Lipoferum 4B Research Articles

Engineering D-glucose utilization in Azospirillum brasilense Sp7 promotes rice root colonization.

Bacteria of the genus Azospirillum include several plant associated bacteria which often promote the growth of their host plants. Although the host range of Azospirillum brasilense Sp7 is much wider than its close relative Azospirillum lipoferum 4B, it lacks the ability to efficiently utilize D-glucose for its growth. By comparing the genomes of both the species, the genes of A. lipoferum 4B responsible for conferring D-glucose utilization ability in A. brasilese Sp7 were identified by cloning individual or a combination of genes in a broad host range expression vector, mobilizing them in A. brasilense Sp7 and examining the ability of exconjugants to use D-glucose as sole carbon source for growth. These genes also included the homologs of genes involved in N-acetyl glucosamine utilization in Pseudomonas aeruginosa PAO1. A transcriptional fusion of the 5 genes encoding glucose-6-phosphate dehydrogenase and 4 components of glucose phosphotransferase system were able to improve D-glucose utilization ability in A. brasilense Sp7. The A. brasilense Sp7 strain engineered with D-glucose utilization ability showed significantly improved root colonization of rice seedling. The improvement in the ability of A. brasilense Sp7 to colonize rice roots is expected to bring benefits to rice by promoting its growth. KEY POINTS: • Genes required for glucose utilization in Azospirillum lipoferum were identified. • A gene cassette encoding glucose utilization was constructed. • Transfer of gene cassette in A. brasilense improves glucose utilization and rice root colonization..

In silico comparative analysis of GGDEF and EAL domain signaling proteins from the Azospirillum genomes

BackgroundThe cyclic-di-GMP (c-di-GMP) second messenger exemplifies a signaling system that regulates many bacterial behaviors of key importance; among them, c-di-GMP controls the transition between motile and sessile life-styles in bacteria. Cellular c-di-GMP levels in bacteria are regulated by the opposite enzymatic activities of diguanylate cyclases and phosphodiesterases, which are proteins that have GGDEF and EAL domains, respectively. Azospirillum is a genus of plant-growth-promoting bacteria, and members of this genus have beneficial effects in many agronomically and ecologically essential plants. These bacteria also inhabit aquatic ecosystems, and have been isolated from humus-reducing habitats. Bioinformatic and structural approaches were used to identify genes predicted to encode GG[D/E]EF, EAL and GG[D/E]EF-EAL domain proteins from nine genome sequences.ResultsThe analyzed sequences revealed that the genomes of A. humicireducens SgZ-5T, A. lipoferum 4B, Azospirillum sp. B510, A. thiophilum BV-ST, A. halopraeferens DSM3675, A. oryzae A2P, and A. brasilense Sp7, Sp245 and Az39 encode for 29 to 41 of these predicted proteins. Notably, only 15 proteins were conserved in all nine genomes: eight GGDEF, three EAL and four GGDEF-EAL hybrid domain proteins, all of which corresponded to core genes in the genomes. The predicted proteins exhibited variable lengths, architectures and sensor domains. In addition, the predicted cellular localizations showed that some of the proteins to contain transmembrane domains, suggesting that these proteins are anchored to the membrane. Therefore, as reported in other soil bacteria, the Azospirillum genomes encode a large number of proteins that are likely involved in c-di-GMP metabolism. In addition, the data obtained here strongly suggest host specificity and environment specific adaptation.ConclusionsBacteria of the Azospirillum genus cope with diverse environmental conditions to survive in soil and aquatic habitats and, in certain cases, to colonize and benefit their host plant. Gaining information on the structures of proteins involved in c-di-GMP metabolism in Azospirillum appears to be an important step in determining the c-di-GMP signaling pathways, involved in the transition of a motile cell towards a biofilm life-style, as an example of microbial genome plasticity under diverse in situ environments.

