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Screening of 10 environmental samples (mainly of rhizospheric origin) for lytic activity against two bacterial phytopathogens, Pseudomonas syringae pv. tomato DC3000 (CFBP2212) and Xanthomonas hortorum pv. vitians (CFBP3979), revealed that four samples harboured phages that were active against one strain. Only one sample, composed of an artisanal nettle liquid manure, contained phages able to lyse both strains. Electron microscopy revealed the presence of tailed bacteriophages, with all phages isolated on the Xanthomonas strain displaying a contractile tail typical of members of the family Myoviridae, whereas phages isolated on the Pseudomonas strain were related to members of the family Siphoviridae and short-tailed members of the family Podoviridae. Sequence analysis of the two Podoviridae-like bacteriophages isolated on Pseudomonas syringae pv. tomato, Pst_GM1 isolated from nettle manure and Pst_GIL1 isolated from infected lettuce leaves, revealed (i) strong homology between the two isolated phages, (ii) a high degree of sequence similarity to various phages isolated from various environments and from different geographical locations, and (iii) similarity of these phages to members of the family Autographiviridae, and more precisely, the genus Ghunavirus. Further investigation of the potential of nettle manure to host phages that could be active against a wider range of strains revealed that it contained phages active against 10 phytopathogens (out of 16 tested). Thus, nettle manure (and likely other plant manures) could represent a valuable source of phages, especially those targeting bacterial phytopathogens, in the same way that anthropized environments such as sewage are widely used as sources of phages active against opportunistic or acute pathogens of humans.

Forty Azospirillum strains were tested for their ability to synthesize N-acyl-homoserine lactones (AHLs). AHL production was detected for four strains belonging to the lipoferum species and isolated from a rice rhizosphere. AHL molecules were structurally identified for two strains: Azospirillum lipoferum TVV3 produces 3O,C8-HSL (N-3-oxo-octanoyl-homoserine-lactone), C8-HSL (N-3-octanoyl-homoserine-lactone), 3O,C10-HSL (N-3-oxo-decanoyl-homoserine-lactone), 3OH,C10-HSL (N-3-hydroxy-decanoyl-homoserine-lactone) and C10-HSL (N-3-decanoyl-homoserine-lactone), whereas A. lipoferum B518 produced 3O,C6-HSL (N-3-oxo-hexanoyl-homoserine-lactone), C6-HSL (N-3-hexanoyl-homoserine-lactone), 3O,C8-HSL, 3OH,C8-HSL and C8-HSL. Genes involved in AHL production were characterized for A. lipoferum TVV3 by generating a genomic library and complementing an AHL-deficient strain with sensor capabilities. Those genes, designated alpI and alpR, were found to belong to the luxI and luxR families, respectively. When cloned in a suitable heterologous host, alpI and alpR could direct the synthesis of the five cognate AHLs present in A. lipoferum TVV3. These two adjacent genes were found to be located on a 85kb plasmid. Southern hybridization experiments with probes alpI/R indicated that genes involved in AHL production in the three other AHL-producing strains were not closely related to alpI and alpR. This study demonstrates that AHL-based quorum-sensing is not widespread among the genus Azospirillum and could be found only in some A. lipoferum strains.

The plant-growth promoting rhizobacterium Azospirillum lipoferum strain 4B generates in vitro a stable phase variant designated 4VI at frequencies of 10−4 to 10−3 per cell per generation. Variant 4VI displays pleitropic modifications, such as the loss of swimming motility and the inability to assimilate certain sugars compared to the wild type. The mechanism underlying phase variation is unknown. To determine whether RecA-mediated processes are involved in phase variation, the recA gene of A. lipoferum 4B was cloned and sequenced and a recA mutant (termed 4BrecA) was constructed by allelic exchange. Strain 4BrecA showed increased sensitivity to UV and MMS compared with 4B and impaired recombinase activity. The ability to generate variants in vitro was not altered; the variants from 4BrecA exhibited all morphological and biochemical features characteristic of the variant generated by strain 4B. However, the frequency of variants generated by 4BrecA was increased by up to 10-fold. So, in contrast with many studies showing the abolition or a large reduction of the frequency of phase variation in recA mutants, this study describes an enhancement of phase variation in the absence of a functional recA.

We report the isolation of nitrogen fixing, phytohormone producing bacteria from sugarcane and their beneficial effects on the growth of micropropagated sugarcane plantlets. Detection of the nitrogen fixing bacteria by ARA-based MPN (acetylene reduction assay-based most probable number) method indicated the presence of up to 106 bacteria per gram dry weight of stem and 107 bacteria per gram dry weight of root of field-grown sugarcane. Two nitrogen fixing bacterial isolates were obtained from stem (SC11, SC20) and two from the roots (SR12, SR13) of field-grown plants. These isolates were identified as Enterobacter sp. strains on the basis of their morphological characteristics and biochemical tests. The isolate SC20 was further characterized by 16S rRNA sequence analysis, which showed high sequence similarity to the sequence of Enterobacter cloacae and Klebsiella oxytoca. All the isolates produced the phytohormone indoleacetic acid (IAA) in pure culture and this IAA production was enhanced in growth medium containing tryptophan. The bacterial isolates were used to inoculate micro-propagated sugarcane in vitro where maximum increase in the root and shoot weight over control was observed in the plantlets inoculated with strain SC20. By using the15N isotope dilution technique, maximum nitrogen fixation contribution (28% of total plant nitrogen) was detected in plantlets inoculated with isolate SC20.

Striga hermonthica (Del.) Benth. is an obligate parasitic weed of tropical cereals whose rhizosphere can also be colonised by bacteria of the genus Azospirillum. A previous study demonstrated that the two organisms (Azospirillum and striga) interacted during cerealroot colonisation. Two strains of A. brasilense isolated from an African sorghum rhizosphere prevented the germination of striga seeds although they were stimulated to germinate by the presence of sorghum roots. Azospirillum cells suspended in a synthetic germination stimulant (GR24) did not inhibit striga seed germination, but did block radicle elongation. Those radicles had an abnormal morphology, and contained no vacuolated cells in the root elongation zone. Lipophilic compounds extracted from the medium of bacteria in the log and stationary growth phases prevented the germination of striga seeds.

Laccase, a p -diphenol oxidase typical of plants and fungi, has been found recently in a proteobacterium, Azospirillum lipoferum . Laccase activity was detected in both a natural isolate and an in vitro-obtained phase variant that originated from the laccase-negative wild type. In this study, the electron transport systems of the laccase-positive variant and its parental laccase-negative forms were compared. During exponential (but not stationary) growth under fully aerobic (but not under microaerobic) conditions, the laccase-positive variant lost a respiratory branch that is terminated in a cytochrome c oxidase of the aa 3 type; this was most likely due to a defect in the biosynthesis of a heme component essential for the oxidase. The laccase-positive variant was significantly less sensitive to the inhibitory action of quinone analogs and fully resistant to inhibitors of the bc 1 complex, apparently due to the rearrangements of its respiratory system. We propose that the loss of the cytochrome c oxidase-containing branch in the variant is an adaptive strategy to the presence of intracellular oxidized quinones, the products of laccase activity.

