Tomato genotype and Azospirillum inoculation modulate the changes in bacterial communities associated with roots and leaves

AIMS: To evaluate the effect of plant variety and Azospirillum brasilense inoculation on the microbial communities colonizing roots and leaves of tomato (Lycopersicon esculentum Mill.) plants. METHODS AND RESULTS: Seeds of cherry and fresh-market tomato were inoculated with A. brasilense BNM65. Sixty days after planting, plants were harvested and the microbial communities of the rhizoplane and phyllosphere were analysed by community-level physiological profiles (CLPP) using BIOLOG EcoPlates and denaturing gradient gel electrophoresis (DGGE) of PCR-amplified 16S rRNA genes. Differences on the rhizoplane and phyllosphere bacterial communities between the two tomato types were detected by principal component analysis of the CLPP; DGGE fingerprints also showed differences at the phyllosphere level. Fresh-market tomato had a more complex phyllosphere bacterial community than cherry tomato, as determined by DGGE profiles. Physiological and genetic changes on phyllosphere and rhizoplane bacterial communities by Azospirillum seed inoculation were evident only on cherry tomato. CONCLUSIONS: Tomato genotype affects the response of native bacterial communities associated with the roots and leaves to A. brasilense seed inoculation. SIGNIFICANCE AND IMPACT OF THE STUDY: The successful implementation of Azospirillum inoculation requires not only the consideration of the interactions between A. brasilense strains and plant genotypes, but also the plant-associated microflora.     

Survival of Azospirillum brasilense in the Bulk Soil and Rhizosphere of 23 Soil Types

The survival of Azospirillum brasilense Cd and Sp-245 in the rhizosphere of wheat and tomato plants and in 23 types of plant-free sterilized soils obtained from a wide range of environments in Israel and Mexico was evaluated. Large numbers of A. brasilense cells were detected in all the rhizospheres tested, regardless of soil type, bacterial strain, the origin of the soil, or the amount of rainfall each soil type received prior to sampling. Survival of A. brasilense in soils without plants differed from that in the rhizosphere and was mainly related to the geographical origin of the soil. In Israeli soils from arid, semiarid, or mountain regions, viability of A. brasilense rapidly declined or populations completely disappeared below detectable levels within 35 days after inoculation. In contrast, populations in the arid soils of Baja California Sur, Mexico, remained stable or even increased during the 45-day period after inoculation. In soils from Central Mexico, viability slowly decreased with time. In all soils, percentages of clay, nitrogen, organic matter, and water-holding capacity were positively correlated with bacterial viability. High percentages of CaCO(inf3) and fine or rough sand had a highly negative effect on viability. The percentage of silt, pH, the percentage of phosphorus or potassium, electrical conductivity, and C/N ratio had no apparent effect on bacterial viability in the soil. Fifteen days after removal of inoculated plants, the remaining bacterial population in the three soil types tested began to decline sharply, reaching undetectable levels 90 days after inoculation. After plant removal, percolating the soils with water almost eliminated the A. brasilense population. Viability of A. brasilense in two artificial soils containing the same major soil components as the natural soils from Israel did was almost identical to that in the natural soils. We conclude that A. brasilense is a rhizosphere colonizer which survives poorly in most soils for prolonged periods of time; that outside the rhizosphere, seven abiotic parameters control the survival of this bacterium in the soil; and that disturbance of the soil (percolation with water or plant removal) directly and rapidly affects the population levels.     

Wheat inoculation with Azospirillum brasilense Sp6 and some mutants altered in nitrogen fixation and indole-3-acetic acid production

Abstract Inoculation of wheat seedlings with Azospirillum brasilense Sp6 produced an increase in the number and length of the lateral roots as a plant response. Inoculation with a Nif⁻ mutant, A. brasilense SpF103, which is producer of indole-3-acetic acid (IAA), yielded a very similar plant response. However, inoculation with a Nif⁻ mutant, A. brasilense SpF57, which is a low producer of IAA, did not elitic any response from the plant. The data suggest that the root system response of wheat seedlings to bacterial inoculation is due mainly to production of auxin-type substances by the microorganism.     

