Physiological and genetic characterization of rice nitrogen fixer PGPR isolated from rhizosphere soils of different crops

Aims

We aimed to identify plant growth-promoting rhizobacteria that could be used to develop a biofertilizer for rice.

Methods

To obtain plant growth-promoting rhizobacteria, rhizosphere soils from different crops (rice, wheat, oats, crabgrass, maize, ryegrass, and sweet potato) were inoculated to rice plants. In total, 166 different bacteria were isolated and their plant growth-promoting traits were evaluated in terms of colony morphology, indole-3-acetic acid production, acetylene reduction activity, and phosphate solubilization activity. Moreover, genetic analysis was carried out to evaluate their phylogenetic relationships based on 16S rRNA sequence data.

Results

Strains of Bacillus altitudinis, Pseudomonas monteilii, and Pseudomonas mandelii formed associations with rice plants and fixed nitrogen. A strain of Rhizobium daejeonense showed nitrogen fixation activity in an in vitro assay and in vivo. Strains of B. altitudinis and R. daejeonense derived from rice rhizosphere soil, strains of P. monteilii and Enterobacter cloacae derived from wheat rhizosphere soil, and a strain of Bacillus pumilus derived from maize rhizosphere soil significantly promoted rice plant growth.

Conclusions

These methods are effective to identify candidate species that could be developed as biofertilizers for target crops.     

Real time PCR detection targeting nifA gene of plant growth promoting bacteria Azospirillum brasilense strain FP2 in maize roots

The plant growth promoting bacteria (PGPB) Azospirillum brasilense has been recommended for use in commercial inoculants in Brazil. Effective methods are necessary to monitor PGPB strains in the rhizosphere. Our purpose was to develop a real time PCR method for detection of A. brasilense strain FP2 in maize seedlings targeting nifA. Primer pairs were designed and their specificity was verified using DNA from 12 different bacterial species. Standard curves were prepared for DNA quantification using serial dilution of A. brasilense DNA extracts. PCR efficiencies and correlation coefficient presented values within the acceptable range for qPCR, mean PCR efficiency was 95 % and correlation coefficient was 0.98, indicating that nifA gene was suitable for the quantitative analysis of the target bacterial genome. Inoculated maize seedlings were grown in vitro or in pots, bacterial DNA copy number per gram of fresh root was quantified 1, 4, 7 and 10 days after inoculation. The developed primers targeting nifA will be useful for monitoring Azospirillum brasilense strain FP2 in crops.     

Enhancement of Wheat Root Colonization and Plant Development by Azospirillum brasilense Cd. Following Temporary Depression of Rhizosphere Microflora

Inoculation of wheat with Azospirillum brasilense, combined with the application of four fungal and bacterium-inhibiting substances to which A. brasilense is resistant in the soil, decreased the rhizosphere population, while it increased wheat root colonization by A. brasilense, even in cases of poor inoculation. The inoculation significantly increased the following wheat plant parameters as well: plant dry weight, number of tillers per plant, spikelet fertility, harvest index, and grain yield. This model may provide a new approach to improve control of root colonization by beneficial bacteria.     

Protection of Tomato Seedlings against Infection by Pseudomonas syringae pv. Tomato by Using the Plant Growth-Promoting Bacterium Azospirillum brasilense

Pseudomonas syringae pv. tomato, the causal agent of bacterial speck of tomato, and the plant growth-promoting bacterium Azospirillum brasilense were inoculated onto tomato plants, either alone, as a mixed culture, or consecutively. The population dynamics in the rhizosphere and foliage, the development of bacterial speck disease, and their effects on plant growth were monitored. When inoculated onto separate plants, the A. brasilense population in the rhizosphere of tomato plants was 2 orders of magnitude greater than the population of P. syringae pv. tomato (10⁷ versus 10⁵ CFU/g [dry weight] of root). Under mist chamber conditions, the leaf population of P. syringae pv. tomato was 1 order of magnitude greater than that of A. brasilense (10⁷ versus 10⁶ CFU/g [dry weight] of leaf). Inoculation of seeds with a mixed culture of the two bacterial strains resulted in a reduction of the pathogen population in the rhizosphere, an increase in the A. brasilense population, the prevention of bacterial speck disease development, and improved plant growth. Inoculation of leaves with the mixed bacterial culture under mist conditions significantly reduced the P. syringae pv. tomato population and significantly decreased disease severity. Challenge with P. syringae pv. tomato after A. brasilense was established in the leaves further reduced both the population of P. syringae pv. tomato and disease severity and significantly enhanced plant development. Both bacteria maintained a large population in the rhizosphere for 45 days when each was inoculated separately onto tomato seeds (10⁵ to 10⁶ CFU/g [dry weight] of root). However, P. syringae pv. tomato did not survive in the rhizosphere in the presence of A. brasilense. Foliar inoculation of A. brasilense after P. syringae pv. tomato was established on the leaves did not alleviate bacterial speck disease, and A. brasilense did not survive well in the phyllosphere under these conditions, even in a mist chamber. Several applications of a low concentration of buffered malic acid significantly enhanced the leaf population of A. brasilense (>10⁸ CFU/g [dry weight] of leaf), decreased the population of P. syringae pv. tomato to almost undetectable levels, almost eliminated disease development, and improved plant growth to the level of uninoculated healthy control plants. Based on our results, we propose that A. brasilense be used in prevention programs to combat the foliar bacterial speck disease caused by P. syringae pv. tomato.     