Maize seeds were inoculated with a commercial inoculant containing 1.3 × 107 Azospirillum lipoferum CRT1 cells. After 24 or 48 h, bacteria were washed from the seed surface. Washed and unwashed seeds were then planted in pots containing perlite and grown for 28 days under greenhouse conditions. Whatever the density of Azospirillum at planting, the number of these bacteria at the end of the experiment was similar (1.9-8.0 × 107 bacteria·plant-1). However, comparison of root surface areas of the plants were different depending on the period of contact between seeds and the density of the inoculum. Twenty-four hours of contact was not sufficient to increase root growth surface areas. Contact for 48 h permitted us to obtain root surface areas comparable with those measured after a continuous contact. These results showed that in order to promote maize root surface areas, an optimal density of Azospirillum is not required during the whole cultural cycle. This optimal density is indispensable only up to the emergence of the radicle.Key words: Azospirillum, maize, inoculation, PGPR.

Les contraintes majeures qui limitent la productivité des agroécosystèmes en milieu arides sont généralement attribuées aux carences minérales [plus particulièrement en phosphore (P) et en azote (N)], à un déficit en eau, aux processus d’érosion hydrique et éolienne, ainsi qu’à des perturbations dans le biofonctionnement du sol (faible diversité génétique et fonctionnelle de la microflore tellurique, etc.). Lorsque ces milieux ne sont pas soumis à une surexploitation, ils présentent une activité biologique élevée. Il a été ainsi suggéré que leur valorisation via des stratégies culturales adéquates pouvait réactiver les processus écologiques impliqués dans le maintien de la fertilité des sols et ainsi assurer une meilleure productivité de l’agro-écosystème. La plupart des plantes de ces régions arides (espèces sauvages ou cultivées) sont associées à des champignons mycorhiziens à arbuscules (CMA). Ces symbiotes fongiques sont reconnus comme étant des composantes microbiennes majeures dans le déroulement des principaux cycles biogéochimiques des sols (C, P et N) et en conséquence dans le développement des plantes en améliorant leur nutrition minérale mais aussi hydrique et leur état sanitaire. Paradoxalement, leur valorisation au sein d’itinéraires culturaux reste encore très limitée, voire inexistante. Ce chapitre a pour objectifs principaux de décrire le rôle des CMA dans le maintien et l’amélioration de la fertilité des sols et sur le développement des plantes tant au niveau de leur nutrition minérale, de leur résistance au stress hydrique que de l’état sanitaire des sols cultivés (ex. : lutte contre Striga spp.). Des stratégies d’ingénierie écologique seront ensuite proposées afin d’optimiser certaines techniques culturales (ex. : agroforesterie) dans le rôle de maintien du potentiel infectieux mycorhizogène des sols en relation avec une pleine expression des propriétés de la symbiose mycorhizienne sur les plantes cultivées, permettant ainsi d’atteindre des rendements culturaux compatibles avec les besoins des populations locales.

The overexploitation of natural resources, resulting in an increased need for arable lands by local populations, causes a serious dysfunction in the soil's biological functioning (mineral deficiency, salt stress, etc.). This dysfunction, worsened by the climatic conditions (drought), requires the implementation of ecological engineering strategies allowing the rehabilitation of degraded areas through the restoration of essential ecological services. The first symptoms of weathering processes of soil quality in tropical and Mediterranean environments result in an alteration of the plant cover structure with, in particular, the pauperization of plant species diversity and abundance. This degradation is accompanied by a weakening of soils and an increase of the impact of erosion on the surface layer resulting in reduced fertility of soils in terms of their physicochemical characteristics as well as their biological ones (e.g., soil microbes). Among the microbial components particularly sensitive to erosion, symbiotic microorganisms (rhizobia, Frankia, mycorrhizal fungi) are known to be key components in the main terrestrial biogeochemical cycles (C, N and P). Many studies have shown the importance of the management of these symbiotic microorganisms in rehabilitation and revegetation strategies of degraded environments, but also in improving the productivity of agrosystems. In particular, the selection of symbionts and their inoculation into the soil were strongly encouraged in recent decades. These inoculants were selected not only for their impact on the plant, but also for their ability to persist in the soil at the expense of the residual native microflora. The performance of this technique was thus evaluated on the plant cover, but its impact on soil microbial characteristics was totally ignored. The role of microbial diversity on productivity and stability (resistance, resilience, etc.) of eco- and agrosystems has been identified relatively recently and has led to a questioning of the conceptual bases of controlled inoculation in sustainable land management. It has been suggested that the environmental characteristics of the area to rehabilitate should be taken into account, and more particularly its degradation level in relation to the threshold of ecological resilience. This consideration should lead to the optimization of the cultural practices to either (i) restore the original properties of an ecosystem in case of slightly degraded environments or (ii) transform an ecosystem in case of highly degraded soils (e.g., mine soils). In this chapter, we discuss, through various examples of experiments conducted in tropical and Mediterranean areas, the performance of different strategies to manage the microbial potential in soils (inoculation of exotic vs. native species, inoculation or controlled management potential microbial stratum via aboveground vegetation, etc.) based on the level of environmental degradation.

International audience; Plant-beneficial effects of bacteria are often underestimated, especially for well-studied strains associated with pathogenicity or originating from other environments. We assessed the impact of seed inoculation with the emblematic bacterial models Agrobacterium tumefaciens C58 (plasmid-cured) or Escherichia coli K-12 on maize seedlings in nonsterile soil. Compared with the noninoculated control, root biomass (with A. tumefaciens or E. coli) and shoot biomass (with A. tumefaciens) were enhanced at 10 days for 'PR37Y15' but not 'DK315', as found with the phytostimulator Azospirillum brasilense UAP-154 (positive control). In roots as well as in shoots, Agrobacterium tumefaciens and E. coli triggered similar (in PR37Y15) or different (in DK315) changes in the high-performance liquid chromatography profiles of secondary metabolites (especially benzoxazinoids), distinct from those of Azospirillum brasilense UAP-154. Genome sequence analysis revealed homologs of nitrite reductase genes nirK and nirBD and siderophore synthesis genes for Agrobacterium tumefaciens, as well as homologs of nitrite reductase genes nirBD and phosphatase genes phoA and appA in E. coli, whose contribution to phytostimulation will require experimental assessment. In conclusion, the two emblematic bacterial models had a systemic impact on maize secondary metabolism and resulted in unexpected phytostimulation of seedlings in the Azospirillum sp.-responsive cultivar.

