青海蚕豆根瘤菌共生固氮效应的研究

蚕豆根瘤菌属于快生型根瘤菌 ,2 4h形成菌落。在盆栽试验中 ,蚕豆根瘤菌Qx -2与蚕豆具有良好共生效应 ,其株高、茎叶干重及含氮量 ,株瘤数 ,根瘤重量以及固氮酶活性等 ,均明显高于不接种对照 ,固氮率提高 69.4 9%。接种根瘤菌是提高蚕豆共生固氮效应的技术措施。     

如东田菁根瘤菌遗传多样性及高效促生菌株筛选

摘要:【目的】研究江苏如东沿海滩涂田菁根瘤菌的遗传多样性和系统发育,筛选高效促生田菁根瘤菌。【方法】采用16S rRNA基因、持家基因(recA、atpD、glnⅡ)、共生基因(nodA、nifH)的系统发育分析,并进行温室盆栽接种试验筛选高效菌株。【结果】分离到的32株田菁根瘤菌分布于Ensifer、Neorhizobium、Rhizobium,并与E. meliloti、N. huautlense、E. sesbaniae、R. pusense 亲缘关系最近。共生基因nodA、nifH的系统进化分析结果基本一致,均与E. saheli 最为相近。7株代表菌株均能耐受5%的NaCl(W/V)浓度,其中YIC5082耐盐性最强,能够耐受6%的NaCl。温室盆栽试验表明7 株代表菌株均能与田菁有效结瘤固氮,其中6株菌显著提高了植株的株高和鲜重。【结论】江苏如东沿海滩涂田菁根瘤菌具有较丰富的遗传多样性,其中N．huautlense、E．meliloti为优势种。该地区的田菁根瘤菌多数具有较好的共生有效性及耐盐性,YIC5077促生效果最为突出,并表现出较强的固氮及结瘤能力,具有良好的应用前景。     

青海一株蚕豆根瘤菌的鉴定及抗旱性评价

【目的】研究青海干旱地区蚕豆根瘤菌的遗传多样性,获得与蚕豆品种共生匹配且具有耐旱性的根瘤菌株,促进蚕豆耐旱根瘤菌在青海干旱地区生产中的应用。【方法】以分离自青海干旱地区一株菌株QHCD22为材料,利用细菌形态学、生理生化指标鉴定、Biolog细菌鉴定系统、16S rRNA基因序列分析、全基因组分析等进行菌种鉴定和系统发育分析,进一步通过PEG6000模拟干旱胁迫、盆栽回接干旱胁迫处理及旱作田间接种验证试验对该菌株的耐旱性进行综合评价。【结果】QHCD22菌株属快生型根瘤菌属(Rhizobium),Rhizobium indicum种。随着PEG6000模拟干旱胁迫程度的加剧,在−0.6 mPa这一更低渗透势时菌株存活数量增高,浊度由61.48%上升到69.42%,表现出较强的耐旱性。盆栽试验表明,接种根瘤菌处理(NA)的株高、植株鲜干比、根瘤数、根瘤鲜重、叶绿素含量(SPAD)、叶片相对含水量(RWC)、脯氨酸含量(PRO)、超氧化物歧化酶活性(SOD)、根系活力(TCC)均高于不接种根瘤菌处理(NN),并且在正常供水条件下,NA处理的各指标也均高于NN处理。旱作田间验证试验表明接种该菌株显著提高固氮酶活性,青海13号蚕豆根瘤固氮酶活性由不接种的42.07 C₂H₄ nmol/(g·h)显著增加到221.78 C₂H₄ nmol/(g·h),青蚕14号蚕豆由40.60 C₂H₄ nmol/(g·h)显著增加到109.78 C₂H₄ nmol/(g·h),马牙蚕豆由33.41 C₂H₄ nmol/(g·h)显著增加到643.15 C₂H₄ nmol/(g·h)。接种根瘤菌对于增加产量具有促进作用,其中青蚕14号的增产效果显著,增产幅度达32.3%。【结论】QHCD22菌株可能为快生型根瘤菌属的一个种Rhizobium indicum,具有一定的耐旱性,研究表明接种根瘤菌可以提高蚕豆的耐旱性,尤其对干旱敏感型蚕豆品种增产效果显著,具有潜在的应用前景。     