Genome-wide survey of two-component signal transduction systems in the plant growth-promoting bacterium Azospirillum

BackgroundTwo-component systems (TCS) play critical roles in sensing and responding to environmental cues. Azospirillum is a plant growth-promoting rhizobacterium living in the rhizosphere of many important crops. Despite numerous studies about its plant beneficial properties, little is known about how the bacterium senses and responds to its rhizospheric environment. The availability of complete genome sequenced from four Azospirillum strains (A. brasilense Sp245 and CBG 497, A. lipoferum 4B and Azospirillum sp. B510) offers the opportunity to conduct a comprehensive comparative analysis of the TCS gene family.ResultsAzospirillum genomes harbour a very large number of genes encoding TCS, and are especially enriched in hybrid histidine kinases (HyHK) genes compared to other plant-associated bacteria of similar genome sizes. We gained further insight into HyHK structure and architecture, revealing an intriguing complexity of these systems. An unusual proportion of TCS genes were orphaned or in complex clusters, and a high proportion of predicted soluble HKs compared to other plant-associated bacteria are reported. Phylogenetic analyses of the transmitter and receiver domains of A. lipoferum 4B HyHK indicate that expansion of this family mainly arose through horizontal gene transfer but also through gene duplications all along the diversification of the Azospirillum genus. By performing a genome-wide comparison of TCS, we unraveled important ‘genus-defining’ and ‘plant-specifying’ TCS.ConclusionsThis study shed light on Azospirillum TCS which may confer important regulatory flexibility. Collectively, these findings highlight that Azospirillum genomes have broad potential for adaptation to fluctuating environments.Electronic supplementary materialThe online version of this article (doi:10.1186/s12864-015-1962-x) contains supplementary material, which is available to authorized users.

Differential responses of Oryza sativa secondary metabolism to biotic interactions with cooperative, commensal and phytopathogenic bacteria.

Profiling of plant secondary metabolite allows to differentiate the different types of ecological interactions established between rice and bacteria. Rice responds to ecologically distinct bacteria by altering its content of flavonoids and hydroxycinnamic acid derivatives. Plants' growth and physiology are strongly influenced by the biotic interactions that plants establish with soil bacterial populations. Plants are able to sense and to respond accordingly to ecologically distinct bacteria, by inducing defense pathways against pathogens to prevent parasitic interactions, and by stimulating the growth of root-associated beneficial or commensal bacteria through root exudation. Plant secondary metabolism is expected to play a major role in this control. However, secondary metabolite responses of a same plant to cooperative, commensal and deleterious bacteria have so far never been compared. The impact of the plant growth-promoting rhizobacteria (PGPR) Azospirillum lipoferum 4B on the secondary metabolite profiles of two Oryza sativa L. cultivars (Cigalon and Nipponbare) was compared to that of a rice pathogen Burkholderia glumae AU6208, the causing agent of bacterial panicle blight and of a commensal environmental bacteria Escherichia coli B6. Root and shoot rice extracts were analyzed by reversed-phase high-performance liquid chromatography (RP-HPLC). Principal component analyses (PCAs) pinpointed discriminant secondary metabolites, which were characterized by mass spectrometry. Direct comparison of metabolic profiles evidenced that each bacterial ecological interaction induced distinct qualitative and quantitative modifications of rice secondary metabolism, by altering the content of numerous flavonoid compounds and hydroxycinnamic acid (HCA) derivatives. Secondary metabolism varied according to the cultivars and the interaction types, demonstrating the relevance of secondary metabolic profiling for studying plant-bacteria biotic interactions.

Plant root transcriptome profiling reveals a strain-dependent response during Azospirillum-rice cooperation.

Cooperation involving Plant Growth-Promoting Rhizobacteria results in improvements of plant growth and health. While pathogenic and symbiotic interactions are known to induce transcriptional changes for genes related to plant defense and development, little is known about the impact of phytostimulating rhizobacteria on plant gene expression. This study aims at identifying genes significantly regulated in rice roots upon Azospirillum inoculation, considering possible favored interaction between a strain and its original host cultivar. Genome-wide analyzes of Oryza sativa japonica cultivars Cigalon and Nipponbare were performed, by using microarrays, seven days post-inoculation with Azospirillum lipoferum 4B (isolated from Cigalon) or Azospirillum sp. B510 (isolated from Nipponbare) and compared to the respective non-inoculated condition. A total of 7384 genes were significantly regulated, which represent about 16% of total rice genes. A set of 34 genes is regulated by both Azospirillum strains in both cultivars, including a gene orthologous to PR10 of Brachypodium, and these could represent plant markers of Azospirillum-rice interactions. The results highlight a strain-dependent response of rice, with 83% of the differentially expressed genes being classified as combination-specific. Whatever the combination, most of the differentially expressed genes are involved in primary metabolism, transport, regulation of transcription and protein fate. When considering genes involved in response to stress and plant defense, it appears that strain B510, a strain displaying endophytic properties, leads to the repression of a wider set of genes than strain 4B. Individual genotypic variations could be the most important driving force of rice roots gene expression upon Azospirillum inoculation. Strain-dependent transcriptional changes observed for genes related to auxin and ethylene signaling highlight the complexity of hormone signaling networks in the Azospirillum-rice cooperation.