Plant growth-promoting activities of fluorescent pseudomonads,isolated from the Iranian soils

Fluorescent pseudomonads are among the most influencing plant growth-promoting rhizobacteria in plants rhizosphere. In this research work the plant growth-promoting activities of 40 different strains of Pseudomonas fluorescens and Pseudomonas putida, previously isolated from the rhizosphere of wheat (Triticum aestivum L.) and canola (Brassica napus L.) and maintained in the microbial collection of Soil and Water Research Institute, Tehran, Iran, were evaluated. The ability of bacteria to produce auxin and siderophores and utilizing P sources with little solubility was determined. Four strains of Wp1 (P. putida), Cfp10 (Pseudomonas sp.), Wp150 (P. putida), and Wp159 (P. putida) were able to grow in the DF medium with ACC. Thirty percent of bacterial isolates from canola rhizosphere and 33% of bacterial isolates from wheat rhizosphere were able to produce HCN. The results indicate that most of the bacteria, tested in the experiment, have plant growth-promoting activities. This is the first time that such PGPR species are isolated from the Iranian soils. With respect to their great biological capacities they can be used for wheat and canola inoculation in different parts of the world, which is of very important agricultural implications.     

Early plant growth and biochemical responses induced by <Emphasis Type="Italic">Azospirillum brasilense</Emphasis> Sp245 lipopolysaccharides in wheat (<Emphasis Type="Italic">Triticum aestivum</Emphasis> L.) seedlings are attenuated by procyanidin B2

This study analyzes the effects of procyanidin B2 on early wheat plant growth and plant biochemical responses promoted by lipopolysaccharides (LPS) derived from the rhizobacteria Azospirillum brasilense Sp245. Measurements of leaf, root length, fresh weight, and dry weight showed in vitro plant growth stimulation 4 days after treatment with A. brasilense as well as LPS. Superoxide anion (O₂ ^·?) and hydrogen peroxide (H₂O₂) levels increased in seedling roots treated with LPS (100 μg mL^?1). The chlorophyll content in leaf decreased while the starch content increased 24 h after treatment in seedling roots. The LPS treatment induced a high increase in total peroxidase (POX) (EC 1.11.1.7) activity and ionically bound cell wall POX content in roots, when compared to respective controls. Early plant growth and biochemical responses observed in wheat seedlings treated with LPS were inhibited by the addition of procyanidin B2 (5 μg mL^?1), a B type proanthocyanidin (PAC), plant-derived polyphenolic compound with binding properties of LPS. All results suggest first that the ionically bound cell wall POX enzymes could be a molecular target of A. brasilense LPS, and second that the recognition or association of LPS by plant cells is required to activate plant responses. This last event could play a critical role during plant growth regulation by A. brasilense LPS.     

