Analysis of R-primes demonstrates that genes for broad host range nodulation of Rhizobium strain NGR234 are dispersed on the Sym plasmid

Summary R-prime plasmids carrying regions of the symbiotic (Sym) plasmid of the broad host range Rhizobium strain NGR234 were isolated in intergeneric matings with Escherichia coli K12. Three R-primes carrying approximately 180 kb (pMN23), 220 kb (pMN31) and 330 kb (pMN49) of Sym DNA were characterized in more detail. Restriction enzyme analysis and hybridization studies showed that these R-primes carried large overlapping regions of the Sym plasmid, and had the symbiotic genes (two copies of nifH, D and K; nodA, B, C and D; region II; host specific nodulation (hsn) genes) located over half of the 470 kb Sym plasmid. Only the largest of these R-primes (pMN49) contained the complete nodulation host range of the original parent strain NGR234. This broad host range was shown to be present on plasmid pMN49 by being expressed in Agrobacterium tumefaciens strain A136. Furthermore the R-prime plasmids were shown to contain different regions of distinctive host specific nodulation (hsn) for tropical legume infection and for the nodulation of the non-legume Parasponia. Nodulation of soybeans, however, required an additional region that was not essential for the nodulation of other tropical legumes. Strain NGR234 was also found to nodulate the stem and roots of the tropical legume Sesbania rostrata at a very low efficiency. However, the R-prime mini Sym plasmid constructions enabled a greater efficiency of nodulation of Sesbania rostrata to occur.     

Macroptilium atropurpureum (siratro) host specificity genes are linked to a nodD-like gene in the broad host range Rhizobium strain NGR234

Summary A 6.7 kb HindIII fragment from the Sym-plasmid of strain NGR234 was found to code a nodD-like gene flanked by two loci which were required for siratro host range. Transfer of the 6.7 kb fragment from NGR234 to R. trifolii strain ANU843 conferred extended host range ability to this strain on siratro plants but not to other plants normally nodulated by strain NGR234. Tn5 mutagenesis of the 6.7 kb fragment showed that insertions located into loci flanking the nodD-like gene abolished the extended host range phenotype. A hybridization probe spanning one of the host specificity loci was shown to hybridize to three specific bands in the NGR234 genome. Complementation and DNA hybridization data showed that the nodD-like gene of strain NGR234 was functionally similar to that in R. trifolii. The introduction to R. trifolii of the 6.7 kb HindIII fragment containing Tn5 insertions located in the nodD-like gene did not abolish the ability to extend the host range of R. trifolii to siratro plants. However, transfer of the 6.7 kb HindIII to R. trifolii derivatives containing Tn5 insertions into either nodA, B or C or other R. trifolii nod genes failed to confer siratro nodulation to these recipients. Reconstruction experiments showed that the 6.7 kb fragment from strain NGR234 and the 14 kb nodulation region of R. trifolii could induce the nodulation of siratro plants when introduced together into Sym-plasmid-cured Rhizobium strains.     

Nodulation of soybean byRhizobium japonicum mutants with altered capsule synthesis

Spontaneous mutants with altered capsule synthesis were isolated from a marked strain of the symbiont,Rhizobium japonicum. Differential centrifugation was used to enrich serially for mutants incapable of forming capsules. The desired mutants were detected by altered colony morphology and altered ability to bind host plant lectin. Three mutants failed to form detectable capsules at any growth phase when cultured in vitro or in association with the host (soybean,Glycine max (L.) Merr.) roots. These mutants were all capable of nodulating and attaching to soybean roots, indicating that the presence of a capsule physically surrounding the bacterium is not required for attachment or for infection and nodulation. Nodulation by several of the mutants was linearly proportional to the amount of acidic exopolysaccharide that they released into the culture medium during the exponential growth phase, indicating that such polysaccharide synthesis is important and perhaps required for nodulation. Two of the mutants appeared to synthesize normal lectin-binding capsules when cultured in association with host roots, but not when cultured in vitro. Nodulation by these mutants appeared to depend on how rapidly after inoculation they synthesized capsular polysaccharide.Abbreviations CPS capsular polysaccharide - EPS exopolysaccharide - FITC fluorescein isothiocyanate Contribution No. 719 of the C.F. Kettering Research Laboratory     

Differential accumulation of hydroxyproline-rich glycoproteins in bean root nodule cells infected with a wild-type strain or a C4-dicarboxylic acid mutant of Rhizobium leguminosarum bv. phaseoli

An antiserum raised against deglycosylated hydroxyproline-rich glycoproteins (HPGPs) from melon (Cucumis melo L.) was used to study the relationship between Rhizobium infection and induction of HRGPs in bean (Phaseolus vulgaris L.) root nodule cells infected with either the wild-type or a C₄-dicarboxylic acid mutant strain of Rhizobium leguminosarum bv. phaseoli. In effective nodules, where fixation of atmospheric dinitrogen is taking place, HRGPs were found to accumulate mainly in the walls of infected cells and in peribacteroid membranes surrounding groups of bacteroids. Internal ramifications of the peribacteroid membrane were also enriched in HRGPs whereas the peribacteroid space as well as the bacteroids themselves were free of these glycoproteins. In mutant-induced root nodules, HRGPs were specifically associated with the electron-dense, laminated structures formed in plastids as a reaction to infection by this mutant. The presence of HRGPs was also detected in the host cytoplasm. The aberrant distribution of HRGPs in infected cells of mutant-induced nodules likely reflects one aspect of the altered host metabolism in relation to peribacteroid-membrane breakdown. The possibility that the antiserum used for HRGP localization may have cross-reacted with ENOD 2 gene products is discussed in relation to amino-acid sequences and sites of accumulation.     