Azospirillum lipoferum 4B and non-motile A. lipoferum 4T have been simultaneously isolated from rice rhizosphere at the same frequency. A. lipoferum 4T showed stable morphological and metabolic traits which are atypical for A. lipoferum species such as lack of motility, carbohydrate metabolism and laccase activity. Inoculation experiments showed that A. lipoferum 4T, but not A. lipoferum 4B, needed rice roots to stabilize in sterile soil. Both strains were able to colonize efficiently rice roots (108 cfu g−1 fresh roots) but motile form 4B remained dominant. In spite of their phenotypical differences, A. lipoferum 4B and 4T co-existed without exclusion in sterile soil (planted or not) and rice rhizosphere. Inoculation of rice roots with A. lipoferum 4B showed that rice rhizosphere enhanced the frequency of appearance of stable non-motile forms (40%). This percentage was weaker in plantlet growth medium (4%). However, these non-motile bacteria kept the same biochemical traits than the motile parental strain 4B (carbohydrates metabolism, laccase activity).

La surexploitation des ressources naturelles, consécutive à un besoin accru en terres arables des populations locales, entraîne de graves dysfonctionnements dans le bio-fonctionnement du sol (carences minérales, stress salins, etc.). Ce dysfonctionnement, aggravé par les aléas climatiques (sécheresse), nécessite la mise en oeuvre de stratégies d'ingénierie écologique permettant une réhabilitation des milieux dégradés via la restauration de services écologiques essentiels. Les premiers symptômes du processus d'altération de la qualité des sols des milieux tropicaux et méditerranéens se traduisent par une altération de la structure de la strate épigée avec, en particulier, une paupérisation de la diversité et de l'abondance des espèces végétales. Cette dégradation est accompagnée par une fragilisation des sols et une augmentation de l'impact de l'érosion sur la couche superficielle, entraînant une baisse de la fertilité des sols au niveau de leurs caractéristiques physico-chimiques, mais aussi biologiques (par exemple, la microflore tellurique). Parmi les composantes microbiennes particulièrement sensibles à ces phénomènes d'érosion, les microorganismes symbiotiques (rhizobia, champignons mycorhiziens) sont connus pour être des composantes clés dans le fonctionnement des principaux cycles biogéochimiques telluriques (C, N et P). De nombreux travaux ont montré l'importance de la gestion de ces microorganismes symbiotiques dans les stratégies de réhabilitation et de revégétalisation des milieux dégradés, mais également pour améliorer la productivité des agro-systèmes. En particulier, la sélection de symbiotes et leur inoculation en masse dans le sol de plantation ou de culture ont été fortement encouragées au cours de ces dernières décennies. Ces inoculants étaient sélectionnés, non seulement pour leur impact sur la plante, mais également pour leur capacité à persister dans le sol, au détriment de la microflore résiduelle native. La performance de cette technique était donc évaluée au niveau du couvert végétal, mais les répercussions au niveau des caractéristiques de la microflore tellurique étaient généralement ignorées. Le rôle de la diversité microbienne sur la productivité et la stabilité (résistance, capacité de résilience, etc.) des éco- et agro-systèmes a été mis en évidence relativement récemment et a conduit à une remise en cause des bases conceptuelles de l'inoculation contrôlée dans la gestion durable des terres. Il a été notamment suggéré que les caractéristiques du milieu à réhabiliter devaient être prises en compte, et plus particulièrement son niveau de dégradation par rapport au seuil de résilience écologique. Cette prise en compte devrait permettre d'optimiser les pratiques culturales pour soit (i) redonner à l'écosystème ses propriétés originelles dans le cas de milieux peu dégradés ou (ii) transformer un écosystème dans le cas de sols hautement dégradés (par exemple, les sols miniers). Dans ce chapitre, nous évoquerons, à travers différents exemples d'expérimentations réalisées en milieu tropical et méditerranéen, les performances des différentes stratégies de gestion du potentiel microbien des sols (inoculation d'espèces exotiques vs des espèces natives, inoculation contrôlée vs la gestion du potentiel microbien via la strate végétale épigée, etc.) suivant le niveau de dégradation du milieu.

International audience; Background and aims Many plant-beneficial microorganisms can influence secondary plant metabolism, but whether these effects add up when plants are coinoculated is unclear. This issue was assessed, under field conditions, by comparing the early impacts of seed inoculation on secondary metabolite profiles of maize at current or reduced mineral fertilization levels. Methods Maize seeds were inoculated singly with selected strains from bacterial genera Pseudomonas and Azospirillum or mycorrhizal genus Glomus, or with these strains combined two by two or all three together. At 16 days, maize root methanolic extracts were analyzed by RP-HPLC and secondary metabolites (phenolics, flavonoids, xanthones, benzoxazionoids, etc.) identified by LC/MS. Results Inoculation did not impact on plant biomass but resulted in enhanced total root surface, total root volume and/or root number in certain inoculated treatments, at reduced fertilization. Inoculation led to qualitative and quantitative modifications of root secondary metabolites, particularly benzoxazinoids and diethylphthalate. These modifications depended on fertilization level and microorganism(s) inoculated. The three selected strains gave distinct results when used alone, but unexpectedly all microbial consortia gave somewhat similar results. Conclusions The early effects on maize secondary metabolism were not additive, as combining strains gave effects similar to those of Glomus alone. This is the first study demonstrating and analyzing inoculation effects on crop secondary metabolites in the field.

Effects of soil management on soil characteristics were investigated on the rhizosphere (RPP) and the non-rhizosphere (NRPP) siol of a re-grass vertisol under #Digitaria decumbens$ and in the soil under continuous cultivation (CC). A low energy technique allowed to separate eight size and density fractions, including macro- and micro-aggregates while preserving soil bacteria. Organic C and N, microbial biomass C and the number of total bacteria (AODC) and of #Azospirillum brasilense$ and their distribution were determined in soil fractions isolated from the CC, NRPP and RPP soils. Soil macroaggregates (>2000 micrometers) were similarly predomminant in the NRPP and RPP soils when the dispersible clay size fraction (2000 micrometers) and from the root fraction, not from the finer separates. The proportion of organic C as microbial biomass C revealed the low turnover of microbial C in the PP situations, especially in the clay size fraction of the NRPP soil. A common shift of AODC toward the finer separates from planted soils (CC and RPP) revealed the influence of living plants on the distribution of soil bacteria. The relative abundance of #A. brasilense$ showed the presence of the active roots of #Digitaria$ in the macroaggregates and their contact with the dispersible clay size fraction of the rhizosphere soil. (Résumé d'auteur)

A mixture of 5-(12-heptadecenyl)-resorcinol as the main compound with four other alkyl resorcinols was isolated from rice root exudate. The chemical characterization was done by HPLC/MS coupling, 1H and 13C NMR.