中国温带地区根瘤菌与山黎豆和棘豆的新共生组合

采用16S rDNAPCR—RFLP,16S－23S IGS RCP—RFLP,TP－RAPD,MLEE,16S rDNA和持家基因序列分析（atpD,recA和glnⅡ）等多种方法研究了50株分离自中国北方地区的山黎豆属（Lathyrus）和棘豆属（Oxytropus）植物根瘤菌的遗传多样性和系统发育关系．结果表明,山黎豆属植物主要与根瘤菌属（Rhizobium）结瘤,而棘豆属植物主要与中华根瘤菌属（Sinorhizobium）结瘤．一个与蚕豆根瘤菌（R．fabae）相近的根瘤菌种群和苜蓿中华根瘤菌（s．meliloti）分别被鉴定为山黎豆和棘豆在本地区的主要共生细菌,而这两个根瘤菌一豆科植物的组合还没有在其他地区被发现．这些结果扩展了与该两属植物共生的细菌谱,并为根瘤菌的生物地理学研究提供了新证据．     

紫云英根瘤菌质粒功能研究

紫云英根瘤菌CH203含有3条质粒(pRHa,97MI);pRHb,168MD;pRHc,251MD为共生质粒),用带蔗糖敏感基因Tn5-sacB进行菌株质粒消除和质粒缺失突变株筛选,获得一系列突变株。与野生型菌相比,质粒pRHa的丢失导致菌株结无效根瘤,质粒pRHb的丢失使菌株失去共生能力,在TY培养基平板上菌落变得粗糙,失去了脂多糖(LPSI)。质粒pRHc(共生质粒)的丢失显然失去其菌株的共生能力,同时使菌株抗酸性明显减弱。质粒回复能恢复突变株的表现特征和共生能力。此外,紫云英根瘤菌CH205含有5条大小不同的质粒(分子量42MD～230MD),该菌株某些质粒的消除能显著增强菌株的结瘤固氮能力。研究结果也表明除共生质粒外,紫云英根瘤菌其它质粒明显影响菌株的共生效应。     

湖北潜江灰潮土中费氏中华根瘤菌(Sinorhizobium fredii)多样性的研究

用5种代表性大豆为捕集植物从湖北潜江灰潮土中经盆栽试验分离筛选出50株费氏中华根瘤菌,对它们的生长速度、耐盐性、pH生长范围、天然抗药性、碳氮源利用和共生效应等同时进行了比较研究,证实了同一土壤环境中费氏中华根瘤菌群体的多样性。     

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     

毛豆根瘤菌转吸氢基因的研究

通过三亲本杂交将携带豌豆根瘤菌吸氢基因的质粒pAL₆₁₈转移到台湾毛豆根瘤菌292-C中,经抗性筛选、质粒检测和吸氢活性测定,得到能稳定遗传的结合株292-C₂和292-C₃。在自生条件下结合株均表现较高的吸氢活性,而受体株292-C不吸氢,在共生条件下结合株表现为高吸氢,而受体株表现为放氢。     

毛豆根瘤菌转吸氢基因的研究

通过三亲本杂交将携带豌豆根瘤菌吸氢基因的质粒ｐＡＬ６１８转移到台湾毛豆根瘤菌２９２－Ｃ中,经抗性筛选、质粒检测和吸氢活性测定,得到能稳定遗传的结合株２９２－Ｃ２和２９２－Ｃ３。在自生条件下结合株均表现较高的吸氢活性,而受体株２９２－Ｃ不吸氢,在共生条件下结合株表现为高吸氢,而受体株表现为放氢。     

石漠化地区根瘤菌的分离鉴定及盆栽抗旱效应

从云南文山峰丛洼地型石漠化地区白刺花(Sopnora riciisolia)、木蓝(Indigofera tinttorial)和三叶草(Trofolium repens)的根瘤中分离到3株细菌，编号为SWFU-B1、SWFU-B2和SWFU-B3,经形态特征、生理生化指标以及16S rDNA序列测定，3株细菌与豌豆根瘤菌(Rhizobium leguminosarum)、安徽根瘤菌(Rhizobium anhuiense)和栖稻根瘤菌(Rhizobium oryzihabitans)的相似性分别是100%、99.93%和99.78%。采用盆栽实验，将3株根瘤菌接种在不同干旱胁迫(正常水分、轻度干旱和中度干旱)下的蚕豆植株上，设置未接种(T0)、接种SWFU-B1(T1)、接种SWFU-B2(T2)和接种SWFU-B3(T3)4种接种处理，研究其对蚕豆株高、茎粗、根瘤数目、叶面积、抗氧化酶活性、渗透调节物质和丙二醛含量的影响。结果表明：随着干旱程度加重，蚕豆植株生长减缓，根瘤数量呈显著下降趋势；在同一水分条件下，与未接种相比，接种组蚕豆生长指标结果较好，接种根瘤菌对蚕豆有显著的促生长效...     