Quantification of Pseudomonas fluorescens strains F113, CHA0 and Pf153 in the rhizosphere of maize by strain-specific real-time PCR unaffected by the variability of DNA extraction efficiency

Pseudomonas fluorescens strains F113 and CHA0 are well-known plant growth-promoting rhizobacteria (PGPR) often used as model strains in biocontrol experiments. To monitor their persistence in large scale field experiments, culture-independent methods are needed. In this study, a strain-specific real-time PCR quantification tool was developed based on sequence-characterized amplified regions (SCAR) for P. fluorescens strains F113, CHA0 and Pf153. Differences in DNA extraction efficiencies from rhizosphere samples were circumvented using plasmid APA9 as internal standard to normalize C_T values after real-time amplification. The detection limits of the real-time PCR assays for all three strains were approximately 10 cells for genomic DNA and 10⁴ cells/g rhizosphere for maize samples grown in different natural soils. Population sizes of the three strains in the rhizosphere of maize measured by the new real-time PCR approaches were similar to those measured by most probable number (MPN)-PCR. A persistence study of the three strains indicated that the strains persisted differently over a period of 5 weeks. In conclusion the newly developed real-time PCR approach is a fast and resource efficient method for monitoring individual biocontrol strains in natural soil, which makes it an apt quantification tool for future large-scale field experiments.     

Inoculation with the Plant-Growth-Promoting Rhizobacterium Azospirillum brasilense Causes Little Disturbance in the Rhizosphere and Rhizoplane of Maize (Zea mays)

Inoculation with Azospirillum brasilense exerts beneficial effects on plant growth and crop yields. In this study, a comparative analysis of maize (Zea mays) root inoculated or not inoculated with A. brasilense strains was performed in two soils. Colonization dynamics of the rhizobacteria were tracked in various root compartments using 16S rRNA-targeted probes and 4′,6′diamidino-2-phenylindole staining, and the structure of bacterial populations in the same samples was analyzed by denaturing gradient gel electrophoresis (DGGE) of polymerase chain reaction products of the 16S rRNA gene. Based on whole cell hybridization, a large fraction of the bacterial community was found to be active in both the rhizoplane–endorhizosphere and rhizosphere soil compartments, in both soil types. A DGGE fingerprint analysis revealed that plant inoculation with A. brasilense had no effect on the structural composition of the bacterial communities, which were also found to be very similar at the root tip and at zones of root branching. However, rhizobacterial populations were strongly influenced by plant age, and their complexity decreased in the rhizoplane–endorhizosphere in comparison to rhizosphere soil. A clone library generated from rhizosphere DNA revealed a highly diverse community of soil and rhizosphere bacteria, including an indigenous Azospirillum-like organism. A large proportion of these clones was only distantly related to known species. Herschkovitz and Lerner contributed equally to this work.     

Enhanced Growth of Wheat and Soybean Plants Inoculated with Azospirillum brasilense Is Not Necessarily Due to General Enhancement of Mineral Uptake

The capacity of Azospirillum brasilense to enhance the accumulation of K⁺, P, Ca²⁺, Mg²⁺, S, Na⁺, Mn²⁺, Fe²⁺, B, Cu²⁺, and Zn²⁺ in inoculated wheat and soybean plants was evaluated by using two different analytical methods with five A. brasilense strains originating from four distinct geographical regions. A Pseudomonas isolate from the rhizosphere of Zea mays seedlings was included as a control. All A. brasilense strains significantly improved wheat and soybean growth by increasing root and shoot dry weight and root surface area. The degree of plant response to inoculation varied among the different strains of A. brasilense. All strains were capable of colonizing roots, but the best root colonizer, Pseudomonas sp., had no effect on plant growth. The numbers of organisms of Brazilian strains Sp-245 and Sp-246 colonizing roots were similar regardless of the host plant. Numbers of organisms for the other strains were directly dependent on the host plant. The main feature characterizing mineral accumulation in inoculated plants was that all inoculation treatments changed the mineral balance of the plants, but in an inconsistent manner. Enhancement of mineral uptake by plants also varied among strains to a great extent and was directly dependent on the strain-plant combination; i.e., a strain capable of increasing accumulation of a particular ion in one plant species or cultivar often lacked the ability to do so in another. Minerals in inoculated plants were not evenly distributed in different plant tissues, and the changes varied among groups of plants within each bacterial strain inoculation treatment. We suggest that, although A. brasilense strains are capable of changing the mineral balance and content of plants, it is unlikely that this ability is a general mechanism responsible for plant improvement by A. brasilense.     

Bacteriophage Prevalence in the Genus Azospirillum and Analysis of the First Genome Sequence of an Azospirillum brasilense Integrative Phage

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.     

