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International audience; Symbioses between the root nodule-forming, nitrogen-fixing actinomycete Frankia and its angiospermous host plants are important in the nitrogen economies of numerous terrestrial ecosystems. Molecular characterization of Frankia strains using polymerase chain reaction/restriction fragment length polymorphism (PCR/RFLP) analyses of the 16S rRNA-ITS gene and of the nifDnifK spacer was conducted directly on root nodules collected worldwide from Casuarina and Allocasuarina trees. In their native habitats in Australia, host species contained seven distinctive sets of Frankia in seven different molecular phylogenetic groups. Where Casuarina and Allocasuarina trees are newly planted outside Australia, they do not normally nodulate unless Frankia is introduced with the host seedling. Nodules from Casuarina trees introduced outside Australia over the last two centuries were found to contain Frankia from only one of the seven phylogenetic groups associated with the host genus Casuarina in Australia. The phylogenetic group of Frankia found in Casuarina and Allocasuarina trees introduced outside Australia is the only group that has yielded isolates in pure culture, suggesting a greater ability to survive independently of a host. Furthermore, the Frankia species in this group are able to nodulate a wider range of host species than those in the other six groups. In baiting studies, Casuarina spp. are compatible with more Frankia microsymbiont groups than Allocasuarina host spp. adapted to drier soil conditions, and C. equisetifolia has broader microsymbiont compatibility than other Casuarina spp. Some Frankia associated with the nodular rhizosphere and rhizoplan, but not with the nodular tissue, of Australian hosts were able to nodulate cosmopolitan Myrica plants that have broad microsymbiont compatibility and, hence, are a potential host of Casuarinaceae-infective Frankia outside the hosts' native range. The results are consistent with the idea that Frankia symbiotic promiscuity and ease of isolation on organic substrates, suggesting saprophytic potential, are associated with increased microsymbiont ability to disperse and adapt to diverse new environments, and that both genetics and environment determine a host's nodular microsymbiont.

Although the biological conversion of nitrite to nitrate is a well-known process, studies of Nitrobacter populations are hindered by their physiological characteristics. This report describes a new method for detecting and counting Nitrobacter populations in situ with the PCR. Two primers from the 16S rRNA gene were used to generate a 397-bp fragment by amplification of Nitrobacter species DNA. No signal was detected from their phylogenetic neighbors or the common soil bacteria tested. Extraction and purification steps were optimized for minimal loss and maximal purity of soil DNA. The detection threshold and accuracy of the molecular method were determined from soil inoculated with 10, 10(2), or 10(3) Nitrobacter hamburgensis cells per g of soil. Counts were also done by the most-probable-number (MPN)-Griess and fluorescent antibody methods. PCR had a lower detection threshold (10(2) Nitrobacter cells per g of soil) than did the MPN-Griess or fluorescent antibody method. When PCR amplification was coupled with the MPN method, the counting rate reached 65 to 72% of inoculated Nitrobacter cells. Tested on nonsterile soil, this rapid procedure was proved efficient.

The genomic diversity among 22 Nitrobacter strains was investigated by determining rRNA gene restriction patterns, DNA hybridization characteristics, and DNA base compositions. The guanine-plus-cytosine contents of the DNAs ranged from 58 to 61 mol%. As determined by DNA hybridization (S1 nuclease method), five DNA genomic groups were differentiated, and these groups formed three genomic species. Genomic species 1, which corresponded to Nitrobacter winogradskyi, was split into three subspecies. Subspecies 1a contained strain W (= ATCC 25391), the type strain of N. winogradskyi; subspecies 1b contained proposed reference strain 6R; and subspecies 1c contained a strain of “Nitrobacter agilis” (strain ATCC 14123). Genomic species 2, which has not been described previously and which contained proposed reference strain LL and four strains that were isolated from lake sediments, was distinct from N. winogradskyi and Nitrobacter hamburgensis. This species is not named in this paper since it could not be differentiated from N. winogradskyi and N. hamburgensis on the basis of phenotypic characteristics. Genomic species 3 corresponded to N. hamburgensis and was distantly related to the other genomic species.

