The drop plate method as an alternative for Azospirillum spp. viable cell enumeration within the consensus protocol of the REDCAI network.

Quality evaluation of commercial inoculants is essential to warrant an adequate crop response to inoculation within a biosecurity framework. In this sense, this work is aimed at standardizing and validating the drop plate method for the enumeration of Azospirillum viable cells as an alternative to the spread plate technique, which is currently proposed in the consensus protocol of the REDCAI network. Between 14 and 25 private and public laboratories participated in three independent trials. We obtained consistent and robust results that allowed to confirm that both techniques are equivalent, concluding that the drop plate method is an alternative enumeration technique that is adequate to be included in the abovementioned consensus protocol.

Towards sustainable yield improvement: field inoculation of soybean with Bradyrhizobium and co-inoculation with Azospirillum in Mozambique.

The effects of sole inoculation of soybean (Glycine max L. Merrill) with Bradyrhizobium and co-inoculation with Bradyrhizobium and Azospirillum on nodulation, plant growth and yields were investigated in the 2013/2014 and 2014/2015 cropping seasons under field conditions in Mozambique. The treatments included (1) Control (non-inoculated control, with symbiosis depending on indigenous rhizobia), (2) Urea (non-inoculated, receiving 200 kg ha-1 of N), (3) Sole inoculation with B. diazoefficiens strain USDA 110, and (4) Co-inoculation with B. diazoefficiens strain USDA 110 and A. brasilense strains Ab-V5 and Ab-V6, evaluated in a randomized complete block design with five replications. Nodule number and dry weight, shoot dry weight, biological and grain yields, grain dry weight, and harvest index were evaluated. In general, both sole inoculation and co-inoculation enhanced nodulation in relation to control. Sole inoculation increased grain yield by 22% (356 kg ha-1), the same enhancement magnitude attained under mineral N treatment, suggesting that Bradyrhizobium inoculation provides ecological and economic sustainability to the soybean crop in Mozambique or other countries with similar agro-climatic conditions. Co-inoculation did not increase grain yields in relation to neither the control nor sole inoculation, indicating that further research with adapted and high yielding soybean varieties along with effective rhizobial strains is required in Mozambique to attune the beneficial Azospirillum-plant cultivar-rhizobia interactions that have been reported in other countries for several legumes, including soybean.

Potentially Mobile Denitrification Genes Identified in Azospirillum sp. Strain TSH58.

Denitrification ability is sporadically distributed among diverse bacteria, archaea, and fungi. In addition, disagreement has been found between denitrification gene phylogenies and the 16S rRNA gene phylogeny. These facts have suggested potential occurrences of horizontal gene transfer (HGT) for the denitrification genes. However, evidence of HGT has not been clearly presented thus far. In this study, we identified the sequences and the localization of the nitrite reductase genes in the genomes of 41 denitrifying Azospirillum sp. strains and searched for mobile genetic elements that contain denitrification genes. All Azospirillum sp. strains examined in this study possessed multiple replicons (4 to 11 replicons), with their sizes ranging from 7 to 1,031 kbp. Among those, the nitrite reductase gene nirK was located on large replicons (549 to 941 kbp). Genome sequencing showed that Azospirillum strains that had similar nirK sequences also shared similar nir-nor gene arrangements, especially between the TSH58, Sp7T, and Sp245 strains. In addition to the high similarity between nir-nor gene clusters among the three Azospirillum strains, a composite transposon structure was identified in the genome of strain TSH58, which contains the nir-nor gene cluster and the novel IS6 family insertion sequences (ISAz581 and ISAz582). The nirK gene within the composite transposon system was actively transcribed under denitrification-inducing conditions. Although not experimentally verified in this study, the composite transposon system containing the nir-nor gene cluster could be transferred to other cells if it is moved to a prophage region and the phage becomes activated and released outside the cells. Taken together, strain TSH58 most likely acquired its denitrification ability by HGT from closely related Azospirillum sp. denitrifiers.IMPORTANCE The evolutionary history of denitrification is complex. While the occurrence of horizontal gene transfer has been suggested for denitrification genes, most studies report circumstantial evidences, such as disagreement between denitrification gene phylogenies and the 16S rRNA gene phylogeny. Based on the comparative genome analyses of Azospirillum sp. denitrifiers, we identified denitrification genes, including nirK and norCBQD, located on a mobile genetic element in the genome of Azospirillum sp. strain TSH58. The nirK was actively transcribed under denitrification-inducing conditions. Since this gene was the sole nitrite reductase gene in strain TSH58, this strain most likely benefitted by acquiring denitrification genes via horizontal gene transfer. This finding will significantly advance our scientific knowledge regarding the ecology and evolution of denitrification.

