已报道的根瘤菌属的DNA(G C)mol％范围(59％-64％)相符。DNA同源性分析表明,XJ96077与苜蓿中华根瘤菌USDA1002^T和042BM的同源性分别达到93％和80％,说明XJ96077归属于苜蓿中华根瘤菌。应用绿色荧光蛋白基因标记XJ96077,得到重组菌株XJ96077(G)。将其接种普通紫花苜蓿,通过激光共聚焦荧光显微镜可以检测到标记基因的表达。接种北引1号大豆上,同样可以清楚地观察到标记基因在根瘤中的表达,从而确证了XJ96077能同时在苜蓿和大豆上结瘤。通过不同品种大豆的结瘤试验,发现XJ96077对大豆品种的结瘤能力不同。     

花生根瘤菌Bradyrhizobium sp.MM6 Ⅲ型分泌系统的结构和功能

【目的】探究花生根瘤菌Bradyrhizobium sp. MM6的Ⅲ型分泌系统(T3SS)的结构及其在根瘤菌与不同宿主建立共生关系中的作用。【方法】同源比对分析菌株MM6的T3SS基因簇的结构特征,并采用三亲本接合转移的方法构建T3SS调节基因ttsI突变菌株;通过蛭石结瘤和石蜡切片实验,比较突变体与野生型的共生固氮表型差异。【结果】经预测,MM6的T3SS基因簇编码区长约34.1 kb,可分为3个区域,包含10个保守结构基因和8个效应蛋白基因,与B. diazoefficiens USDA110相应基因的序列相似性为83%–93%;成功构建了MM6的ttsI突变株;ttsI突变株与野生型分别与花生(S523和Y45)、野大豆和大豆中黄57结瘤,ttsI突变体在花生中的总瘤数显著增加(P0.05),根瘤中含菌细胞更多;ttsI突变体在野大豆中平均每株植物增加4个根瘤,根瘤中含菌细胞更多,地上部干重相比野生型MM6显著增加(P0.05);在大豆中黄57中,野生型MM6能形成红色的有效根瘤,ttsI突变体不结瘤,且植株叶片发黄,地上部干重相比野生型MM6显著降低(P0.05)。【结论】MM6的T3SS在花生和野大豆共生体系中起着有害的作用,而在大豆中黄57的共生体系中起着有利的作用。     

花生根瘤菌Bradyrhizobium sp.MM6 Ⅲ型分泌系统的结构和功能

【目的】探究花生根瘤菌Bradyrhizobium sp.MM6的Ⅲ型分泌系统(T3SS)的结构及其在根瘤菌与不同宿主建立共生关系中的作用。【方法】同源比对分析菌株MM6的T3SS基因簇的结构特征,并采用三亲本接合转移的方法构建T3SS调节基因ttsI突变菌株;通过蛭石结瘤和石蜡切片实验,比较突变体与野生型的共生固氮表型差异。【结果】经预测,MM6的T3SS基因簇编码区长约34.1 kb,可分为3个区域,包含10个保守结构基因和8个效应蛋白基因,与B.diazoefficiens USDA110相应基因的序列相似性为83%–93%;成功构建了MM6的ttsI突变株;ttsI突变株与野生型分别与花生(S523和Y45)、野大豆和大豆中黄57结瘤,ttsI突变体在花生中的总瘤数显著增加(P<0.05),根瘤中含菌细胞更多;ttsI突变体在野大豆中平均每株植物增加4个根瘤,根瘤中含菌细胞更多,地上部干重相比野生型MM6显著增加(P<0.05);在大豆中黄57中,野生型MM6能形成红色的有效根瘤,ttsI突变体不结瘤,且植株叶片发黄,地上部干重相比野生型MM6显著降低(P<0.05)。【结论】MM6的T3SS在花生和野大豆共生体系中起着有害的作用,而在大豆中黄57的共生体系中起着有利的作用。     

接种S.fredii WGF03及突变体对大豆土壤微生物群落结构的影响

为了研究接种S.fredii WGF03及其exo D基因突变体对大豆结瘤及土壤的微生物群落影响,进一步了解exo D基因的功能,在大豆盛花期摘取每株大豆的根瘤并计数,利用PCR-DGGE电泳结合测序技术分析土壤的微生物群落。结果表明,大豆接种S.fredii WGF03后,根瘤数比不接种组增加191.67%。而大豆接种驻exo D突变体的根瘤数最少,比不接种组减少了16.67%。与空白相比,种植大豆后土壤细菌的种类和数量明显增加;接种不同根瘤菌后,细菌种类及细菌多样性也有变化;测序结果显示,土壤中细菌以Proteobacteria为主,占45.5%,土壤中土著根瘤菌为Bradyrhizobium。总之,S.fredii WGF03能够促进大豆结瘤,种植作物比接种根瘤菌对土壤细菌群落的影响更大。     