DNA sequences from the intergenic spacer (IGS) region of the ribosomal operon were amplified by the polymerase chain reaction (PCR) technique using two primers derived from 16S and 23S rRNA conserved sequences. The PCR products, cleaved by 4 base cutting restriction enzymes, were used to differentiate Nitrobacter strains. This method offered a convenient alternative to serological testing for characterization of Nitrobacter isolates and enabled a large number of strains to be genotypically characterized easily and rapidly. This method was successfully used to characterize natural populations of Nitrobacter from various soils and a lake. A diversity was demonstrated in various soils, and in a lake both in freshwater and in sediments. Strains closely related to both WL and LL were found in these eco-systems. It seems that the diversity of Nitrobacter populations was not associated with global environments but may be related to the presence of locally coexisting niches.

Three N2-fixing yellow bacteria (5AJ, 5ATR, and 29AJ) isolated from the rhizosphere of rice, previously found to be related to Flavobacterium capsulatum by DNA–rRNA hybridization, were compared on the basis of 115 morphological, physiological, biochemical, and nutritional characters. The bacteria were nonfermentative, irregular motile rods with polar flagella. They exhibited a gram-negative reaction and yellow pigmentation. A numerical analysis using these phenotypic characteristics showed that the bacteria were more closely related to Pseudomonas paucimobilis than to F. capsulatum. A relationship to the P. paucimobilis cluster was also supported by the presence of the pigment nostoxanthin. This pigment was also found in F. capsulatum and could be used as a distinctive marker for this group of yellow bacteria. Their phenotypic properties, protein patterns, and serological tests (ELISA) showed that two of the isolates (5AJ and 5ATR) were identical and had originated from the same parental strain. On the other hand, the serological tests indicated that both N2-fixing yellow bacteria (5AJ or 5ATR and 29AJ) differed from the reference strains, F. capsulatum and P. paucimobilis. Key words: N2-fixing bacteria, taxonomic study, nostoxanthin, Pseudomonas.

Three15N isotopic dilution methods (15N natural abundance, labelled mineral fertilizer, and organic matter) were used to determine the proportion of N derived from different available sources in seedlines ofAlnus glutinosa andPopulus nigra planted together or in monoculture under natural climatic conditions. The proportion of N derived from N2 fixation in associated alders was appreciably higher than that determined in monoculture. The reduction of soil N uptake by associated alders contributed to an increase in total plant N and biomass production in associated poplars. When slightly N-labelled organic matter (alder leaf litter) was incorporated into the soil, 10–15% of its initial N content was recovered in poplar tissues, showing that this N source makes an important contribution to the N yield of associated non-fixing plants. There were no significant differences between the results obtained by15N natural abundance and those obtained by labelled fertilizer methods, suggesting that the δ15N method could be used to evaluate annual N budgets in natural ecosystems.

The degree of phenotypic variation of the bacterial strains USDA 125-Sp, USDA 138 and USDA 138-SmBradyrhizobium japonicum a long time after introduction was studied in three experimental fields. A total of 54 phenotypic characters were analyzed by constructing a dendrogram based on an hierarchic classification. Strong similarities (92.6, 94 and 95%) were found between the isolates introduced into soil 8, 10 and 13 years ago and between their respectiveB. japonicum parental clones. The dendrogrammic analysis detected a small amount of phenotypic drift, however, between soil isolates and parental clones belonging to the same serogroup (selective effects were found to have generated 0 to 3.9% variation for the USDA 125-Sp inoculum introduced 8 years ago, and 3.2-3.5% after 10 and 13 years, respectively, for the USDA 138 and USDA 138-Sm bacterial inocula) and within the serogroup 125 soil isolates (2.7%). We found a similar evolution of serogroup 125 isolates when compared with parental clones conserved on slant agar at 4°C. When a drift was observed, the isolates from soil presented a lower activity for several enzymes and lower diversity compared with the parental clones.

A set of oligonucleotides has been developed to study the competitivity of two Frankia strains in the nodulation of the roots of two host plant species: Alnus glutinosa and Alnus incana. Two 20 mer-oligonucleotides, complementary to highly conserved sequences inside the nifH gene, were used as primers for the polymerase chain reaction (PCR) system in order to amplify microsymbiont DNA extracted from actinorhizae. PCR products were analyzed using two strain-specific 15-mer oligonucleotides identified in the amplified region. Hybridization data indicate that strain ACoN24d is more competitive than strain ArI3 in the nodulation of both hosts.