Use of the gusA gene marker in a competition study of the Rhizobium strains nodulating the common bean (Phaseolus vulgaris) in Senegal soils

Indigenous rhizobial population is among the factors which influence increased crop yield through inoculation with elite strains. In this study, we compared in greenhouse conditions the competitiveness of Rhizobium strain ISRA 355 for nodulation of the common bean (Phaseolus vulgaris) cultivated in different unsterile Senegal soils in terms of pH, N and C contents. The strain ISRA 355 produced a stable GUS+ transconjugant which was used for competition with indigenous soil rhizobia in six localities. At Bayakh, the transconjugant ISRA 355gusA was less competitive than the indigenous rhizobial strains, whereas in the other localities, it was more competitive since it occupied more than 90% of the nodules. Thus the Rhizobium strain ISRA 355 should be used for successfully inoculating the common bean in Senegal soils.     

Ability of selected accessions of Phaseolus vulgaris L. to restrict nodulation by particular rhizobia

Plant genotypes that limit nodulation by indigenous rhizobia while nodulating normally with inoculant-strain nodule occupancy in Phaseolus vulgaris. In this study, eight of nine Rhizobium tropici strains and six of 15 Rhizobium etli strains examined, showed limited ability to nodulate and fix nitrogen with the two wild P. vulgaris genotypes G21117 and G10002, but were effective in symbiosis with the cultivated bean genotypes Jamapa and Amarillo Gigante. Five of the R. etli strains restricted in nodulation by G21117 and G10002 produced an alkaline reaction in yeast mannitol medium. In a competition experiment in which restricted strains were tested in 1:1 mixtures with the highly effective R. etli strain CIAT632, the restricted strains produced a low percentage of the nodules formed on G2117, but produced over 40% of the nodules formed on Jamapa. The interaction of the four Rhizobium strains with the two bean genotypes, based on the percentage of nodules formed, was highly significant (P<0.001).     

Degradation of aromatic compounds byRhizobium spp.

Summary The ability of rhizobia to utilize catechol, protocatechuic acid, salicylic acid, p-hydroxybenzoic acid and catechin was investigated. The degradation pathway of p-hydroxybenzoate byRhizobium japonicum, R. phaseoli, R. leguminosarum, R. trifolii andRhizobium sp. isolated from bean was also studied.R. leguminosarum, R. phaseoli andR. trifolii metabolized p-hydroxybenzoate to protocatechuate which was cleaved by protocatechuate 3,4-dioxygenasevia ortho pathway.R. japonicum degraded p-hydroxybenzoate to catechol which was cleaved by catechol 1,2-dioxygenase.Rhizobium sp., a bean isolate, dissimilatedp-hydroxybenzoate to salicylate. Salicylate was converted to gentisic acid prior to ring cleavage. The rhizobia convertedp-hydroxybenzoate to Rothera positive substance. Catechol and protocatechuic acid were directly cleaved by the species.R. japonicum converted catechin to protocatechuic acid.     

Genetic diversity of fast-growing rhizobia that nodulate soybean ( Glycine max L. Merr)

The fast-growing Rhizobium sp. strain NGR234, isolated from Papua New Guinea, and 13 strains of Sinorhizobium fredii, isolated from China and Vietnam, were fingerprinted by means of RAPD, REP, ERIC and ARDRA. ERIC, REP and RAPD markers revealed a considerable genetic diversity among fast-growing rhizobia. Chinese isolates showed higher levels of diversity than those strains isolated from Vietnam. ARDRA analysis revealed three different genotypes among fast-growing rhizobia that nodulate soybean, even though all belonged to a subcluster that included Sinorhizobium saheli and Sinorhizobium meliloti. Among S. fredii rhizobia, two strains, SMH13 and HH303, might be representatives of other species of nitrogen-fixing organisms. Although restriction analysis of the nifD–nifK intergenic DNA fragment confirmed the unique nature of Rhizobium sp. strain NGR234, several similarities between Rhizobium sp. strain NGR234 and S. fredii USDA257, the ARDRA analysis and the full sequence of the 16S rDNA confirmed that NGR234 is a S. fredii strain. In addition, ARDRA analysis and the full sequence of the 16S rDNA suggested that two strains of rhizobia might be representatives of other species of rhizobia.     