Effects of site and plant species on rhizosphere community structure as revealed by molecular analysis of microbial guilds

The bacterial and fungal rhizosphere communities of strawberry (Fragaria ananassa Duch.) and oilseed rape (Brassica napus L.) were analysed using molecular fingerprints. We aimed to determine to what extent the structure of different microbial groups in the rhizosphere is influenced by plant species and sampling site. Total community DNA was extracted from bulk and rhizosphere soil taken from three sites in Germany in two consecutive years. Bacterial, fungal and group-specific (Alphaproteobacteria, Betaproteobacteria and Actinobacteria) primers were used to PCR-amplify 16S rRNA and 18S rRNA gene fragments from community DNA prior to denaturing gradient gel electrophoresis (DGGE) analysis. Bacterial fingerprints of soil DNA revealed a high number of equally abundant faint bands, while rhizosphere fingerprints displayed a higher proportion of dominant bands and reduced richness, suggesting selection of bacterial populations in this environment. Plant specificity was detected in the rhizosphere by bacterial and group-specific DGGE profiles. Different bulk soil community fingerprints were revealed for each sampling site. The plant species was a determinant factor in shaping similar actinobacterial communities in the strawberry rhizosphere from different sites in both years. Higher heterogeneity of DGGE profiles within soil and rhizosphere replicates was observed for the fungi. Plant-specific composition of fungal communities in the rhizosphere could also be detected, but not in all cases. Cloning and sequencing of 16S rRNA gene fragments obtained from dominant DGGE bands detected in the bacterial profiles of the Rostock site revealed that Streptomyces sp. and Rhizobium sp. were among the dominant ribotypes in the strawberry rhizosphere, while sequences from Arthrobacter sp. corresponded to dominant bands from oilseed rape bacterial fingerprints.     

Assessing the impact of the biological control agent Bacillus thuringiensis on the indigenous microbial community within the pepper plant phyllosphere

Although biological control agents (BCAs) have been used extensively for controlling insects and pathogens of plants, little is known regarding the effects of such agents on the indigenous microbial communities within the plant phyllosphere. We assessed the effect of the BCA Bacillus thuringiensis (Bt) on the microbial communities within the pepper plant phyllosphere using culture-independent methodologies. Phospholipid fatty acid (PLFA) analysis suggested that the bacterial and fungal biomass were not significantly affected following Bt application. However, principal component analysis of PLFA data indicated that Bt did change the phyllosphere microbial community structure significantly. 16S rRNA gene-directed PCR with denaturing gradient gel electrophoresis (DGGE) also suggested a significant change in the phyllosphere bacterial community structure following Bt inoculation. Phylogenetic analysis of excised DGGE bands suggested a change in bacterial phyla; bands from untreated samples predominantly belonged to the Firmicutes, while Gammaproteobacteria abounded in the treated samples.     

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 (10(7) versus 10(5) 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 (10(7) versus 10(6) 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 (10(5) to 10(6) 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 (>10(8) 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.     

Enhanced micropropagation response and biocontrol effect of Azospirillum brasilense Sp245 on Prunus cerasifera L. clone Mr.S 2/5 plants

Inoculation with Azospirillum brasilense Sp245 exerts beneficial effects on micropropagated plants of Prunus cerasifera L. clone Mr.S 2/5, as seen in the results of a comparative analysis of inoculated and non-inoculated explants, during both the rooting and acclimatation phases. The presence of Azospirillum brasilense Sp245 increased root system, root hair biomass production and apical activity. Although the presence of the bacteria had a positive effect on rooting, the addition of indolebutyric acid (IBA) to Murashige and Skoog (MS) medium was seen as indispensable in order to promote the rooting of explants. Aside from the promotion of plant growth, A. brasilense Sp245 protects plants against pathogen attacks, such as Rhizoctonia spp., with a plant survival rate of nearly 100% vs. 0% as seen in the negative control. The biocontrol effect of A. brasilense Sp245 on the fungal rhizospheric community has been confirmed by denaturing gradient gel electrophoresis (DGGE) profiles of the rhizospheric microbial community. This study indicates that A. brasilense Sp245 could be employed as a tool in plant biotechnology.     