用AFLP技术检测慢生型花生根瘤茵竞争结瘤的研究

以5株慢生型花生根瘤菌和天府3号花生为材料,用AFLP技术研究了慢生型花生根瘤菌Spr2—9、Spr3—3、Spr3—5、Spr4—5和Spr7—1的遗传特性和竞争结瘤能力。结果显示,供试条件下,传代次数对菌株的遗传性状无明显影响,28C培养条件下,花生根瘤菌连续传96代,其AFLP指纹未发生明显变化;37C培养,仅Spr3—3和Spr3—5能够存活并正常生长,其AFLP指纹也未发生明显改变,然而其它菌株不能生长。将供试慢生型花生根瘤菌分别接种天府3号花生。光照培养30d后,随机各取4个根瘤,从根瘤中提取类菌体DNA进行AFLP分析,各根瘤类菌体DNA的AFLP指纹图谱与该菌株纯培养物AFLP指纹相同。将5个菌株混合接种天府3号花生,不同菌株的占瘤率存在差异,Spr3—3和Spr3—5的竞争结瘤能力最强,两菌株的占瘤率之和为85．4％;Spr4—5的占瘤率为12．2％;Spr7—1为2．4％;而Spr2—9的竞争结瘤能力最差。本试验结果说明,AFLP技术用于根瘤菌生态和竞争结瘤能力研究,具有下列优点：简易、快速、准确;直接取豆科植物的根瘤提取DNA,进行原位研究;在不改变菌株遗传特性,即不使用突变株的前提下,可以直接测定已知菌株的竞争结瘤能力。     

大豆-根瘤菌共生体系光合与固氮关系研究

水培大豆和田间生长的大豆,接种根瘤菌 Rhizobium B16-11C 后植株全氮含量、叶片叶绿素含量和净光合速率及种子产量都明显增加。比较 Clark 大豆的结瘤品系和不结瘤品系获类似结果。摘除根瘤后3天内叶片净光合速率无明显变化。大豆植株遮阴、去叶或切掉地上部导致根瘤活性明显下降。但去豆荚不能提高根瘤固氮的比活性。根瘤活性的日变化不能用根瘤蔗糖、淀粉含量或周围温度的变化来解释,其控制因子尚待深入研究。     

Hydrogen evolution and uptake by nodules of soybeans inoculated with different strains of Rhizobium japonicum

Hydrogen evolved by nitrogenase may be recycled by a hydrogenase present in some legume nodules. Anoka and Portage cultivars of soybeans were inoculated with each of 8 and 24 strains, respectively, of Rhizobium japonicum and surveyed for H2 evolution and C2H2 reduction rates nodule weight, and plant dry weight. Six of the strains (3Ilb 110, USDA 122, USDA 136, 3Ilb 6, 3Ilb 142, and 3Ilb 143) which exhibited no H2 evolution in air were shown to take up H2. The relative efficiencies of nitrogenase energy utilization based on C2H2 reduction rates of nodules relative efficiences of nitrogenase energy utilization based on C2H2 reduction rates of nodules ranged from 0.96 to 1.0 for the six strains. Nodules formed by strain WA 5099-1-1 evolved small amounts of H2 in air and had a relative efficiency of 0.92. Nodules formed by the remaining 25 strains had relative efficiencies ranging from 0.41 to 0.80. A H2-evolving (3Ilb 123) and non-H2-evolving (3Ilb 143) strain were tested on seven soybean cultivars to determine the effect on the expression of hydrogenase. Nodules formed by strain 3Ilb 143 exhibited an efficiency of 1.0 on the following cultivars: Amsoy 71, Anoka, Bonus, Clark 63, Kent, Peking, and Portage. Relative efficiencies from 0.63 to 0.77 were determined for the five cultivars nodulated by strain 3Ilb 123. From the experiments with these cultivars, the capacity to recycle H2 produced from the nitrogenase system appears to be determined by the R. japonicum strain.     

Effect of Rhizobium sp. BARIRGm901 inoculation on nodulation,nitrogen fixation and yield of soybean (Glycine max) genotypes in gray terrace soil

Soybean plants require high amounts of nitrogen, which are mainly obtained from biological nitrogen fixation. A field experiment was conducted by soybean (Glycine max) genotypes, growing two varieties (Shohag and BARI Soybean6) and two advanced lines (MTD10 and BGM02026) of soybean with or without Rhizobium sp. BARIRGm901 inoculation. Soybean plants of all genotypes inoculated with Rhizobium sp. BARIRGm901 produced greater nodule numbers, nodule weight, shoot and root biomass, and plant height than non-inoculated plants. Similarly, inoculated plants showed enhanced activity of nitrogenase (NA) enzyme, contributing to higher nitrogen fixation and assimilation, compared to non-inoculated soybean plants in both years. Plants inoculated with Rhizobium sp. BARIRGm901 also showed higher pod, stover, and seed yield than non-inoculated plants. Therefore, Rhizobium sp. BARIRGm901 established an effective symbiotic relationship with a range of soybean genotypes and thus increased the nodulation, growth, and yield of soybean grown in gray terrace soils in Bangladesh.     