Genome wide profiling ofAzospirillum lipoferum4B gene expression during interaction with rice roots

Azospirillum-plant cooperation has been mainly studied from an agronomic point of view leading to a wide description of mechanisms implicated in plant growth-promoting effects. However, little is known about genetic determinants implicated in bacterial adaptation to the host plant during the transition from free-living to root-associated lifestyles. This study aims at characterizing global gene expression of Azospirillumlipoferum 4B following a 7-day-old interaction with two cultivars of Oryza sativa L. japonica (cv. Cigalon from which it was originally isolated, and cv. Nipponbare). The analysis was done on a whole genome expression array with RNA samples obtained from planktonic cells, sessile cells, and root-adhering cells. Root-associated Azospirillum cells grow in an active sessile-like state and gene expression is tightly adjusted to the host plant. Adaptation to rice seems to involve genes related to reactive oxygen species (ROS) detoxification and multidrug efflux, as well as complex regulatory networks. As revealed by the induction of genes encoding transposases, interaction with root may drive bacterial genome rearrangements. Several genes related to ABC transporters and ROS detoxification display cultivar-specific expression profiles, suggesting host specific adaptation and raising the question of A.lipoferum 4B/rice cv. Cigalon co-adaptation.

A bioinformatics approach to the determination of genes involved in endophytic behavior in Burkholderia spp.

The vast majority of plants harbor endophytic bacteria that colonize a portion of the plant's interior tissues without harming the plant. Like plant pathogens, endophytes gain entry into their plants hosts through various mechanisms. Bacterial endophytes display a broad range of symbiotic interactions with their host plants. The molecular bases of these plant-endophyte interactions are currently not fully understood. In the present study, a set of genes possibly responsible for endophytic behavior for genus Burkholderia was predicted and then compared and contrasted with a number (nine endophytes from different genera) of endophytes by comparative genome analysis. The nine endophytes included Burkholderia phytofirmans PsJN, Burkholderia spp. strain JK006, Azospirillum lipoferum 4B, Enterobacter cloacae ENHKU01, Klebsiella pneumoniae 342, Pseudomonas putida W619, Enterobacter spp. 638, Azoarcus spp. BH72, and Serratia proteamaculans 568. From the genomes of the analyzed bacterial strains, a set of bacterial genes orthologs was identified that are predicted to be involved in determining the endophytic behavior of Burkholderia spp. The genes and their possible functions were then investigated to establish a potential connection between their presence and the role they play in bacterial endophytic behavior. Nearly all of the genes identified by this bioinformatics procedure encode function previously suggested in other studies to be involved in endophytic behavior.

Plant secondary metabolite profiling evidences strain-dependent effect in the Azospirillum–Oryza sativa association

Azospirillum is a plant growth-promoting rhizobacterium (PGPR) able to enhance growth and yield of cereals such as rice, maize and wheat. The growth-promoting ability of some Azospirillum strains appears to be highly specific to certain plant species and cultivars. In order to ascertain the specificity of the associative symbiosis between rice and Azospirillum, the physiological response of two rice cultivars, Nipponbare and Cigalon, inoculated with two rice-associated Azospirillum was analyzed at two levels: plant growth response and plant secondary metabolic response. Each strain of Azospirillum (Azospirillum lipoferum 4B isolated from Cigalon and Azospirillum sp. B510 isolated from Nipponbare) preferentially increased growth of the cultivar from which it was isolated. This specific effect is not related to a defect in colonization of host cultivar as each strain colonizes effectively both rice cultivars, either at the rhizoplane (for 4B and B510) and inside the roots (for B510). The metabolic profiling approach showed that, in response to PGPR inoculation, profiles of rice secondary metabolites were modified, with phenolic compounds such as flavonoids and hydroxycinnamic derivatives being the main metabolites affected. Moreover, plant metabolic changes differed according to Azospirillum strain×cultivar combinations; indeed, 4B induced major secondary metabolic profile modifications only on Cigalon roots, while B510, probably due to its endophytic feature, induced metabolic variations on shoots and roots of both cultivars, triggering a systemic response. Plant secondary metabolite profiling thereby evidences the specific interaction between an Azospirillum strain and its original host cultivar.