Mimosine produced by the tree-legume Leucaena provides growth advantages to some Rhizobium strains that utilize it as a source of carbon and nitrogen

Growth of most Rhizobium strains is inhibited by mimosine, a toxin found in large quantities in the seeds, foliage and roots of plants of the genera Leucaena and Mimosa. Some Leucaena-nodulating strains of Rhizobium can degrade mimosine (Mid⁺) and are less inhibited by mimosine in the growth medium than the mimosine-nondegrading (Mid^-) strains. Ten Mid⁺ strains were identified that did not degrade 3-hydroxy-4-pyridone (HP), a toxic intermediate of mimosine degradation. However, mimosine was completely degraded by these strains and HP was not accumulated in the cells when these strains were grown in a medium containing mimosine as the sole source of carbon and nitrogen. The mimosine-degrading ability of rhizobia is not essential for nodulation of Leucaena species, but it provides growth advantages to Rhizobium strains that can utilize mimosine, and it suppresses the growth of other strains that are sensitive to this toxin.     

Genomic insight into the taxonomy of Rhizobium genospecies that nodulate Phaseolus vulgaris

Due to the wide cultivation of bean (Phaseolus vulgaris L.), rhizobia associated with this plant have been isolated from many different geographical regions. In order to investigate the species diversity of bean rhizobia, comparative genome sequence analysis was performed in the present study for 69 Rhizobium strains mainly isolated from root nodules of bean and clover (Trifolium spp.). Based on genome average nucleotide identity, digital DNA:DNA hybridization, and phylogenetic analysis of 1,458 single-copy core genes, these strains were classified into 28 clusters, consistent with their species definition based on multilocus sequence analysis (MLSA) of atpD, glnII, and recA. The bean rhizobia were found in 16 defined species and nine putative novel species; in addition, 35 strains previously described as Rhizobium etli, Rhizobium phaseoli, Rhizobium vallis, Rhizobium gallicum, Rhizobium leguminosarum and Rhizobium spp. should be renamed. The phylogenetic patterns of symbiotic genes nodC and nifH were highly host-specific and inconsistent with the genomic phylogeny. Multiple symbiovars (sv.) within the Rhizobium species were found as a common feature: sv. phaseoli, sv. trifolii and sv. viciae in Rhizobium anhuiense; sv. phaseoli and sv. mimosae in Rhizobium sophoriradicis/R. etli/Rhizobium sp. III; sv. phaseoli and sv. trifolii in Rhizobium hidalgonense/Rhizobium acidisoli; sv. phaseoli and sv. viciae in R. leguminosarum/Rhizobium sp. IX; sv. trifolii and sv. viciae in Rhizobium laguerreae. Thus, genomic comparison revealed great species diversity in bean rhizobia, corrected the species definition of some previously misnamed strains, and demonstrated the MLSA a valuable and simple method for defining Rhizobium species.     

Factors affecting growth of Bdellovibrio on Rhizobium

The extent of decline in the population density of Rhizobium sp. exposed to Bdellovibrio was markedly reduced in the presence of montmorillonite, kaolinite or vermiculite but not by a soil clay fraction. Increasing levels of montmorillonite reduced the numbers of vibrios that appeared in a two-membered culture and allowed for greater survival of the rhizobia. Bdellovibrio and not Rhizobium sp. was retained when mixed with the three clay minerals, but no appreciable retention was evident with the soil clay fraction. Suspensions of colloidal soil organic matter protected the hosts from parasitism, although aqueous extracts of soil did not affect the relationship. Cells from old Rhizobium sp. cultures were attacked only after a lag phase, but rhizobia that had been stored were more rapidly lysed than cells tested immediately after removal from the growth medium. The possible significance of these findings to the survival of rhizobia in soils containing Bdellovibrio is discussed.     