一株连香树根际促生细菌LWK2的分离鉴定及其全基因组序列分析

【背景】植物根际促生细菌是一类位于植物根际并能对植物生长产生促进作用的有益菌,在微生物肥料领域具有重要的应用价值。【目的】对濒危植物连香树根际的植物根际促生细菌进行分离筛选和连香树接种效应评价,挑选对连香树生长促进作用最为显著的菌种进行促生特性分析、菌种鉴定及全基因组序列测定与促生相关基因分析。【方法】利用相应筛选培养基对连香树根际土壤中解有机磷、溶无机磷和解钾细菌进行分离筛选,通过根际接种验证各菌株对连香树实生苗的促生能力。从中选取促生作用最为显著的细菌,进行解钾能力、产吲哚乙酸(indole-3-acetic acid,IAA)和1-氨基环丙烷-1-羧酸(1-aminocyclopropane-1-carboxylate,ACC)脱氨酶能力测定。利用菌体形态观察、16S rRNA基因序列分析及全基因组序列的平均核苷酸一致性比对进行菌种鉴定。最后利用基因组功能注释和比较基因组学分析对该菌株中的植物促生及重金属抗性相关基因进行解析。【结果】从连香树根际土壤中共筛选得到3株解有机磷细菌、2株溶无机磷细菌和2株解钾细菌,其中解钾细菌LWK2对连香树实生苗的生长促进作用最为显著。该菌株能够产...     

Impact of seed-applied fungicide and insecticide on Azospirillum brasilense survival and wheat growth-promoting ability

The use of Azospirillum brasilense as a crop inoculant has increased in recent years. Thus, the compatibility of the inoculation technology with seed treatments using pesticides needs to be evaluated. In this study, we evaluated the effect of an insecticide and fungicide formulation on A. brasilense strain FP2 population by culturing and culture-independent approaches. In addition, we evaluated the impact of these pesticides on the ability of A. brasilense to promote plant growth by monitoring biometric traits (root and shoot dry mass and length) of wheat grown in Greenhouse conditions. Seed pesticide dressings, mainly fungicide, led to a significant mortality of A. brasilense over time. The ability of A. brasilense to promote wheat growth also decreased due to pesticide treatments combined with sowing delay. Considering that pesticides confer fitness advantages to the wheat in field condition, our results suggest that sowing within the first 4 h after inoculation maintain the beneficial effects of A. brasilense on wheat growth promotion. Furthermore, we conclude that inoculation and treatment of seeds with pesticides may be compatible techniques when carried out immediately before sowing.     

Analysis of Fungal Diversity in the Wheat Rhizosphere by Sequencing of Cloned PCR-Amplified Genes Encoding 18S rRNA and Temperature Gradient Gel Electrophoresis

Like bacteria, fungi play an important role in the soil ecosystem. As only a small fraction of the fungi present in soil can be cultured, conventional microbiological techniques yield only limited information on the composition and dynamics of fungal communities in soil. DNA-based methods do not depend on the culturability of microorganisms, and therefore they offer an attractive alternative for the study of complex fungal community structures. For this purpose, we designed various PCR primers that allow the specific amplification of fungal 18S-ribosomal-DNA (rDNA) sequences, even in the presence of nonfungal 18S rDNA. DNA was extracted from the wheat rhizosphere, and 18S rDNA gene banks were constructed in Escherichia coli by cloning PCR products generated with primer pairs EF4-EF3 (1.4 kb) and EF4-fung5 (0.5 kb). Fragments of 0.5 kb from the cloned inserts were sequenced and compared to known rDNA sequences. Sequences from all major fungal taxa were amplified by using both primer pairs. As predicted by computer analysis, primer pair EF4-EF3 appeared slightly biased to amplify Basidiomycota and Zygomycota, whereas EF4-fung5 amplified mainly Ascomycota. The 61 clones that were sequenced matched the sequences of 24 different species in the Ribosomal Database Project (RDP) database. Similarity values ranged from 0.676 to 1. Temperature gradient gel electrophoresis (TGGE) analysis of the fungal community in the wheat rhizosphere of a microcosm experiment was carried out after amplification of total DNA with both primer pairs. This resulted in reproducible, distinctive fingerprints, confirming the difference in amplification specificity. Clear banding patterns were obtained with soil and rhizosphere samples by using both primer sets in combination. By comparing the electrophoretic mobility of community fingerprint bands to that of the bands obtained with separate clones, some could be tentatively identified. While 18S-rDNA sequences do not always provide the taxonomic resolution to identify fungal species and strains, they do provide information on the diversity and dynamics of groups of related species in environmental samples with sufficient resolution to produce discrete bands which can be separated by TGGE. This combination of 18S-rDNA PCR amplification and TGGE community analysis should allow study of the diversity, composition, and dynamics of the fungal community in bulk soil and in the rhizosphere.     