Strain selection for improvement of Bradyrhizobium japonicum competitiveness for nodulation of soybean

A Bradyrhizobium japonicum USDA 110-derived strain able to produce wider halos in soft-agar medium than its parental strain was obtained by recurrent selection. It was more chemotactic than the wild type towards mannitol and three amino acids. When cultured in minimal medium with mannitol as a single carbon-source, it had one thick subpolar flagellum as the wild type, plus several other flagella that were thinner and sinusoidal. Root adsorption and infectivity in liquid media were 50-100% higher for the selected strain, but root colonization in water-unsaturated vermiculite was similar to the wild type. A field experiment was then carried out in a soil with a naturalized population of 1.8 x 10(5) soybean-nodulating rhizobia g of soil(-1). Bradyrhizobium japonicum strains were inoculated either on the soybean seeds or in the sowing furrows. Nodule occupation was doubled when the strains were inoculated in the sowing furrows with respect to seed inoculation (significant with P<0.05). On comparing strains, nodule occupation with seed inoculation was 6% or 10% for the wild type or selected strains, respectively, without a statistically significant difference, while when inoculated in the sowing furrows, nodule occupation increased to 12% and 22%, respectively (differences significant with P<0.05).     

Expression of nir, nor and nos denitrification genes from Bradyrhizobium japonicum in soybean root nodules

Expression of Bradyrhizobium japonicum wild-type strain USDA110 nirK , norC and nosZ denitrification genes in soybean root nodules was studied by in situ histochemical detection of β -galactosidase activity. Similarly, P_nirK- lacZ , P_norC- lacZ , and P_nosZ- lacZ fusions were also expressed in bacteroids isolated from root nodules. Levels of β -galactosidase activity were similar in both bacteroids and nodule sections from plants that were solely N₂-dependent or grown in the presence of 4 m M KNO₃. These findings suggest that oxygen, and not nitrate, is the main factor controlling expression of denitrification genes in soybean nodules. In plants not amended with nitrate, B. japonicum mutant strains GRK308, GRC131, and GRZ25, that were altered in the structural nirK , norC and nosZ genes, respectively, showed a wild-type phenotype with regard to nodule number and nodule dry weight as well as plant dry weight and nitrogen content. In the presence of 4 m M KNO₃, plants inoculated with either GRK308 or GRC131 showed less nodules, and lower plant dry weight and nitrogen content, relative to those of strains USDA110 and GRZ25. Taken together, the present results revealed that although not essential for nitrogen fixation, mutation of either the structural nirK or norC genes encoding respiratory nitrite reductase and nitric oxide reductase, respectively, confers B. japonicum reduced ability for nodulation in soybean plants grown with nitrate. Furthermore, because nodules formed by each the parental and mutant strains exhibited nitrogenase activity, it is possible that denitrification enzymes play a role in nodule formation rather than in nodule function.     

Phloem Glutamine and the Regulation of O2 Diffusion in Legume Nodules

The aim of the present study was to test the hypothesis that the N content or the composition of the phloem sap that supplies nodulated roots may play a role in the feedback regulation of nitrogenase activity by increasing nodule resistance to O2 diffusion. Treating shoots of lupin (Lupinus albus cv Manitoba) or soybean (Glycine max L. Merr. cv Maple Arrow) with 100 [mu]L L-1 NH3 caused a 1.3-fold (lupin) and 2.6-fold (soybean) increase in the total N content of phloem sap without altering its C content. The increase in phloem N was due primarily to a 4.8-fold (lupin) and 10.5-fold (soybean) increase in the concentration of glutamine N. In addition, there was a decline in both the apparent nitrogenase activity and total nitrogenase activity that began within 4 h and reached about 54% of its initial activity within 6 h of the start of the NH3 treatment. However, the potential nitrogenase activity values in the treated plants were not significantly different from those of the control plants. These results provide evidence that changes in the N composition of the phloem sap, particularly the glutamine content, may increase nodule resistance to O2 diffusion and, thereby, down-regulate nodule metabolism and nitrogenase activity by controlling the supply of O2 to the bacteria-infected cells.     