Nitrobacter is the most common nitrite-oxidizing genus isolated from natural environments. In soil, several Nitrobacter strains can coexist. It is now well known that in pure cultures these strains are able to use organic carbon for their growth with highly variable efficiencies. Our purpose was to examine the role of nitrite or organic carbon as factors governing the evolution of the structure, the density and the activity of a Nitrobacter community in soil. This study has been performed on a recreated nitrite-oxidizing community consisting of 3 Nitrobacter strains co-inoculated into sterile soil and differing in their metabolic potentialities: a preferentially autotrophic strain (N. agilis strain AG), a preferentially mixotrophic strain (Nitrobacter genomic species 2 strain LL) and a preferentially heterotrophic strain (N. hamburgensis strain X14). Whatever the enrichments performed (0.271 or 1.353 mg NO2−-N g−1 dry soil or 1 mg sodium acetate g−1 dry soil), we observed a first phase of multiplication of the total Nitrobacter cells until day 7 or 15, and then, a stabilization of the community until the end of the experiment (day 48). The growth and level of stabilization of this Nitrobacter community as well as the competition between strains in the first phase were predominantly controlled by soil nitrite concentration, resulting in a dominance of the more autotrophic strains. The stabilization of the community resulted from a drastic decrease in nitrite-oxidation activity due not only to a direct nitrite limitation but also probably to a limiting O2 effect of the cells' ability to utilize nitrite. The competition was then predominantly monitored by strains' ability to utilize organic carbon for aerobic or anaerobic respiration. The autotrophy/heterotrophy shift strongly modified the result of the competition obtained at the end of the growth stage by inducing drastic declines of the more autotrophic strain and allowing continuous growth of the more heterotrophic strain. This suggests that heterotrophy being more tolerant than autotrophy to environmental changes, organic carbon could be a more permanent structuring factor of the Nitrobacter community than nitrite in soil.

Nitrification has been essentially studied as a chemical process and the studies of the microbial population have been slightly worked out. This work constitutes a first attempt to study the diversity of Nitrobacter strains with the aim of working out the genetic structure of natural populations. The Nitrobacter population structure was compared between freshwater and sediments of the same lake. Nitrobacter isolates were identified and characterized by DNA/DNA hybridization, restriction pattern of rRNA genes, PCR/RFLP analysis of the ribosomal intergenic spacer and plasmid patterns. It was shown that this lake population was split into 3 subpopulations: a specific freshwater subpopulation, a specific sediment subpopulation and a nonspecific one. A 60 MDa plasmid was detected in 3,3% of freshwater isolates. A 37 MDa plasmid was detected in 59% of sediments isolates. This 37 MDa plasmid was not associated with a genomic group, but associated with a localization: the sediments. These results suggest that this 37 MDa plasmid could be implicated in the adaptation to sediment environment.

Annual N2-fixation in virgin forest ecosystems has been measured using a15N natural abundance (δ15N) procedure. This method was compared to a15N labelled fertilizer isotopic dilution method. For young alders (5–6 years old), δ15N of leaves gave results in good agreement with the isotopic dilution of fertilizer method. Since δ15N variability was expected according to plant physiology, for alder trees, leaves were collected at various heights after the end of the growing season, and, to take account of isotopic variations coming from derived inputs, δ15N of leaves of a large number of other plants in the same are were measured to give control values. Following this procedure, the δ15N method gave reliable evaluation of the nitrogen supply, by through N2-fixation, to alders, which were found to maintain high nitrogen fixing capacity in a sequence ranging from first stage of establishment of climactic formation. Moreover, the same method is reported to discriminate various origins ofAlnus glutinosa grown in natural conditions, possibly in relation to the genetic diversity of this species.

An enzyme recently described for Strepiomyces sp. protoplast formation was used to digest Frankia sp. cell walls. This new enzyme, achromopeptidase, allowed the use of an in situ method to detect large plasmids. Plasmids ranging in size from 7 to 190 kilobases were detected in some Frankia strains.

Southern blots of Frankia total DNAs were hybridized with nifHDK probes from Rhizobium meliloti, Klebsiella pneumoniae and Frankia strain Arl3. Differences between strains were noted in the size of the hybridizing restriction fragments. These differences were more pronounced among Elaeagnus-compatible strains than among Alnus- or Casuarina-compatible strains. Gene banks constructed for Frankia strains EUN1f, HRN18a, CeD and ACoN24d were used to isolate nif-hybridizing restriction fragments for subsequent mapping and comparisons. The nifH zone had the highest sequence conservation and the nifH and nifD genes were found to be contiguous. The complete nucleotide sequence of the nifH open reading frame (ORF) from Frankia strain Arl3 is 861 bp in length and encodes a polypeptide of 287 amino acids. Comparisons of these nucleic acid and amino acid sequences with other published nifH sequences suggest that Frankia is most similar to Anabaena and Azotobacter spp. and K. pneumoniae and least similar to the Gram-positive Clostridium pasteurianum and to the archaebacterium Methanococcus voltae.