DNA/DNA hybridization, plasmid content and partial 16S rDNA sequence were determined to confirm the relationship between two Azospirillum strains, 4B and 4T, isolated from the same rice rhizosphere. The partial 16S rDNA sequence was determined for 9 strains belonging to 5 Azospirillum species which included Azospirillum lipoferum strains 4B and 4T, and was compared to a set of ribosomal sequences from other bacteria of the alpha subdivision of the Proteobacteria. Four Azospirillum species sequences were found to form a coherent group, whereas A. halopraeferens was found to be more divergent. The five Azospirillum species were closely related to Magnetospirillum and to a lesser extent to Rhodospirillum, with which they share some morphological features. The partial 16S rDNA sequences of the two strains 4B and 4T were identical and confirmed the closeness of these two strains. Plasmid content and DNA/DNA hybridization data (S1 nuclease and ΔTm) grouped Azospirillum strains 4B and 4T with the other strains of species Azospirillum lipoferum (USA5a, 59b, BR17). Other biochemical and genetic characters confirmed the grouping of Azospirillum strains 4B and 4T together with A. lipoferum.

In non-motile forms of Azospirillum lipoferum isolated from the rhizosphere of rice, polyphenol oxidase activity was observed which correlated with production of a dark-brown pigment. Using a combination of substrate/inhibitor specificity tests, intracellular enzyme extracts of non-motile strains were clearly demonstrated to have a laccase activity by oxidising various o- and p-diphenols. This work is the first report on laccase activity in Azospirillum.

International audience; • Most Azospirillum plant growth-promoting rhizobacteria (PGPR) benefit plant growth through source effects related to free nitrogen fixation and ⁄ or phytohormone production, but little is known about their potential effects on plant physiology. These effects were assessed by comparing the early impacts of three Azospirillum inoculant strains on secondary metabolite profiles of two different maize (Zea mays) cultivars. • After 10 d of growth in nonsterile soil, maize methanolic extracts were analyzed by reverse-phase high-performance liquid chromatography (RP-HPLC) and secondary metabolites identified by liquid chromatography ⁄ mass spectrometry (LC ⁄ MS) and nuclear magnetic resonance (NMR). • Seed inoculation resulted in increased shoot biomass (and also root biomass with one strain) of hybrid PR37Y15 but had no stimulatory effect on hybrid DK315. In parallel, Azospirillum inoculation led to major qualitative and quantitative modifications of the contents of secondary metabolites, especially benzoxazinoids, in the maize plants. These modifications depended on the PGPR strain · plant cultivar combination. • Thus, Azospirillum inoculation resulted in early, strain-dependent modifications in the biosynthetic pathways of benzoxazine derivatives in maize in compatible interactions. This is the first study documenting a PGPR effect on plant secondary metabolite profiles, and suggests the establishment of complex interactions between Azospirillum PGPR and maize.

Erratum paru dans Plant and soil (2009) 325 (1-2) p. 351-352 http://dx.doi.org/10.1007/s11104-009-0100-4. Diversity, structure and productivity of above-ground compartment of terrestrial ecosystems have been generally considered as the main drivers of the relationships between diversity and ecosystem functioning. More recently it has been suggested that plant population dynamics may be linked with the development of the below-ground community. The biologically active soil zone where root-root and rootmicrobe communications occur is named "Rhizosphere" where root exudates play active roles in regulating rhizosphere interactions. Root exudation can regulate the soil microbial community, withstand herbivory, facilitate beneficial symbioses, modify the chemical and physical soil properties and inhibit the growth of competing plant species. In this review, we explore the current knowledge assessing the importance of root exudates in plant interactions, in communications between parasitic plants and their hosts and how some soil microbial components could regulate plant species coexistence and change relationships between plants. This review will be focussed on several well documented biological processes regulating plant-plant communications such as exotic plant species invasions, negative root-root communication (allelopathy) and parasitic plant / host plant interactions and how some soil microbial components can interfere with signal traffic between roots. The reported data show that the overall effect of one plant to another results from multiple interacting mechanisms where soil microbiota can be considered as a key component.

The nitrogen-fixing bacteria, Azospirillum lipoferum and Azospirillum brasilense , contain several large plasmids. Cryptic plasmids of about 150 MDa have only been detected in A. lipoferum strains. The 150 MDa plasmid of A. lipoferum 4B used as probe hybridized with plasmids of comparable size in all A. lipoferum strains. Total DNA of six A. lipoferum strains were digested and hybridized with the p150 probe. Restriction fragment length polymorphism (RFLP) patterns differ from that of strain 4B except for one strain isolated from the same rhizosphere. However, a high number of hybridizing bands in each strain was close to 150 MDa, showing a high degree of similarity between 150 MDa plasmids. This strongly suggests that A. lipoferum 150 MDa plasmids, though not identical, have evolved from a common ancestor and likely constitute a family of plasmids sharing unidentified common functions.

The reliability of Tn 5 as labelling tool was investigated in soil microcosm. The occurence of a selective in soil microcosm. The occurence of resistances encoded by Tn 5 nptII gene was assesed by kanamycin and neomycin amendment. The bioassay developed to monitor the persistence of the soil-added kanamycin did not detect the antibiotic activity in soil extract. A nptII-engineered Escherichia coli strain showed no enhanced survival in aminoglycoside amended soil. Tn 5-marker properties were investigated within indigenous bacteria to determine the specificity of labelling to follow the fate of recombinant DNA. Kanamycin and neomycin resistant population levels made Tn 5 aminoglycoside-resistance phenotype non-sensitive enough to select a soil dissemination of the labelled DNA. The unexpected occurrence of homologous sequences among soil organisms also prevented Tn 5 from being a specific DNA marker. By contrast, colony hybridization did not reveal homology to nptII suggesting its use as a reliable gene transfer indicator.

International audience; A quorum-quenching approach was exploited in order to identify functions regulated by quorum-sensing (QS) in the plant growth-promoting bacterium Azospirillum lipoferum. The AttM lactonase from Agrobacterium tumefaciens was shown to enzymatically inactivate N-acyl homoserine lactones (AHLs) produced by two A. lipoferum strains. The targeted analysis of several phenotypes revealed that in strain B518, a rice endophyte, AHL inactivation abolished pectinase activity, increased siderophore synthesis and reduced indoleacetic acid production (in stationary phase) but no effect was observed on cellulase activity or on swimming and swarming motilities. None of the tested phenotypes appeared to be under QS regulation in strain TVV3 isolated from the rice rhizosphere. Moreover, AHL inactivation had no deleterious effect on the phytostimulatory effect of the two strains in vitro. A global proteomic approach revealed little modification of protein patterns when comparing attM-expressing TVV3 and the wild-type strain, but numerous proteins appeared to be regulated by the AHL-mediated QS system in strain B518. Several proteins identified by MSeMS analysis were revealed to be implicated in transport (such as OmaA) and chemotaxis (ChvE). Altogether, the results indicate that in A. lipoferum, QS regulation is strain-specific and is dedicated to regulating functions linked to rhizosphere competence and adaptation to plant roots.