Microscopic studies of cell divisions induced in alfalfa roots by Rhizobium meliloti

We have used spot-inoculation and new cytological procedures to observe the earliest events stimulated in alfalfa (Medicago sativa L.) roots by Rhizobium meliloti. Roots were inoculated with 1–10 nl of concentrated bacteria, fixed in paraformaldehyde, and after embedding and sectioning stained with a combination of acridine orange and DAPI (4-6-diamidino-2-phenylindole hydrochloride). Normal R. meliloti provoke cell dedifferentiation and mitosis in the inner cortex of the root within 21–24 h after inoculation. This activation of root cells spreads progressively, leading to nodule formation. In contrast, the R. meliloti nodA and nodC mutants do not stimulate any activation or mitosis. Thus the primary and earliest effect of Rhizobium nod gene action is plant cellular activation. A rapid, whole-mount visualization by lactic acid shows that the pattern of nodule form varies widely. Some R. meliloti strains were found to be capable of stimulating on alfalfa roots both normal nodules and a hybrid structure intermediate between a nodule and a lateral root.     

Marking of a Sym plasmid of Rhizobium with Tn7

Summary Transposon Tn7 was inserted into wide host range plasmid pSUP202 and used as a suicide plasmid vehicle for transposon mutagenesis in Rhizobium leguminosarum. Tn7 is transposed with high frequency into the self-transmissible plasmid pJB5JI without affecting the transfer, nodulation and nitrogen fixation functions. Tn7 transposition provides a useful tool for marking symbiotic plasmids.     

Selection of salt-tolerant Rhizobium isolates of Acacia nilotica

Among 35 Rhizobium isolates of Acacia nilotica, from different agro-climatic zones, two, ANG4 and ANG5, tolerated up to 850 mm NaCl and one, ANG3, was sensitive to NaCl above 250 mm. Nodulation and nitrogenase activity of the three isolates decreased with increasing concentration of salt up to 150 mm. Nodulation by ANG3 was 15% at 75 mm NaCl and nil at 100 mm. With ANG4 and ANG5, nodulation was only slightly decreased at 150 mm NaCl. Nitrogenase activity associated with plants inoculated with ANG3 was halved at 25 mm NaCl compared with salt-free controls, whereas isolates ANG4 and ANG5 retained 25% and 15% activity, respectively, even at 100 mm NaCl. Salt-tolerant Rhizobium isolates can therefore nodulate and fix N₂ in saline soils.     

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。     

Corresponding 16S rRNA gene segments in Rhizobiaceae and Aeromonas yield discordant phylogenies

Previous evidence has indicated that the 16S rRNA genes in certain species of Aeromonas may have a history of lateral transfer and recombination. A comparative analysis of patterns of 16S nucleotide sequence polymorphism among species of Rhizobium and Agrobacterium was conducted to determine if there is similar evidence for chimeric 16S genes in members of the Rhizobiaceae. Results from phylogenetic analyses and comparison of patterns of nucleotide sequence polymorphism in portions of rhizobial 16S genes revealed the same type of segment-dependent polymorphic site partitioning that was previously reported for Aeromonas. These results support the hypothesis that certain 16S segments in rhizobia may have a history of lateral transfer and recombination.Abbreviations 16S rRNA 16S ribosomal ribonucleic acid - 16S the 16S rRNA gene     

Assessment of nodulation of Mucuna pruriens by promiscuous indigenous rhizobia in the moist savanna zone of Nigeria

Leucaena leucocephala failed to nodulate effectively with promiscuous indigenous rhizobia with which Mucuna pruriens nodulated effectively. Mucuna pruriens was adequately established and well nodulated due to the presence of favourable climatic and edaphic factors which enhanced its establishment in the humid/moist savanna zone of Nigeria. The microsymbiont for M. pruriens seems to thrive more in an alkaline rhizosphere. Introduction of M. pruriens into the farming system in Nigeria may serve as a low input agricultural system, which is not only sustainable but also economically viable.     

Phylogeny of symbiotic cyanobacteria within the genus <Emphasis Type="Italic">Nostoc</Emphasis> based on 16S rDNA sequence analyses

A phylogenetic analysis of selected symbiotic Nostoc strain sequences and available database 16S rDNA sequences of both symbiotic and free-living cyanobacteria was carried out using maximum likelihood and Bayesian inference techniques. Most of the symbiotic strains fell into well separated clades. One clade consisted of a mixture of symbiotic and free-living isolates. This clade includes Nostoc sp. strain PCC 73102, the reference strain proposed for Nostoc punctiforme. A separate symbiotic clade with isolates exclusively from Gunnera species was also obtained, suggesting that not all symbiotic Nostoc species can be assigned to N. punctiforme. Moreover, isolates from Azolla filiculoides and one from Gunnera dentata were well nested within a clade comprising most of the Anabaena sequences. This result supports the affiliation of the Azolla isolates with the genus Anabaena and shows that strains within this genus can form symbioses with additional hosts. Furthermore, these symbiotic strains produced hormogonia, thereby verifying that hormogonia formation is not absent in Anabaena and cannot be used as a criterion to distinguish it from Nostoc.The GenBank accession numbers for the cyanobacterial 16S rRNA gene sequences determined in this study are AY742447-AY742454.