巴西固氮螺菌Yu62由水稻和烟草根部向茎、叶的迁移运动(英文)

巴西固氮螺菌(Azospirillum brasilence)是重要的植物促生内生菌之一。用gfp基因标记固氮螺菌后接种无菌的水稻和烟草幼苗的根部,限菌培养一定时间后,用共聚焦激光显微镜观察,结果表明:除了根内部有发荧光的螺菌定殖外,螺菌还分布在茎、叶的表皮细胞,皮层细胞和维管系统组织的细胞和细胞间隙。从根、茎、叶器官分离固氮螺菌,都存在有较高的螺菌群体密度。这一结果证明螺菌在植物内存在着从根部向茎、叶顶端的迁移现象。这一发现为研究巴西固氮螺菌在宿主植物体内的迁移运动的机制、与植物细胞间的分子相互作用及其对植物的促生作用奠定了生态学和细胞形态学的基础,也为实际应用提供了进一步的科学依据,具有重要的科学和实践意义。     

Growth of Quailbush in Acidic, Metalliferous Desert Mine Tailings: Effect of Azospirillum brasilense Sp6 on Biomass Production and Rhizosphere Community Structure

Mine tailing deposits in semiarid and arid environments frequently remain devoid of vegetation due to the toxicity of the substrate and the absence of a diverse soil microbial community capable of supporting seed germination and plant growth. The contribution of the plant growth promoting bacterium (PGPB) Azospirillum brasilense Sp6 to the growth of quailbush in compost-amended, moderately acidic, high-metal content mine tailings using an irrigation-based reclamation strategy was examined along with its influence on the rhizosphere bacterial community. Sp6 inoculation resulted in a significant (2.2-fold) increase in plant biomass production. The data suggest that the inoculum successfully colonized the root surface and persisted throughout the 60-day experiment in both the rhizosphere, as demonstrated by excision and sequencing of the appropriate denaturing gradient gel electrophoresis (DGGE) band, and the rhizoplane, as indicated by fluorescent in situ hybridization of root surfaces. Changes in rhizosphere community structure in response to Sp6 inoculation were evaluated after 15, 30, and 60 days using DGGE analysis of 16S rRNA polymerase chain reaction amplicons. A comparison of DGGE profiles using canonical correspondence analysis revealed a significant treatment effect (Sp6-inoculated vs. uninoculated plants vs. unplanted) on bacterial community structure at 15, 30, and 60 days (p?<?0.05). These data indicate that in an extremely stressed environment such as acid mine tailings, an inoculated plant growth promoting bacterium not only can persist and stimulate plant growth but also can directly or indirectly influence rhizobacterial community development.     

巴西固氮螺菌Yu62由水稻和烟草根部向茎、叶的迁移运动

巴西固氮螺菌(Azospirillrm brasilence)是重要的植物促生内生菌之一.用gfp基因标记固氮螺菌后接种无菌的水稻和烟草幼苗的根部,限制培养一定时间后,用共聚焦激光显微镜观察,结果表明:除了根部有发荧光的螺菌定殖外,螺菌还分布在茎、叶的表皮细胞,皮层细胞和维管系统组织的细胞间隙.从根、茎、叶器官分离固氮螺菌,都存在有较高的螺菌群体密度.这一结果证明螺菌在植物内存在着从根部向茎、叶顶端的迁移现象.这一发现为研究巴西固氮螺菌在窠主植物体内的迁移运动的机制、与植物细胞间的分子相互作用及其对植物的促生作用奠定了生态学和细胞形态学的基础,也为实际应用提示了进一步的科学依据,具有重要的科学和实践意义.     

AM真菌对青枯菌和根际细菌群落结构的影响

利用传统的平板培养与DGGE相结合的技术手段,研究了接种AM真菌对番茄根际土壤中的青枯菌和细菌群落结构的影响。结果表明,菌根根际土壤中的细菌总量和总DNA量都高于非菌根根际土壤,其中前者的青枯菌种群数量比后者低60倍;DGGE图谱也证实了AM真菌对青枯菌的抑制效应,还揭示出接种AM真菌对根际土壤中细菌群落结构所产生的复杂的影响。文章对AM真菌抑制青枯菌的机制进行了探讨。     

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.     