The prevalence of bacteriophages was investigated in 24 strains of four species of plant growth-promoting rhizobacteria belonging to the genus Azospirillum . Upon induction by mitomycin C, the release of phage particles was observed in 11 strains from three species. Transmission electron microscopy revealed two distinct sizes of particles, depending on the identity of the Azospirillum species, typical of the Siphoviridae family. Pulsed-field gel electrophoresis and hybridization experiments carried out on phage-encapsidated DNAs revealed that all phages isolated from A. lipoferum and A. doebereinerae strains had a size of about 10 kb whereas all phages isolated from A. brasilense strains displayed genome sizes ranging from 62 to 65 kb. Strong DNA hybridizing signals were shown for most phages hosted by the same species whereas no homology was found between phages harbored by different species. Moreover, the complete sequence of the A. brasilense Cd bacteriophage (ΦAb-Cd) genome was determined as a double-stranded DNA circular molecule of 62,337 pb that encodes 95 predicted proteins. Only 14 of the predicted proteins could be assigned functions, some of which were involved in DNA processing, phage morphogenesis, and bacterial lysis. In addition, the ΦAb-Cd complete genome was mapped as a prophage on a 570-kb replicon of strain A. brasilense Cd, and a region of 27.3 kb of ΦAb-Cd was found to be duplicated on the 130-kb pRhico plasmid previously sequenced from A. brasilense Sp7, the parental strain of A. brasilense Cd.

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.

International audience; Striga hermonthica (Del.) Benth is an obligate root hemi-parasite of several cereals. Its effect on cereal crops is the main constraint for food production in sub-Saharan Africa. Various control methods have been already proposed, but the infestation by these parasitic plants persists. An appropriated method for Striga management adapted for the African farmer is very much needed. In this study, amendment of soil infested by this phytoparasite with Cubitermes mound powder is proposed as chemical amendment and natural microbial inoculum, to promote plant growth and reduce damage by S. hermonthica on sorghum (Sorghum bicolor L.). The influence of Cubitermes mound powder on the development of several microbial groups (arbuscular mycorrhizal fungi, actinomycetes, saprophytic fungi) was investigated in a pot experiment with sorghum cultured in a sandy soil infested by S. hermonthica. In the amended soil, sorghum growth and mycorrhizal colonization of sorghum plants were significantly greater than in the control treatment. Mycorrhizal colonization was negatively correlated with the number of emerged Striga plants per pot and positively correlated with sorghum growth. The relationship with substrate-induced respiration (SIR) responses showed that amended soil was characterized by its response to hydroxybutyric acid (catabolic marker of mycorrhizal colonization) and non-amended soil by its response to phenylalanine. We noted that the number of emerged Striga plants in amended pots was significantly decreased. Since Cubitermes mound suspensions did not affect Striga seed germination under axenic conditions, it suggests that the amendment with Cubitermes powder reduces S. hermonthica infestation indirectly, i.e. via its effect on the indigenous soil microflora. Overall, it appears that management of Cubitermes mounds is a promising strategy to consider for effective protection of sorghum from Striga infestation.

The effect of direct inoculation of seeds with the plant growth promoting rhizobacteria (PGPR) Azospirillum lipoferum CRT1 was assessed on maize (Zea mays) grown for 35 days after sowing (d.a.s.) in controlled conditions (greenhouse) in a luvisol soil from south-eastern France. WhinRhizo® software was used to describe the following changes in the root system morphology for each plant: distribution and average root diameter, root surface and the number of tips. The stress at breakage and stiffness of the roots in tension were also determined. Evaluation of biochemical components of roots was achieved by direct Attenuated Total Reflectance (or reflection) (ATR)-Fourier transform infrared (FTIR) on root section. Inoculated roots exhibited significantly larger numbers of tips and extending surface to rhizosphere when compared to controls. Measured mechanical parameters of inoculated roots showed a slight increase in rupture stress up to the largest diameter (1.2 mm) when compared to controls. Stiffness (Young’s modulus) values were nearly constant for inoculated plants with higher values than for non-inoculated plants at day 26 and day 35. Using Principal Components Analysis of ATR-FTIR profiles, the polysaccharide enrichment of inoculated roots compared to controls was found at day 35. Noticeable absorbance at wavenumber specific to aromatic ether (lignin) was observed in control plants. All these data had a pattern of immature root properties, when maize was inoculated with Azospirillum lipoferum CRT1. Observed modifications of root development are possibly conducive to unseen beneficial effects, like water retention, resistance to mechanical stress, or root litter quality. Studies on more mature plants are required to assess if the differences between inoculated and control plants would persist or become accentuated with time until harvest.

Forty Azospirillum strains were tested for their ability to synthesize N-acyl-homoserine lactones (AHLs). AHL production was detected for four strains belonging to the lipoferum species and isolated from a rice rhizosphere. AHL molecules were structurally identified for two strains: Azospirillum lipoferum TVV3 produces 3O,C(8)-HSL (N-3-oxo-octanoyl-homoserine-lactone), C(8)-HSL (N-3-octanoyl-homoserine-lactone), 3O,C(10)-HSL (N-3-oxo-decanoyl-homoserine-lactone), 3OH,C(10)-HSL (N-3-hydroxy-decanoyl-homoserine-lactone) and C(10)-HSL (N-3-decanoyl-homoserine-lactone), whereas A. lipoferum B518 produced 3O,C(6)-HSL (N-3-oxo-hexanoyl-homoserine-lactone), C(6)-HSL (N-3-hexanoyl-homoserine-lactone), 3O,C(8)-HSL, 3OH,C(8)-HSL and C(8)-HSL. Genes involved in AHL production were characterized for A. lipoferum TVV3 by generating a genomic library and complementing an AHL-deficient strain with sensor capabilities. Those genes, designated alpI and alpR, were found to belong to the luxI and luxR families, respectively. When cloned in a suitable heterologous host, alpI and alpR could direct the synthesis of the five cognate AHLs present in A. lipoferum TVV3. These two adjacent genes were found to be located on a 85 kb plasmid. Southern hybridization experiments with probes alpI/R indicated that genes involved in AHL production in the three other AHL-producing strains were not closely related to alpI and alpR. This study demonstrates that AHL-based quorum-sensing is not widespread among the genus Azospirillum and could be found only in some A. lipoferum strains.