Alleviation of Mercury Toxicity in Wheat by the Interaction of Mercury-Tolerant Plant Growth-Promoting Rhizobacteria

Heavy metal contamination of agricultural soils has increased along with industrialization. Mercury is a toxic heavy metal and a widespread pollutant in the ecosystem. Mercury-tolerant and plant growth-promoting rhizobacteria (PGPR) HG 1, HG 2, and HG 3 were isolated from the rhizosphere of plants growing in a mercury-contaminated site. These isolates were able to grow in the presence of mercury ranging from 10 to 200 µM in minimal medium and 25 to 500 µM in LB medium. The strains were characterized by morphological, biochemical, and plant growth-promoting traits. In the present study, these PGPR strains were analyzed for their involvement in metal stress tolerance in Triticum aestivum (wheat). Two bacterial strains, namely, Enterobacter ludwigii (HG 2) and Klebsiella pneumoniae (HG 3), showed better growth promotion of T. aestivum seedlings under metal stress. Different growth parameters like, water content and biochemical properties were analyzed in the PGPR-inoculated wheat plants under 75 µM HgCl₂. Shoot length, root length, shoot dry weight, root dry weight and relative water content (RWC) were significantly higher in inoculated plants compared to uninoculated plants under stress condition. Proline content, electrolyte leakage, and malondialdehyde content (shoots and roots) were significantly lower in inoculated plants with respect to uninoculated plants under mercury stress. Therefore, it could be assumed that all these parameters collectively improve plant growth under mercury stress conditions in the presence of PGPR. Hence, these PGPRs can serve as promising candidates for increasing plant growth and also have immense potential for bioremediation of mercury-contaminated soils.     

Water stress amelioration and plant growth promotion in wheat plants by osmotic stress tolerant bacteria

Soil microorganisms with potential for alleviation of abiotic stresses in combination with plant growth promotion would be extremely useful tools in sustainable agriculture. To this end, the present study was initiated where forty-five salt tolerant bacterial isolates with ability to grow in high salt medium were obtained from the rhizosphere of Triticum aestivum and Imperata cylindrica. These bacteria were tested for plant-growth-promoting rhizobacteria traits in vitro such as phosphate solubilization, siderophore, ACC deaminase and IAA production. Of the forty-five isolates, W10 from wheat rhizosphere and IP8 from blady grass rhizosphere, which tested positive in all the tests were identified by morpholological, biochemical and 16SrDNA sequencing as Bacillus safensis and Ochrobactrum pseudogregnonense respectively and selected for in vivo studies. Both the bacteria could promote growth in six varieties of wheat tested in terms of increase in root and shoot biomass, height of plants, yield, as well as increase in chlorophyll content. Besides, the wheat plants could withstand water stress more efficiently in presence of the bacteria as indicated by delay in appearance of wilting symptoms increases in relative water content of treated water stressed plants in comparison to untreated stressed ones, and elevated antioxidant responses. Enhanced antioxidant responses were evident as elevated activities of enzymes such as catalase, peroxidase, ascorbate peroxidase, superoxide dismutase and glutathione reductase as well as increased accumulation of antioxidants such as carotenoids and ascorbate. Results clearly indicate that the ability of wheat plants to withstand water stress is enhanced by application of these bacteria which also function as plant growth promoting rhizobacteria.     

缓解花生连作障碍的根际促生菌分离及功能鉴定

【目的】长期连作障碍严重降低花生生产的产量及品质,根际促生菌可有效降解土壤中自毒化感物质、抑制植物病原菌生长及促进植物生长,从而有效缓解连作障碍问题。筛选优化具有缓解花生连作障碍能力的多功能根际益生微生物,验证其益生作用能力,为根际促生菌株在连作障碍中的应用提供理论依据及技术支持。【方法】采集连作12年地块花生根际土壤,利用以酚酸为唯一碳源的筛选培养基获得具有酚酸自毒化感物质降解及利用能力的根际促生菌,通过16S rRNA基因测序进行系统发育分析,确定根际促生菌菌株的分类地位,并验证其对植物病原菌生长抑制能力及解磷、解钾、产植物激素吲哚乙酸能力。【结果】从连作12年的花生发病土壤中获得7株可高效降解酚酸类自毒物质且降解底物多样的根际微生物菌株,经16S rRNA测序比对分别为克雷伯氏菌B02 (Klebsiella sp. B02)、克雷伯氏菌B07(Klebsiella sp. B07)、克雷伯氏菌B15 (Klebsiella sp. B15)、芽孢杆菌B28 (Bacillus sp. B28)、不动杆菌P09(Acinetobacter sp. P09)、布鲁氏杆菌VA05 (...     