Competition of Rhizobium japonicum Strains in Early Stages of Soybean Nodulation

The effects of preexposure of soybean (Glycine max L. Merrill) roots to Rhizobium japonicum strains and subsequent establishment of other strains in the nodules were investigated by using combinations of effective strains (USDA 110 and USDA 138) and effective-ineffective strains (USDA 110 and SM-5). Strain USDA 110 was a better competitor than either USDA 138 or SM-5 on cultivars Lee and Peking. However, when either of the two less-competitive strains was inoculated into 2-day-old seedlings before USDA 110 was, their nodule occupancy increased significantly on both cultivars. With USDA 138 as the primary inoculum and USDA 110 delayed for 6, 48, and 168 h, the incidence of USDA 138 nodules increased on cultivar Peking from 6% (at zero time) to 28, 70, and 82% and on cultivar Lee from 17% (at zero time) to 32, 88, and 95% for the three time delays, respectively. Preexposure of 2-week-old roots of cultivar Lee to USDA 138 had essentially the same effect: the incidence of USDA 138 nodules increased from 23% at zero time to 89 and 97% when USDA 110 was delayed for 24 and 72 h, respectively. When the ineffective strain SM-5 was used as the primary inoculum, followed by USDA 110 72 h later, the percentage of nodules containing SM-5 increased from 7 to 76%. These results indicate that the early events in the nodulation process of soybeans are perhaps the most critical for competition among R. japonicum strains.     

Symbiotic properties of antibiotic-resistant mutants of Rhizobium galegae

Mutagenesis provoked by exposure to increased concentration of antibiotics of five indigenous Rhizobium galegae strains resulted in the generation of several antibiotic-resistant mutants. The mutants differed from the wild type and one from another in respect to the nodulation capacity, the nitrogenase activity, the nodule ultrastructure, and the plant growth response. Galega plants inoculated with mutants resistant to streptomycin and rifampicin formed nodules with higher nitrogenase activity and accumulated more shoot dry biomass than plants inoculated with the parent strains. Resistance to kanamycin and nalidixic acid was associated with significant decrease of nitrogenase activity. A correlation between nitrogen-fixing efficiency and nodule infected cell ultrastructure was found. When the bacteroids occupied about 10 times higher area in infected cells of nodule than peribacteroid spaces and host cytosol had electron dense and homogenous structure, the nitrogenase activity was the highest. This revised version was published online in August 2006 with corrections to the Cover Date.     

Effect of inoculation and leaf litter amendment on establishment of nodule-forming Frankia populations in soil

High-N(2)-fixing activities of Frankia populations in root nodules on Alnus glutinosa improve growth performance of the host plant. Therefore, the establishment of active, nodule-forming populations of Frankia in soil is desirable. In this study, we inoculated Frankia strains of Alnus host infection groups I, IIIa, and IV into soil already harboring indigenous populations of infection groups (IIIa, IIIb, and IV). Then we amended parts of the inoculated soil with leaf litter of A. glutinosa and kept these parts of soil without host plants for several weeks until they were spiked with [(15)N]NO(3) and planted with seedlings of A. glutinosa. After 4 months of growth, we analyzed plants for growth performance, nodule formation, specific Frankia populations in root nodules, and N(2) fixation rates. The results revealed that introduced Frankia strains incubated in soil for several weeks in the absence of plants remained infective and competitive for nodulation with the indigenous Frankia populations of the soil. Inoculation into and incubation in soil without host plants generally supported subsequent plant growth performance and increased the percentage of nitrogen acquired by the host plants through N(2) fixation from 33% on noninoculated, nonamended soils to 78% on inoculated, amended soils. Introduced Frankia strains representing Alnus host infection groups IIIa and IV competed with indigenous Frankia populations, whereas frankiae of group I were not found in any nodules. When grown in noninoculated, nonamended soil, A. glutinosa plants harbored Frankia populations of only group IIIa in root nodules. This group was reduced to 32% +/- 23% (standard deviation) of the Frankia nodule populations when plants were grown in inoculated, nonamended soil. Under these conditions, the introduced Frankia strain of group IV was established in 51% +/- 20% of the nodules. Leaf litter amendment during the initial incubation in soil without plants promoted nodulation by frankiae of group IV in both inoculated and noninoculated treatments. Grown in inoculated, amended soils, plants had significantly lower numbers of nodules infected by group IIIa (8% +/- 6%) than by group IV (81% +/- 11%). On plants grown in noninoculated, amended soil, the original Frankia root nodule population represented by group IIIa of the noninoculated, nonamended soil was entirely exchanged by a Frankia population belonging to group IV. The quantification of N(2) fixation rates by (15)N dilution revealed that both the indigenous and the inoculated Frankia populations of group IV had a higher specific N(2)-fixing capacity than populations belonging to group IIIa under the conditions applied. These results show that through inoculation or leaf litter amendment, Frankia populations with high specific N(2)-fixing capacities can be established in soils. These populations remain infective on their host plants, successfully compete for nodule formation with other indigenous or inoculated Frankia populations, and thereby increase plant growth performance.