Genotypic and phenotypic characterization of rhizobia that nodulate snap bean (Phaseolus vulgaris L.) in Egyptian soils

Snap bean fields in 12 of the 25 governorates of Egypt were surveyed to determine the distribution and taxonomy of snap bean-nodulating rhizobia. Nodulation rates in the field were very low, indicating that Egyptian soils do not have sufficient numbers of snap bean-compatible Rhizobium spp. A total of 87 rhizobial isolates were assayed on the most commonly grown cultivars in order to identify the most effective strains. The five most effective isolates (R11, R13, R28, R49 and R52) were fast-growing and utilized a wide range of carbon and nitrogen sources. A phylogenetic assignment of these strains by analysis of the 16S ribosomal RNA gene suggested that all fell within the Rhizobium etli–Rhizobium leguminosarum group. Strains R11, R49 and R52 all clustered with other identified R. etli strains, while strains R13 and R28 were more distinct. The distinctness of R13 and R28 was supported by physiological characteristics, such as their ability to utilize citrate, erythritol, dulcitol and lactate. Strains R13 and R28 also yielded the highest plant nitrogen content of all isolates.The highly effective strains isolated in this study, in particular strains R13 and R28, are promising candidates for improving crop yields. The data also suggested that these two strains represented a novel sub-group within the R. etli–R. leguminosarum group. As snap bean is a crop of great economic value to Egypt, the identification of highly effective rhizobial strains adapted to Egyptian soils, such as strains R13 and R28, is of great interest.     

Phenotypic and genetic diversity among rhizobia isolated from three Hedysarum species: H. spinosissimum,H. coronarium and H. flexuosum

Cultural, physiological and biochemical properties of 18 strains of rhizobia isolated from root nodules of the forage legume H. spinosissimum were compared with those of rhizobia from the related species H. coronarium (15 strains) and H. flexuosum (four strains). On the basis of 43 characteristics the 37 strains of Hedysarum rhizobia could be divided into two groups by numerical analysis. The H. spinosissimum rhizobia formed the first group and the second group comprised the strains from H. coronarium and H. flexuosum. The reference Rhizobium leguminosarum bv. viceae strain 250A was clustered with the rhizobia from H. coronarium and H. flexuosum. By contrast Bradyrhizobium sp. (Arachis) reference strain 280A was not clustered with any of the strains tested, indicating that the H. spinosissimum rhizobia differ from both Rhizobium and Bradyrhizobium. Serological data also discriminate between H. spinosissimum and H. coronariumrhizobia but not between the latter and H. flexuosum strains. The strains tested exhibit a high degree of specificity for nodulation and nitrogen fixation. We also determined the16SrRNA gene sequence of H. spinosissimum rhizobia (four strains), H. coronarium (two strains) and H. flexuosum (two strains) and found that the four H. spinosissimum isolates share a 98% identity among each other in this region but they showed less than 92% identity to the H. coronarium and H. flexuosum isolates. The H. spinosissimum isolates were closely related to both Mesorhizobium loti and M. ciceri, sharing 97% identity with each species.     

Phage sensitivity and host range of Rhizobium strains isolated from root nodules of temperate legumes

Twenty-five Rhizobium strains were isolated from root nodules of Astragalus spp. (10), Hedysarum alpinum (7), Glycyrrhiza pallidiflora (3) and Ononis arvensis (5). The sensitivity of these strains to bacteriophages of Rhizobium loti, R. meliloti, R. galegae and R. leguminosarum was studied. Phages specific to R. loti strains were shown to induce the phage lysis of several Astragalus, Hedysarum and Ononis rhizobia. Ten R. loti strains tested for nodulation abilities on the plant hosts under investigation were able to develop nitrogen-fixing nodules on the Ononis arvensis roots. On the other hand, rhizobia from Ononis and Glycyrrhiza could form an effective symbiosis with Lotus corniculatus plants, so these bacteria are considered to belong to the Rhizobium loti taxon. Bacterial strains isolated from Astragalus and Hedysarum were observed to cross-nodulate their plant hosts as well as Oxytropis campestris, Glycyrrhiza uralensis and Ononis arvensis plants, whereas they could not nodulate Lotus plants. It is concluded that these Rhizobium strains comprise a cross-inoculation group related to Rhizobium loti. ei]{gnR O D}{fnDixon}