Genome Sequence of Azospirillum brasilense CBG497 and Comparative Analyses of Azospirillum Core and Accessory Genomes provide Insight into Niche Adaptation

Bacteria of the genus Azospirillum colonize roots of important cereals and grasses, and promote plant growth by several mechanisms, notably phytohormone synthesis. The genomes of several Azospirillum strains belonging to different species, isolated from various host plants and locations, were recently sequenced and published. In this study, an additional genome of an A. brasilense strain, isolated from maize grown on an alkaline soil in the northeast of Mexico, strain CBG497, was obtained. Comparative genomic analyses were performed on this new genome and three other genomes (A. brasilense Sp245, A. lipoferum 4B and Azospirillum sp. B510). The Azospirillum core genome was established and consists of 2,328 proteins, representing between 30% to 38% of the total encoded proteins within a genome. It is mainly chromosomally-encoded and contains 74% of genes of ancestral origin shared with some aquatic relatives. The non-ancestral part of the core genome is enriched in genes involved in signal transduction, in transport and in metabolism of carbohydrates and amino-acids, and in surface properties features linked to adaptation in fluctuating environments, such as soil and rhizosphere. Many genes involved in colonization of plant roots, plant-growth promotion (such as those involved in phytohormone biosynthesis), and properties involved in rhizosphere adaptation (such as catabolism of phenolic compounds, uptake of iron) are restricted to a particular strain and/or species, strongly suggesting niche-specific adaptation.

Physical organization and phylogenetic analysis of acdR as leucine-responsive regulator of the 1-aminocyclopropane-1-carboxylate deaminase gene acdS in phytobeneficial Azospirillum lipoferum 4B and other Proteobacteria

The phytostimulatory alphaproteobacterium Azospirillum lipoferum 4B exhibits the plant-beneficial gene acdS, which enables deamination of the ethylene precursor 1-aminocyclopropane-1-carboxylic acid (ACC). Here, we show that acdS is in the vicinity of acdR, a homolog to leucine-responsive regulator lrp, in A. lipoferum 4B and most other acdS+ Proteobacteria. Unlike in Beta- and Gammaproteobacteria, acdS (and acdR) is preferentially located on symbiotic islands and plasmids in Alphaproteobacteria. In A. lipoferum 4B, acdS was mapped on a 750-kb plasmid that is lost during phenotypic variation, whereas other phytobeneficial genes such as nifH (associative nitrogen fixation) are maintained. In Proteobacteria, the phylogenies of acdR and acdS were largely but not totally congruent, despite physical proximity of the genes, regardless of whether DNA or deduced protein sequences were used. Potential Lrp, cAMP receptor protein (CRP) and fumarate-nitrate reduction regulator (FNR) binding sites were evidenced in the acdS promoter regions of strain 4B and most of 46 other acdS+ Proteobacteria. Indeed, transcriptional and enzymatic analyses done in vitro pointed to the involvement of Lrp- and FNR-like transcriptional up-regulation of ACC deaminase activity in A. lipoferum 4B. This is the first synteny, phylogenetic, and functional analysis of factors modulating acdS expression in Azospirillum plant growth-promoting rhizobacterium.

Effects of the Inoculation of Burkholderia vietnamensis and Related Endophytic Diazotrophic Bacteria on Grain Yield of Rice

During a survey of endophytic diazotrophic bacteria associated with different rice varieties in Tamilnadu, some "endophytes" were obtained. Thirteen bacterial isolates from surface-sterilized roots and shoots were obtained in pure culture, which produced indole acetic acid (IAA) and reduced acetylene to ethylene. Polymerase chain reaction (PCR) amplification confirmed the presence of nif-H gene in all the isolates. Morphological, biochemical, and molecular characteristics indicated that all of them belonged to the genus Burkholderia One of them, MGK3, was consistently more active in reducing acetylene, and 16S rDNA sequences of isolate MGK3 confirmed its identification as Burkholderia vietnamiensis. Colonization of rice root was confirmed by strain MGK3 marked with gusA gene. The inoculated roots showed a blue color, which was most intense at the points of lateral root emergence and at the root tip. Transverse sections of roots, 15 days after inoculation, revealed beta-glucuronidase (GUS) activity within many of the cortical intercellular spaces next to the stele and within the aerenchyma. Nitrogen fixation was quantified by using (15)N isotope dilution method with two different cultivars grown in pot and field experiments. Higher nitrogen fixation was observed in variety Ponni than in ADT-43, where nearly 42% (field) and 40% (pot) of the nitrogen was derived from the atmosphere (% Ndfa). Isolate MGK3 was used to inoculate rice seedlings in a comparison with four other diazotrophs, viz., Gluconacetobacter diazotrophicus LMG7603, Herbaspirillum seropedicae LMG6513, Azospirillum lipoferum 4B LMG4348, and B. vietnamiensis LMG10929. They were used to conduct two pot and four field inoculation experiments. MGK3 alone, and combined with other diazotrophs, performed best under both pot and field conditions: combined inoculation produced yield increases between 9.5 and 23.6%, while MGK3 alone increased yield by 5.6 to 12.16% over the uninoculated control treatment.