Spatial and temporal analysis of the microbial community in slow sand filters used for treating horticultural irrigation water

An experimental slow sand filter (SSF) was constructed to study the spatial and temporal structure of a bacterial community suppressive to an oomycete plant pathogen, Phytophthora cryptogea. Passage of water through the mature sand column resulted in complete removal of zoospores of the plant pathogen. To monitor global changes in the microbial community, bacterial and fungal numbers were estimated on selective media, direct viable counts of fungal spores were made, and the ATP content was measured. PCR amplification of 16S rRNA genes and denaturing gradient gel electrophoresis (DGGE) were used to study the dynamics of the bacterial community in detail. The top layer (1 cm) of the SSF column was dominated by a variable and active microbial population, whereas the middle (50 cm) and bottom (80 cm) layers were dominated by less active and diverse bacterial populations. The major changes in the microbial populations occurred during the first week of filter operation, and these populations then remained to the end of the study. Spatial and temporal nonlinear mapping of the DGGE bands provided a useful visual representation of the similarities between SSF samples. According to the DGGE profile, less than 2% of the dominating bands present in the SSF column were represented in the culturable population. Sequence analysis of DGGE bands from all depths of the SSF column indicated that a range of bacteria were present, with 16S rRNA gene sequences similar to groups such as Bacillus megaterium, Cytophaga, Desulfovibrio, Legionella, Rhodococcus rhodochrous, Sphingomonas, and an uncharacterized environmental clone. This study describes the characterization of the performance, and microbial composition, of SSFs used for the treatment of water for use in the horticultural industry. Utilization of naturally suppressive population of microorganisms either directly or by manipulation of the environment in an SSF may provide a more reproducible control method for the future.     

Nested PCR-denaturing gradient gel electrophoresis approach to determine the diversity of sulfate-reducing bacteria in complex microbial communities

Here, we describe a three-step nested-PCR-denaturing gradient gel electrophoresis (DGGE) strategy to detect sulfate-reducing bacteria (SRB) in complex microbial communities from industrial bioreactors. In the first step, the nearly complete 16S rRNA gene was amplified using bacterial primers. Subsequently, this product was used as a template in a second PCR with group-specific SRB primers. A third round of amplification was conducted to obtain fragments suitable for DGGE. The largest number of bands was observed in DGGE patterns of products obtained with primers specific for the Desulfovibrio-Desulfomicrobium group, indicating a large diversity of these SRBs. In addition, members of other phylogenetic SRB groups, i.e., Desulfotomaculum, Desulfobulbus, and Desulfococcus-Desulfonema-Desulfosarcina, were detected. Bands corresponding to Desulfobacterium and Desulfobacter were not detected in the bioreactor samples. Comparative sequence analysis of excised DGGE bands revealed the identity of the community members. The developed three-step PCR-DGGE strategy is a welcome tool for studying the diversity of sulfate-reducing bacteria.     

Spatial and Temporal Analysis of the Microbial Community in Slow Sand Filters Used for Treating Horticultural Irrigation Water

An experimental slow sand filter (SSF) was constructed to study the spatial and temporal structure of a bacterial community suppressive to an oomycete plant pathogen, Phytophthora cryptogea. Passage of water through the mature sand column resulted in complete removal of zoospores of the plant pathogen. To monitor global changes in the microbial community, bacterial and fungal numbers were estimated on selective media, direct viable counts of fungal spores were made, and the ATP content was measured. PCR amplification of 16S rRNA genes and denaturing gradient gel electrophoresis (DGGE) were used to study the dynamics of the bacterial community in detail. The top layer (1 cm) of the SSF column was dominated by a variable and active microbial population, whereas the middle (50 cm) and bottom (80 cm) layers were dominated by less active and diverse bacterial populations. The major changes in the microbial populations occurred during the first week of filter operation, and these populations then remained to the end of the study. Spatial and temporal nonlinear mapping of the DGGE bands provided a useful visual representation of the similarities between SSF samples. According to the DGGE profile, less than 2% of the dominating bands present in the SSF column were represented in the culturable population. Sequence analysis of DGGE bands from all depths of the SSF column indicated that a range of bacteria were present, with 16S rRNA gene sequences similar to groups such as Bacillus megaterium, Cytophaga, Desulfovibrio, Legionella, Rhodococcus rhodochrous, Sphingomonas, and an uncharacterized environmental clone. This study describes the characterization of the performance, and microbial composition, of SSFs used for the treatment of water for use in the horticultural industry. Utilization of naturally suppressive population of microorganisms either directly or by manipulation of the environment in an SSF may provide a more reproducible control method for the future.     