Restoration of Growth of Durum Wheat (Triticum durum var. waha) Under Saline Conditions Due to Inoculation with the Rhizosphere Bacterium Azospirillum brasilense NH and Extracts of the Marine Alga Ulva lactuca

Inoculation with the rhizosphere bacterium Azospirillum brasilense NH, originally isolated from salt-affected soil in northern Algeria, greatly enhanced growth of durum wheat (Triticum durum var. waha) under saline soil conditions. Important plant parameters like the rate of germination, stem height, spike length, dry weight of roots and shoots, chlorophyll a and b content, proline and total sugar contents, 1000-seed weight, seed number per spike, and weight of seeds per spike were measured. At salt stress conditions (160 and 200 mM NaCl) A. brasilense NH restored almost completely vegetative growth and seed production. The combination with extracts of the marine alga Ulva lactuca resulted in even more improved salt tolerance of durum wheat. Proline and total sugar accumulation, a sign of physiological plant stress under inhibitory salt conditions, was reduced in plants inoculated with A. brasilense NH with and without addition of algal extracts. Inoculation with the salt-sensitive A. brasilense strain Sp7 could not restore salt-affected plant growth at 200 mM NaCl. Furthermore, it could be demonstrated by fluorescence in situ hybridization and confocal laser scanning microscopy that A. brasilense NH is able to colonize roots of durum wheat endophytically under salt-stressed conditions. Thus, the salt-tolerant rhizobacterium A. brasilense NH could effectively provide alone or in combination with extracts of U. lactuca a promising solution to overcome salt inhibition which is a major threat hindering productive wheat cultivation in arid saline soils.     

Development of a strain-specific genomic marker for monitoring a Bacillus subtilis biocontrol strain in the rhizosphere of tomato

A strain-specific molecular marker enabling the detection and tracking of the biological control agent Bacillus subtilis 101, when released into the environment, was developed. Random amplified polymorphic DNA (RAPD) technique was used to differentiate this from other B. subtilis strains. A differentially amplified fragment obtained from RAPD profiles was sequenced and characterized as sequence-characterized amplified region (SCAR) marker, and four primer pairs were designed and evaluated for their specificity towards this strain. The sensibility of the selected SCAR primer pair was evaluated by qualitative PCR and Southern blotting, and the detection limit was assessed around 10(2) CFU (g dry wt soil)(-1), thus providing a reliable tool for the traceability of this B. subtilis strain in greenhouse or field trials. A plating assay coupled to PCR with the SCAR primer pair was then used as a detection method in microcosm experiments for monitoring the population of B. subtilis 101 in the rhizosphere of tomato, grown under two different soil conditions, i.e. nonsterile peat-based substrate and sandy-loam agricultural soil, respectively. The data of rhizosphere colonization indicated that the soil conditions significantly affected the rhizosphere establishment of strain 101.     

Effects of Azospirillum brasilense indole-3-acetic acid production on inoculated wheat plants

The production of phytohormones by plant-growth promoting rhizobacteria is considered to be an important mechanism by which these bacteria promote plant growth. In this study the importance of indole-3-acetic acid (IAA) produced by Azospirillum brasilense Sp245 in the observed plant growth stimulation was investigated by using Sp245 strains genetically modified in IAA production. Firstly wild-type A. brasilense Sp245 and an ipdC knock-out mutant which produces only 10% of wild-type IAA levels (Vande Broek et al., J Bacteriol 181:1338–1342, 1999) were compared in a greenhouse inoculation experiment for a number of plant parameters, thereby clearly demonstrating the IAA effect in plant growth promotion. Secondly, the question was addressed whether altering expression of the ipdC gene, encoding the key enzyme for IAA biosynthesis in A. brasilense, could also contribute to plant growth promotion. For that purpose, the endogenous promoter of the ipdC gene was replaced by either a constitutive or a plant-inducible promoter and both constructs were introduced into the wild-type strain. Based on a greenhouse inoculation experiment it was found that the introduction of these recombinant ipdC constructs could further improve the plant-growth promoting effect of A. brasilense. These data support the possibility of constructing Azospirillum strains with better performance in plant growth promotion.