Construction of a recA mutant of Azospirillum lipoferum and involvement of recA in phase variation

The plant-growth promoting rhizobacterium Azospirillum lipoferum strain 4B generates in vitro a stable phase variant designated 4V I at frequencies of 10 −4 to 10 −3 per cell per generation. Variant 4V I 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 4B recA) was constructed by allelic exchange. Strain 4B recA 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 4B recA exhibited all morphological and biochemical features characteristic of the variant generated by strain 4B. However, the frequency of variants generated by 4B recA 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.

A phase variant of Azospirillum lipoferum lacks a polar flagellum and constitutively expresses mechanosensing lateral flagella.

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.

Population dynamics of a motile and a non-motile Azospirillum lipoferum strain during rice root colonization and motility variation in the rhizosphere

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).

Population dynamics of a motile and a non-motile Azospirillum lipoferum strain during rice root colonization and motility variation in the rhizosphere

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 (10 8 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).

Whey as a growth medium for two spp. of Azospirillum grown in batch culture

Summary The growth curves of Azospirillum lipoferum 4B and Azospirillum brasilense NO 40 grown on raw whey, deproteinized whey and malate media were investigated in batch culture. A maximal cell density of 14 × 108 and 4.5 × 10 8 cell/ml were attained after 72 h of the growth of Azospirillum brasilense NO40 and Azospirillum lipoferum 4B on deproteinized whey medium respectively. Raw whey as well as deproteinized whey was successfully utilized by both strains in a similar manner to that of malate. The highest growth of A. lipoferum 4B and A. brasilense NO40 was detected when 20 ml/l of raw whey or deproteinized whey were used as a carbon source in the basal medium. Prospective economic balance for the capital cost of malate and whey or deproteinized whey growth media was also discussed.

Similarities between large plasmids of Azospirillum lipoferum

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.

Similarities between large plasmids ofAzospirillum lipoferum

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.

Mobilization and transfer of Azospirillum lipoferum plasmid by the Tn5-Mob transposon into a plasmid-free Agrobacterium tumefaciens strain.

Azospirillum lipoferum 4B harbors five cryptic plasmids. Several suicide plasmids were used to transfer Tn5-Mob to A. lipoferum 4B. Tn5-Mob insertion mutations of this strain could be obtained at frequencies of 10(-8)-10(-7) per recipient cell. One hundred Tn5-Mob A. lipoferum 4B mutants were used in bacterial matings with a plasmid-free Agrobacterium tumefaciens recipient strain. This is the first report of mobilization, transfer, and replication of an Azospirillum plasmid in Agrobacterium tumefaciens. One transconjugant was found which had lost an indigenous plasmid.

Stimulation of root exudation of rice seedlings by Azospirillum strains: carbon budget under gnotobiotic conditions

The association of rice seedlings (cv. Delta) with different strains of Azospirillum was studied under monoxenic conditions in the dark. Axenic 3-day-old seedlings were obtained on a C- and N-free medium and inoculated with 6 · 107 bacteria per plant in a closed vial. Seven days later, different components of a carbon budget were evaluated on them and on sterile controls: respired CO2, carbon of shoot and roots, bacterial and soluble carbon in the medium. Two strains (A. lipoferum 4B and A. brasilense A95) isolated from the rhizosphere of rice caused an increase in exudation, + 36% and + 17% respectively compared with sterile control. Shoot carbon incorporation and respiration were reduced by inoculation. A third strain (A. brasilense R07) caused no significant change in exudation. A. lipoferum B7C isolated from maize did not stimulate rice exudation either. We further investigated a possible effect of nitrogen fixation on this phenomenon: inhibition of nitrogen fixation by 10% C2H2 did not modify the extent of C exudation by rice associated with A. lipoferum 4B or with the non-motile A. lipoferum 4T.