Responses of Sorghum and Pennisetum Species to the N(2)-Fixing Bacterium Azospirillum brasilense

Three field inoculation experiments, two in Florida and one in New Mexico, were conducted with Azospirillum brasilense Cd. Each of the Florida experiments evaluated two crop species. One species in each of the Florida experiments responded to inoculation with a significant dry matter yield increases of 11 to 24% and nitrogen yield increases of 9 to 39%. No inoculation response was noted in the New Mexico experiment. The responding species were Sorghum bicolor (L.) Moench (sorghum) and the interspecific hybrid between Pennisetum americanum (L.) K. Schum. (pearl millet) and P. purpureum Schumach. (napiergrass). Nonresponding species were pearl millet (Florida) and Sorghum sudanense (Piper) Staph. (New Mexico). Acetylene reduction activity of inoculated plots in Florida was low, showing no increase over the natural uninoculated background rates and, in one case, was negatively correlated with yield. Acetylene reduction activity was not measured in New Mexico. In Florida, A. brasilense populations were found to decline from 5 x 10 to 5 x 10 bacteria g of soil in about 3 weeks (quadratic regressions). Continued decline to less than 10 by week 5 indicated that the inoculated bacteria did not become established in the soil in high numbers. The A. brasilense population declined at about the same rate in the New Mexico experiment. The erractic inoculation responses in these experiments are similar to those observed in earlier work at the University of Florida. The lack of acetylene reduction activity response to inoculation and the rapid population decline of the inoculated bacteria suggest that N(2) fixation is not the major mechanism causing yield responses after inoculation.     

Maintenance of soil functioning following erosion of microbial diversity

The paradigm that soil microbial communities, being very diverse, have high functional redundancy levels, so that erosion of microbial diversity is less important for ecosystem functioning than erosion of plant or animal diversity, is often taken for granted. However, this has only been demonstrated for decomposition/respiration functions, performed by a large proportion of the total microbial community, but not for specialized microbial groups. Here, we determined the impact of a decrease in soil microbial diversity on soil ecosystem processes using a removal approach, in which less abundant species were removed preferentially. This was achieved by inoculation of sterile soil microcosms with serial dilutions of a suspension obtained from the same non-sterile soil and subsequent incubation, to enable recovery of community size. The sensitivity to diversity erosion was evaluated for three microbial functional groups with known contrasting taxonomic diversities (ammonia oxidizers < denitrifiers < heterotrophs). Diversity erosion within each functional group was characterized using molecular fingerprinting techniques: ribosomal intergenic spacer analysis (RISA) for the eubacterial community, denaturing gradient gel electrophoresis (DGGE) analysis of nirK genes for denitrifiers, and DGGE analysis of 16S rRNA genes for betaproteobacterial ammonia oxidizers. In addition, we simulated the impact of the removal approach by dilution on the number of soil bacterial species remaining in the inoculum using values of abundance distribution of bacterial species reported in the literature. The reduction of the diversity of the functional groups observed from genetic fingerprints did not impair the associated functioning of these groups, i.e. carbon mineralization, denitrification and nitrification. This was remarkable, because the amplitude of diversity erosion generated by the dilution approach was huge (level of bacterial species loss was estimated to be around 99.99% for the highest dilution). Our results demonstrate that the vast diversity of the soil microbiota makes soil ecosystem functioning largely insensitive to biodiversity erosion even for functions performed by specialized groups.     