Azospirillum is a plant growth-promoting rhizobacterium associated with roots of monocots, including important crops, such as wheat, corn, and rice. Both in greenhouse and in field trials, Azospirillum was shown to exert beneficial effects on plant growth and crop yields, under various soil and climatic conditions (15). The actual benefit from biological nitrogen fixation has been questioned, and plant growth promotion by Azospirillum seems to be due mainly to production of phytohormones (9, 15). The most abundant phytohormone produced is indole-3-acetic acid, allowing an increase in the number of lateral roots and root hairs; this results in a higher absorption of water and minerals from the soil (9). Bacterial populations, especially in soil or in the rhizosphere, have to endure fluctuating environmental conditions. Bacteria have evolved different strategies to adapt to these environments. Phase variation is one adaptive process by which bacteria undergo frequent, usually reversible phenotypic changes resulting from genetic or epigenetic alterations at specific genetic loci (29). This process is used by several bacterial species to generate intrapopulation diversity that increases bacterial fitness and is important in niche adaptation or to escape host defenses (reviewed in references 46 and 55). In contrast to spontaneous mutations, which occur at a frequency of approximately 10−8 to 10−6 mutations per growing cell per generation, phase variation occurs at frequencies higher than 10−5 switches per cell per generation (29). Various mechanisms control phase variation. These include DNA inversion or duplication, deletion, transposition, homologous recombination, slipped-strand mispairing, and differential methylation (reviewed in reference 55). Phase variation in pathogenic bacteria—for example, switching of type IV pili in Neisseria gonorrhoeae (27), differential expression of surface layer proteins in Campylobacter fetus (16), and loss of virulence in the phytopathogen Ralstonia solanacearum (43)—has been extensively studied. However, phase variation is not restricted to pathogenic bacteria. Indeed, it also occurs during rhizosphere colonization of various plants by several strains of beneficial plant-associated Pseudomonas (1, 48). For instance, the regulation of biocontrol traits (production of antifungal metabolites, chitinases, and biosurfactants) by phase variation was reported for Pseudomonas spp. strains (54). Azospirillum lipoferum 4B, a strain isolated from a rice rhizosphere, generates in vitro at high frequencies (10−4 to 10−3 per cell per generation) a stable phase variant named 4VI (3, 30). Variant colonies are readily distinguishable from wild-type colonies by the differential absorption of dyes incorporated into the growth medium. The 4VI variant exhibits pleiotropic modifications; it gained assimilation of certain sugars but lost the ability to assimilate other sugars (3), to reduce triphenyl tetrazolium chloride, to bind some dyes, to swim (4), and to reduce nitrous oxide (our unpublished results). A. lipoferum 4T, a nonswimming strain displaying all of the features of the 4VI variant, and strain A. lipoferum 4B have been isolated simultaneously from rice rhizosphere at the same frequency (8). A. lipoferum 4T retains the ability to efficiently colonize rice roots (2). Like 4VI, A. lipoferum 4T was found to be genetically very close to A. lipoferum 4B (3, 8, 28), suggesting that A. lipoferum 4T could in fact be a 4VI variant of strain 4B generated within the soil ecosystem. We recently showed that inactivating recA in strain 4B resulted in a higher frequency of generation of variants (57), contrasting with many studies of other bacteria showing either no effect or the abolition of phase variation process in recA mutants. This finding suggests the possibility that phase variation is accompanied by genetic rearrangements. As of now, the molecular mechanism underlying these nonreversible changes in A. lipoferum 4B remains to be determined. In this study, the objective was to determine whether genomic rearrangements take place during phase variation of strain 4B and related Azospirillum strains. The current work shows that genomic rearrangements are concomitant with phase variation in three species of Azospirillum.

International audience; Heavy nitrogen fertilisation is often implemented in maize cropping systems, but it can have negative environmental effects. Nitrogen-fixing, phytohormone-producing Azospirillum plant growth-promoting rhizobacteria (PGPR) have been proposed as crop inoculants to maintain high yield when decreasing nitrogen fertilisation. In this context, agronomic and ecological effects of the inoculation of maize seeds with the PGPR Azospirillum lipoferum CRT1 were studied in two consecutive years. The inoculant was recovered from maize at 105 CFU g-1 root or higher. Inoculation enhanced root growth and development based on results of root biomass, rooting depth and/or parameters describing root system architecture, and a transient positive effect on shoot height was observed in the first year. Inoculation did not increase yield, but reducing mineral nitrogen fertilisation had only a minor effect on yield. This suggests that the lack of positive effect of the PGPR on yield was due to the fact that the whole field was heavily fertilised in years prior to the start of the experiment. Soil nitrogen levels decreased during the 2 years of the study, and the inoculant had no effect on residual soil nitrogen levels at harvest. Inoculation had no impact on Fusarium symptoms and concentration of the mycotoxin deoxynivalenol in maize kernels, but both were influenced by the interaction between inoculation and nitrogen fertilisation level. Inoculation did not influence meso/macrofaunal soil populations, but had a small but significant effect (smaller than the effect of added nitrogen) on decomposition, nitrogen mineralisation and mesofaunal colonisation of maize leaves (in litter bags). Overall, the ecological impact of seed inoculation with the PGPR A. lipoferum CRT1 was small, and its magnitude was smaller than that of chemical nitrogen fertilisation.

Nitrogen-fixing plant growth-promoting rhizobacteria (PGPR) from the genus Pseudomonas have received little attention so far. In the present study, a nitrogen-fixing phytohormone-producing bacterial isolate from kallar grass (strain K1) was identified as Pseudomonas sp. by rrs (16S ribosomal RNA gene) sequence analysis. rrs identity level was high with an uncharacterized marine bacterium (99%), Pseudomonas sp. PCP2 (98%), uncultured bacteria (98%), and Pseudomonas alcaligenes (97%). Partial nifH gene amplified from strain K1 showed 93% and 91% sequence similarities to those of Azotobacter chroococcum and Pseudomonas stutzeri, respectively. The effect of Pseudomonas strain K1 on rice varieties Super Basmati and Basmati 385 was compared with those of three non-Pseudomonas nitrogen-fixing PGPR (Azospirillum brasilense strain Wb3, Azospirillum lipoferum strain N4 and Zoogloea strain Ky1) used as single-strain inoculants. Pseudomonas sp. K1 was detected in the rhizosphere of inoculated plants by enrichment culture in nitrogen-free growth medium, which was followed by observation under the microscope as well as by PCR using a rrs-specific primer. For both rice varieties, an increase in shoot biomass and/or grain yield over that of noninoculated control plants was recorded in each inoculated treatment. The effect of Pseudomonas strain K1 on grain yield was comparable to those of A. brasilense Wb3 and Zoogloea sp. Ky1 for both rice varieties. These results show that nitrogen-fixing pseudomonads deserve attention as potential PGPR inoculants for rice.

The physical organization of phytobeneficial genes was investigated in the plant growth-promoting rhizobacterium Azospirillum lipoferum 4VI by hybridization screening of a bacterial artificial chromosome (BAC) library. Pulsed-field gel electrophoresis gave an estimated 5.7-Mb genome size for strain 4VI and a coverage level of 9 for the BAC library. The phytobeneficial genes nifH (associative nitrogen fixation) and ipdC (synthesis of the phytohormone indoleacetic acid) are chromosomal, but no BAC clone containing both genes was found, pointing to the absence of any genetic island containing nifH and ipdC. A 11.8-kb fragment containing nifH was analyzed. Neighboring genes implicated in nitrogen fixation (nifH, draT, draG) or not (arsC, yafJ and acpD) were organized as in A. brasilense. In contrast, the region located downstream of acpD contained four housekeeping genes (i.e. genes encoding DapF-, MiaB- and FtsY-like proteins, as well as gene amn) and differed totally from the one found in A. brasilense.