A nif gene probe from Rhizobium meliloti was used to isolate a recombinant bacteriophage from a Frankia sp. ArI3 gene bank. There is a large homology between nifD and nifH genes of R. meliloti or Klebsiella pneumoniae and Frankia DNA sequences. Approximately 4.5 kb to the right of nifK, we have localized a DNA region hybridizing to a R. meliloti probe containing nifA and nifB genes. The extent of the homology was greater for nifB than for nifA.

One hundredFrankia strains isolated from variousAlnus species in a single alder stand were tested for plasmid presence. Plasmid DNA was observed in five of the frankiae strains and was analyzed. We found that plasmids with a similar molecular weight exhibited in fact minor divergences in restriction patterns. The genetic diversity among the five isolates which contained plasmids and seven isolates which contained no plasmid DNA were examined by using restriction endonucleas digestions, Southern hybridization ofnifHDK,nifAB genes, andFrankia cryptic DNA fragments determined ‘at random’. Results indicate that genomic DNA digestion patterns and Southern hybridizations to anifHDK probe were not able to discriminate between closely related frankiae. On the other hand, plasmid presence, Southern hybridization to anifAB proble or to a crypticFrankia probe allowed us to delineate groupings of these isolates.

Bradyrhizobium japonicum USDA 125-Sp, USDA 138, and USDA 138-Sm had been used as inoculants for soybean ( Glycine max (L.) Merr.) in soils previously free of B. japonicum. At 8 to 13 years after their release, these strains were reisolated from soil samples. A total of 115 isolates were obtained through nodules, and seven colonies were obtained directly by a serological method. The stability of the inoculants was confirmed by comparing the reisolated cultures with their respective parental strains which had been preserved by being lyophilized or stored on a yeast extract-mannitol agar slant at 4°C. Comparisons were made on morphological and serological characters, carbon compound utilization (8 tested), intrinsic antibiotic resistance (9 tested), and enzymatic activity (19 tested). Mucous and nonmucous isolates of serogroup 125 were analyzed for symbiotic effectiveness and restriction fragment hybridization with a DNA probe. Our data suggest that the B. japonicum inoculants have survived for up to 13 years in the soils without significant mutation except for two reisolates with a slightly increased kanamycin resistance level.

We report that in DNA from one Frankia sp. strain at least some of the nif genes are located on a large indigenous plasmid of 190 kb. Using the cloned nitrogenase structural genes from Klebsiella pneumoniae as hybridization probes, homology was detected with a 5.6 kb EcoRI fragment from the Frankia sp. plasmid. This 5.6 kb fragment was cloned; used as a probe it hybridized to the nif K, D and H genes from Rhizobium meliloti and Klebsiella pneumoniae. The nif probes also hybridized on total DNA blots to a 10 kb EcoRI fragment that is not present on plasmid DNA, suggesting that the nif genes could be located on more than one replicon.

A method based on simultaneous short-term exposure to 14CO2 and 15N2 is described for studying nitrogen fixation and distribution in legumes relative to carbon assimilation and use. Equipment designed to accomodate experiments under natural conditions with very little disturbance of the N2 fixing association is used. It permits continuous measurement and regulation of variables such as air temperature, humidity and CO2 concentration as well as soil aeration. Measurements of distribution and use of assimilates, respiration of nodulated roots, quantitative N2 fixation and the distribution and fate of fixed N as a function of time lead to a precise estimation of C and N budgets for each labelling period. When experiments are done at several phenological stages they give a new insight into the complex C and N interrelations in legume symbiosis. A series of trials throughout the growth period of Glycine max (L.) Merr. cv. Hodgson demonstrated the sensitivity of the method. The development of the plants from vegetative to reproductive stages was accompanied by a complete change in the distribution patterns of current assimilates and products of nitrogen fixation. Maximum sink strength moved from the leaves to the pods and seeds which ended up receiving 70% of the incoming C and 35% of the fixed N. The fact that up to 85% of fixed N in the plants was in the reproductive organs at maturity can be accounted for by remobilisation from vegetative parts. The respiration of nodulated roots utilized 33% of carbon translocated to below-ground plant parts before nitrogen fixation started, but as much as 50% during the period of optimal fixation. The advantages and limitations of the isotopic method described are critically discussed as a prelude to future investigations.