Analysis of the Dynamics of Bacterial Communities in the Rhizosphere of the Chrysanthemum via Denaturing Gradient Gel Electrophoresis and Substrate Utilization Patterns

In order to gain a better understanding of the spatial and temporal dynamics of bacterial communities of the rhizosphere of the chrysanthemum, two complementary methods were used: a molecular bacterial community profiling method, i.e., 16S rRNA gene-based PCR followed by denaturing gradient gel electrophoresis (DGGE), and an agar plate method in which 11 sole-carbon-source utilization tests were used. The DGGE patterns showed that the bacterial communities as determined from direct rhizosphere DNA extracts were largely stable along developing roots of the chrysanthemum, with very little change over time or between root parts of different ages. The patterns were also similar to those produced with DNA extracts obtained from bulk soil samples. The DGGE patterns obtained by using microbial colonies from dilution plates as the source of target DNA were different from those found with the direct DNA extracts. Moreover, these patterns showed differences among plant replicates but also among replicate plates. Results obtained with the sole-carbon-source utilization tests indicated that the metabolic profile of the bacterial communities in the rhizosphere of the root tip did not change substantially during plant growth. This suggests selective development of specific bacterial populations by the presence of a root tip. On the other hand, the metabolic profile of bacterial communities in the rhizosphere of the root base changed during plant growth. With eight sole-carbon-source utilization tests, a significant effect of the development stage of the plant on the number of bacteria which were able to grow on these carbon sources was observed.     

Analysis of actinomycete communities by specific amplification of genes encoding 16S rRNA and gel-electrophoretic separation in denaturing gradients.

A group-specific primer, F243 (positions 226 to 243, Escherichia coli numbering), was developed by comparison of sequences of genes encoding 16S rRNA (16S rDNA) for the detection of actinomycetes in the environment with PCR and temperature or denaturing gradient gel electrophoresis (TGGE or DGGE, respectively). The specificity of the forward primer in combination with different reverse ones was tested with genomic DNA from a variety of bacterial strains. Most actinomycetes investigated could be separated by TGGE and DGGE, with both techniques giving similar results. Two strategies were employed to study natural microbial communities. First, we used the selective amplification of actinomycete sequences (E. coli positions 226 to 528) for direct analysis of the products in denaturing gradients. Second, a nested PCR providing actinomycete-specific fragments (E. coli positions 226 to 1401) was used which served as template for a PCR when conserved primers were used. The products (E. coli positions 968 to 1401) of this indirect approach were then separated by use of gradient gels. Both approaches allowed detection of actinomycete communities in soil. The second strategy allowed the estimation of the relative abundance of actinomycetes within the bacterial community. Mixtures of PCR-derived 16S rDNA fragments were used as model communities consisting of five actinomycetes and five other bacterial species. Actinomycete products were obtained over a 100-fold dilution range of the actinomycete DNA in the model community by specific PCR; detection of the diluted actinomycete DNA was not possible when conserved primers were used. The methods tested for detection were applied to monitor actinomycete community changes in potato rhizosphere and to investigate actinomycete diversity in different soils.     

Improvement in bioavailability of tricalcium phosphate to Cymbopogon martinii var. motia by rhizobacteria, AMF and Azospirillum inoculation

The interactive effects of phosphate solubilizing bacteria, N2 fixing bacteria and arbuscular mycorrhizal fungi (AMF) were studied in a low phosphate alkaline soil amended with tricalcium insoluble source of inorganic phosphate on the growth of an aromatic grass palmarosa (Cymbopogon martinii). The microbial inocula consisted of the AM fungus Glomus aggregatum, phosphate solubilizing rhizobacteria Bacillus polymyxa and N2 fixing bacteria Azospirillum brasilense. These rhizobacteria behaved as "mycorrhiza helper" and enhanced root colonization by G. aggregatum in presence of tricalcium phosphate at the rate of 200 mg kg(-1) soil (P1 level). Dual inoculation of G. aggregatum and B. polymyxa yielded 21.5 g plant dry weight (biomass), while it was 21.7 g in B. polymyxa and A. brasilense inoculated plants as compared to 14.9 g of control at the same level. Phosphate content was maximum (0.167%) in the combined treatment of G. aggregatum, B. polymyxa and A. brasilense at P1 level, however acid phosphatase activity was recorded to be 4.75 pmol mg(-1) min(-1) in G. aggregatum, B. polymyxa and A. brasilense treatment at P0 level. This study indicates that all microbes inoculated together help in the uptake of tricalcium phosphate which is otherwise not used by the plants and their addition at 200 mg kg(-1) of soil gave higher productivity to palmarosa plants.