Two variants have been isolated from the wild-type Azospirillum lipoferum strain 4B. The first variant, 4V(I), spontaneously emerged from the wild-type at frequencies in the order of 10(-4) to 10(-3) per cell generation. Compared to the wild-type, the 4V(I) variant gained (production of a carotenoid-like pigment, assimilation of certain carbohydrates) and lost (swimming motility, reduction of triphenyl tetrazolium chloride, acid production from certain sugars) apparently unrelated phenotypic characteristics. Only from the 4V(I) variant, a second atypical stable form, variant 4V(II), which acquired laccase activity and ability to produce melanin, appeared under very specific conditions, namely growth at extremely low oxygen concentrations. Neither of the variants was able to revert to the parental phenotype. The results suggest that atypical non-motile laccase-positive isolates of A. lipoferum that are found in the rice rhizosphere originate from wild-type (motile, laccase-negative) cells via a two-step phenotypic switching event, a non-motile laccase-negative variant being an intermediate phase.

Activity of bacterial Azospirillum lipoferum laccase on phenolic derivatives was studied by spectrophotometry, HPLC and GC/MS. Phenolic compounds of the syringic type (aldehyde, acid or acetophenone) were transformed into 2,6-dimethoxy-1,4-benzoquinone (2,6-DMBQ). Comparison has been made with the fungal Pyricularia oryzae laccase. Transformation of other phenolic acid derivatives, related to lignin metabolism, have been studied with both bacterial and fungal enzymes, using spectrophotometric analysis.

Azospirillum lipoferum and Pyricularia oryzae laccases were compared, using several substrates and inhibitors. Sixteen phenolic or nonphenolic compounds were found to be substrates of both fungal and bacterial laccases. In the presence of different phenol oxidase inhibitors, P. oryzae and A. lipoferum laccase activities had similar properties.

Azospirillum lipoferum 4T has original properties such as nonmotility, melanin synthesis, and laccase activity. Following random Tn 5 mutagenesis in A. lipoferum 4T, we obtained 10 mutants which were affected in melanization and laccase activity. The class 1 mutants, with intermediate levels of laccase activity, showed some coloration; the class 2 mutants, which were completely negative for laccase activity, were also colorless. The Tn 5 localization on the chromosome or on the cryptic 300-MDa plasmid of A. lipoferum 4T was proven by hybridization for all class 1 mutants or for most class 2 mutants, respectively.

The effects of 3 and 19 yr natural fallows on soil structure and soil bacteria were assessed by aggregate size fractionation of a tropical sandy soil (0–10 cm). Bulk unfractionated soil and fractions including particulate coarse organic residues (COR), and four fractions (>2000, 50–2000, 2–50 and 50 μm), suggesting that these microhabitats were favourable to active N2 fixers. In contrast, more than 70% of the N2 fixing microorganisms and 90% of the recovered Azospirillum were isolated from the dispersible clay fraction (0–2 μm). Diversity of Azospirillum species was assessed by hybridisation with specific genetic probes on colonies within each fraction. This approach revealed the abundance of Azospirillum irakense in the 3 yr fallow soil fractions only and a selective effect of fallow on Azospirillum brasilense/Azospirillum amazonense genomic species in the 19 yr fallow soil. These changes would remain hidden, if the investigation had been restricted to the unfractionated soil.

The present study deals with the isolation of plant growth promoting rhizobacteria (PGPR) from rice (variety NIAB IRRI-9) and the beneficial effects of these inoculants on two Basmati rice varieties. Nitrogen-fixing activity (acetylene-reduction activity) was detected in the roots and submerged shoots of field-grown rice variety NIAB IRRI-9. Estimation of the population size of diazotrophic bacteria by ARA-based MPN (acetylene reduction assay-based most probable number) in roots and shoots indicated about 105–106counts/g dry weight at panicle initiation and grain filling stages. Four bacterial isolates from rice roots and shoots were obtained in pure culture which produced phytohormone indoleacetic acid (IAA) in the growth medium. Among these, three isolates S1, S4, and R3 reduced acetylene to ethylene in nitrogen-free semi-solid medium. Morphological and physiological characteristics of the isolates indicated that three nitrogen-fixing isolates S1, S4, and R3 belonged to the genus Enterobacter, while the non-fixing isolate R8 belonged to the genus Aeromonas. 16S rRNA sequence of one isolate from root (R8) and one isolate from shoot (S1) was obtained which confirmed identification of the isolates as Aeromonas veronii and Enterobacter cloacae, respectively. The 1517-nucleotide-long sequence of the isolate R8 showed 99% similarity with Aeromonas veronii (accession No. AF099023) while partial 16S rRNA sequence (two stretches of total 1271 nucleotide length) of S1 showed 97% similarity with the sequence of Enterobacter cloacae (accession No. AJ251469). The seedlings of two rice varieties Basmati 385 and Super Basmati were inoculated with the four bacterial isolates from rice and one Azospirillum brasilense strain Wb3, which was isolated from wheat. In the rice variety Basmati 385, maximum increase in root area and plant biomass was obtained in plants inoculated with Enterobacter S1 and Azospirillum Wb3, whereas in the rice variety Super Basmati, inoculation with Enterobacter R3 resulted in maximum increase of root area and plant biomass. Nitrogen fixation was quantified by using15N isotopic dilution method. Maximum fixation was observed in Basmati 385 with the inoculants Azospirillum Wb3 and Enterobacter S1 where nearly 46% and 41% of the nitrogen was derived from atmosphere (%Ndfa), respectively. In general, higher nitrogen fixation was observed in variety Basmati 385 than in Super Basmati, and different bacterial strains were found more effective as inoculants for the rice varieties Basmati 385 and Super Basmati.Key words: phytohormones, nitrogen fixation, Enterobacter, Aeromonas.

Several specific primers for the nifH gene were tested with different pure telluric N2-fixing strains. A PolF/PolR primer set provided successful amplification of 19 representative N2-fixing strains. Three restriction enzymes, HaeIII, NdeII and MnlI, chosen for restriction fragment length polymorphism (RFLP) analyses, were the most discriminating for the study of nifH gene diversity as they resulted in differences between strains at the species level. Amplification by selected primers and RFLP were applied to assess the genetic diversity of the nifH gene pool in soil. Pair soils, one under cultivation, the second under permanent pasture, were found to harbor a contrasting diversity of nifH genes. Pure strain profiles could not be recognized in the nifH soil patterns. Using the simple procedure described, it was shown that the structure of nitrogen fixers in soil was influenced by soil functioning.

International audience; Thirteen of the most abundant Gram-negative bacteria which are able to grow in N-depleted culture conditions were isolated from the rhizoplane and endorhizosphere of canola (Brassica napus) and identified by 16S rDNA sequence analysis. Eight of these bacteria induced a significant increase in root dry weight ranging from 11 to 52%. Phylogenetic positioning based on 16S rDNA sequences indicated that at least four genera are represented, Pseudomonas, Variovorax, Agrobacterium and Phyllobacterium. The most important direct plant growth-promoting-rhizobacteria effect was found with both isolates belonging to the Phyllobacterium.