The nodulation ability of variousFrankia strains isolated from the nodules of Casuarina were tested on two Casuarina species (C. equisetifolia andC. glauca), and onHippophae rhamnoides. We found that the isolates could be separated into two groups, some of them being unable to reinfect the Casuarina host-plant but infective onH. rhamnoides. Other isolates effectively nodulated the original Casuarina host-plant. The second purpose of this study was to examine the genetic diversity among the Casuarina-isolated strains using well-characterized symbiotic genes as hybridization probes. We found a relationship between nodulation characteristics and hybridization patterns.

Dinitrogen-fixing activity (acetylene reduction and 15 N 2 fixation) was found in an oily sludge originating from a petroleum refinery. Two representative dinitrogen-fixing bacterial strains were isolated from this oily waste. Their nitrogenase activity was effective when they were cultivated on sterilized sludge or simple carbon substrates (organic acid salts, sugars). Using the classical methods, these strains could not be unambiguously related to other diazotrophic taxa. The landfarming process is widely used for oily sludge disposal; this study shows that oily sludges are more than a simple carbon input into the soil but that they must also be considered as real sources of dinitrogen-fixing and probably degradative microorganisms.

To use the 15N natural abundance method to evaluate the symbiotic nitrogen fixation by actinorhizal trees, it is necessary to determine the isotopic identity of assimilated nitrogen from two sources: the soil and the air. This study reports an isotopic value of fixed nitrogen by two alder species (Alnus incana (L.) Moench and Alnus glutinosa (L.) Gaertn. growing on nitrogen-free medium in greenhouse experiments. The δ15N value of the aerial parts was −2. This value was stable with time and did not depend on the Frankia strains used. This value could be used to estimate the nitrogen fixation in the natural ecosystem. Other parameters such as the mobilization of nitrogen reserves and the choice of the reference plant must be investigated to apply this method. The nodules of these two alder species were enriched in 15N relative to the rest of the plant but there was no relationship between symbiotic effectiveness of Frankia strains and 15N enrichment of nodules. On the other hand, for naturally growing trees, an enrichment in 15N was found primarily in the vesicles of nodules that are the sites of nitrogen fixation.

Bardin René. Introduction à l'étude écologique de quelques groupements végétaux de Haute-Durance. In: Bulletin mensuel de la Société linnéenne de Lyon, 36ᵉ année, n°3, mars 1967. pp. 122-126.

The taxonomic position of nitrogen-fixing strains that were isolated from rhizosphere macerates of rice cultivated in the Binh Thanh region of Vietnam was determined by using polyphasic taxonomy. We determined the phylogenetic relationships of these organisms by performing DNA-rRNA hybridization experiments with a labeled rRNA probe from the type strain of Burkholderia cepacia, and we found that they belong to a single rRNA complex. Other members of this rRNA complex were also studied, and the N2-fixing strains were found to be closely related to B. cepacia. In addition, all members of the rRNA complex containing B. cepacia were studied by performing auxanographic and DNA-DNA hybridization experiments. Phenotypically and genotypically, the N2-fixing isolates constitute a single cluster together with two strains of clinical origin. These organisms constitute a new Burkholderia species, for which the name Burkholderia vietnamiensis is proposed; the type strain of this species is TVV75 (= LMG 10929). All members of this species can fix nitrogen. On the basis of our polyphasic taxonomy results and previously published data we concluded that the genus Burkholderia should be restricted to the following species: B. cepacia (the type species), Burkholderia mallei, Burkholderia pseudomallei, B. vietnamiensis, Burkholderia gladioli, Burkholderia caryophylli, Burkholderia plantarii, Burkholderia glumae, Burkholderia vandii, Burkholderia cocovenenans comb. nov., and Burkholderia andropogonis comb. nov. On the basis of genotypic and phenotypic results [Alcaligenes] eutrophus, [Burkholderia] solanacearum, and [Burkholderia] pickettii belong to two other clusters whose internal structures must be studied further.