International audience; Azospirillum lipoferum is a soil bacterium known for its ability to colonize roots and to promote plant growth. Recently, a laccase-like polyphenol oxidase activity has been reported in a strain isolated from the rhizosphere of rice. Purification to apparent homogeneity of A. lipoferum laccase was achieved after cell disruption by sonication, protein solubilization by Triton X-100, acetone and protamine precipitation, hydroxyapatite column chromatography in the presence of urea (5 M) and NaCl (2 M), and finally by conventional hydroxyapatite column chromatography. After this final step, laccase was in an inactive form but could be reactivated by adding sodium dodecyl sulfate (SDS). Based on gel electrophoresis results, we suggest that native A. lipoferum laccase is composed of a catalytic polypeptide chain with low molecular mass (16.3 kDa) and one or two regulatory/structural chains with high molecular mass (81.5 kDa). The purified enzyme was found to be thermostable up to 70°C for 10 min, had an optimal pH of 6.0, and was inhibited by tropolone, a known inhibitor of metal-containing enzymes. Although the natural substrate of this enzyme is unknown, its biochemical characterization may facilitate further investigations on the ecological role of this laccase in the process of root colonization by A. lipoferum.

Flagellation of a nonswimming variant of the mixed flagellated bacterium Azospirillum lipoferum 4B was characterized by electron microscopy, and polyclonal antibodies were raised against polar and lateral flagellins. The variant cells lacked a polar flagellum due to a defect in flagellin synthesis and constitutively expressed lateral flagella. The variant cells were able to respond to conditions that restricted the rotation of lateral flagella by producing more lateral flagella, suggesting that the lateral flagella, as well as the polar flagellum, are mechanosensing.

Les racines de certaines especes vegetales hebergent une microflore specifique qui favorise la croissance de la plante; une propriete exploitee depuis plusieurs annees pour ameliorer les rendements agricoles.

Azospirillum lipoferum CRT1 has been isolated from a maize rhizosphere by Fages and Mulard in 1988. Pot experiments carried out on maize showed a strong beneficial effect on root development and shoot dry weight. These results led them to test the efficiency of the strain in field experiments. Fages (1994) reported results from different field trials. The main result was a better absorption of nitrogen fertilizer. Moreover, grain yield improvement was frequently observed (particularly for suboptimal nitrogen doses). It was concluded that these improvements were probably due to a better root development. Strain CRT1 is now integrated in a peat-based inoculant for maize, named Azogreen-m® and marketed by LIPHA group.

In order to develop a reliable and specific tool for the detection of Azospirillum lipoferum CRT1, randomly cloned DNA fragments from this strain were used as hybridization probes to differentiate A. lipoferum CRT1 from 29 closely related Azospirillum strains. Two cloned fragments hybridizing only with DNA from A. lipoferum CRT1 (CRT1-5 and CRT1-7) were considered as specific probes of this strain. CRT1-7 fragment (1.4 kb) was further tested for purity control of the inoculant Azogreen-m by colony hybridization. The sequence of the CRT1-7 fragment has been determined and compared with those present in databases: no significant similarity with other sequences was detected. This probe permitted us to count specifically A. lipoferum CRT1 cells on maize roots during a field trial. During the first two weeks, A. lipoferum CRT1 remained at 107 CFU plant−1. Afterwards, bacterial concentration sharply decreased. We could not detect any CRT1 cells on maize roots 28 days after sowing. Concurrently, three plant parameters were estimated (plant height, primary root length and root fresh weight). The results showed that A. lipoferum CRT1 growth promotion effect began early on (from day 14) in plant development and increased in spite of a rapid decrease of bacterial density.

A new, simple method for isolating Azospirillum strains from the roots and the rhizosphere of rice is described. The method is based on the capacity of Azospirillum, a nitrogen-fixing bacterial genus, to grow in nutrient-deficient liquid media such as distilled water, KCl (8.5 g/L), or soil extract medium. The enrichment efficiency of the deficient media was compared with classical N-free malate medium. Serial dilutions from 10−1 to 10−10 of rice root macerates and rhizosphere soil were incubated at 28 °C in these media without agitation for 15 days. Pellicle formation was regularly examined under a microscope to detect the spiral motility of Azospirillum cells. Tubes with putative azospirilla were plated on nutrient agar. Most probable numbers of Azospirillum in root macerates were 2 × 103/g dry root weight in KCl, 2 × 104/g dry root weight in H2O, 2 × 106/g dry root weight in malate, and 3 × 106/g dry root weight in the soil extract medium. Soil extract medium repressed the most abundant bacterial populations and facilitated isolation of azospirilla from a population representing less than 0.001% of the total microflora. The isolates had vibrioid to S-shaped cells with one polar flagellum when grown in liquid medium and fixed nitrogen according to the acetylene reduction assay. Their identification as Azospirillum lipoferum was based on biochemical tests and hybridization with a 16S rRNA probe specific for this species and was confirmed by the detection of a 150-MDa plasmid.Key words: rice, rhizosphere, soil extract, isolation, identification, Azospirillum.

Resume Les plantes du genre Striga sont des parasites obligatoires des cereales dans les regions tropicales du globe, ou elles representent un fleau contre lequel il est difficile de lutter. L'utilisation, en lutte biologique, de bacteries du sol ayant la meme specifite d'hote que ces parasites est envisagee. Quatre souches bacteriennes appartenant au genre Azospirillum et a l'espece A. brasilense ont ete isolees d'un sol sous sorgho et testees pour leurs effets sur la germination de la plante parasite ( Striga hermonthica ) et sur la croissance de la cereale ( Sorghum vulgare ). Parmi ces souches, deux inhibent completement la germination des graines du parasite. En outre, l'une de ces deux souches augmente la croissance de la cereale (effet PGPR).

16S rRNA sequences were used to measure phylogenetic and evolutionary relationships among bacteria belonging to the alpha subdivision of the proteobacteria. Partial 16S rDNA sequences were determined for the five bacterial species belonging to the genus Azospirillum and compared with ribosomal sequences from other bacteria belonging to the alpha subdivision of the proteobacteria. The five Azospirillum species, A. lipoferum, A. brasilense, A. irakense, A. amazonense and A. halopraeferens, were grouped into a cluster which has been found to be related to Magnetospirillum magnetotacticum.

Partial sequences of the 16S rRNA molecules of nine strains belonging to four Azospirillum species were used to design species-specific oligonucleotide probes. Azospirillum strains sequences were analyzed and three homologous fragments containing 16 nucleotides were determined. These three probes were found to be characteristic of A. lipoferum (Al), A. irakense (Ai), and A. brasilense/amazonense species (Aba) and of few nontarget organisms. The specificity of these three probes was tested both against sequences in the GenBank data base and in numerous colony hybridization experiments. As a few non-target organisms hyridized with the different Azospirillum probes, the use of these probes in bulk soil hybridization is not permitted. However, their use together with specific isolation techniques is validated.Key words: Azospirillum, bacterial isolation, hybridization, oligonucleotide probe, sequence analysis, 16S rRNA.