Effects of colonization of a bacterial endophyte, Azospirillum sp. B510, on disease resistance in tomato.

A plant growth-promoting bacteria, Azospirillum sp. B510, isolated from rice, can enhance growth and yield and induce disease resistance against various types of diseases in rice. Because little is known about the interaction between other plant species and this strain, we have investigated the effect of its colonization on disease resistance in tomato plants. Treatment with this strain by soil-drenching method established endophytic colonization in root tissues in tomato plant. The endophytic colonization with this strain-induced disease resistance in tomato plant against bacterial leaf spot caused by Pseudomonas syringae pv. tomato and gray mold caused by Botrytis cinerea. In Azospirillum-treated plants, neither the accumulation of SA nor the expression of defense-related genes was observed. These indicate that endophytic colonization with Azospirillum sp. B510 is able to activate the innate immune system also in tomato, which does not seem to be systemic acquired resistance.

Activation of a calcium-dependent protein kinase involved in the Azospirillum growth promotion in rice.

Rice seedlings (Oryza sativa) inoculated with the plant growth-promoting rhizobacteria Azospirillum brasilense FT326 showed an enhanced development of the root system 3 days after inoculation. Later on, a remarkable enlargement of shoots was also evident. An increase in the Ca2+-dependent histone kinase activity was also detected as a result of inoculation. The biochemical characterization and Western-blot analysis of the kinase strongly supports the hypothesis that it belongs to a member of the rice CDPK family. The fact that the amount of the protein did not change upon inoculation seems to indicate that a posttranslational activation is responsible for the change in the enzymatic activity. An in-gel kinase experiment identified a 46 kDa CDPK like protein kinase as a putative component of the signal transduction pathway triggered by Azospirillum inoculation. To our knowledge, this is the first report on the possible involvement of a Ca2+-dependent protein kinase in promotion of rice plants growth by A. brasilense.

Evaluation of the possibility to use the plant-microbe interaction to stimulate radioactive 137Cs accumulation by plants in a contaminated farm field in Fukushima, Japan.

Field experiments in a contaminated farmland in Nihonmatsu city, Fukushima were conducted to assess the effectiveness of the plant-microbe interaction on removal of radiocesium. Before plowing, 93.3% of radiocesium was found in the top 5 cm layer (5,718 Bq kg DW(-1)). After plowing, Cs radioactivity in the 0-15 cm layer ranged from 2,037 to 3,277 Bq kg DW(-1). Based on sequential extraction, the percentage of available radiocesium (water soluble + exchangeable) was fewer than 10% of the total radioactive Cs. The transfer of (137)Cs was investigated in three agricultural crops; komatsuna (four cultivars), Indian mustard and buckwheat, inoculated with a Bacillus or an Azospirillum strains. Except for komatsuna Nikko and Indian mustard, inoculation with both strains resulted in an increase of biomass production by the tested plants. The highest (137)Cs radioactivity concentration in above-ground parts was found in Bacillus-inoculated komatsuna Nikko (121 Bq kg DW(-1)), accompanied with the highest (137)Cs TF (0.092). Furthermore, komatsuna Nikko-Bacillus and Indian mustard-Azospirillum associations gave the highest (137)Cs removal, 131.5 and 113.8 Bq m(-2), respectively. Despite the beneficial effect of inoculation, concentrations of (137)Cs and its transfer to the tested plants were not very high; consequently, removal of (137)Cs from soil would be very slow.

Stable cesium uptake and accumulation capacities of five plant species as influenced by bacterial inoculation and cesium distribution in the soil.

The effects of inoculation with Bacillus and Azospirillum strains on growth and cesium accumulation of five plant species, Komatsuna, Amaranth, sorghum, common millet and buckwheat, grown on cesium-spiked soil were assessed for potential use in cesium remediation. Pot experiments were performed using "artificially" Cs-contaminated soil. Three treatments were applied based on Cs location in the soil. For a soil height of 15 cm in the pots, Cs was added as follows: in the top five cm to imitate no ploughing condition; in the bottom five cm simulating inverted ploughing; and uniformly distributed Cs reproducing normal plowing. Generally, inoculation of Cs-exposed plants significantly enhanced growth and tolerance to this element. Transfer factor (ratio of Cs concentration in the plant tissues to that in surrounding soil) was strongly influenced by Cs distribution, with higher values in the top-Cs treatment. Within this treatment, inoculation of Komatsuna with Bacillus and Azospirillum strains resulted in the greatest transfer factors of 6.55 and 6.68, respectively. Cesium content in the shoots was high in the Azospirillum-inoculated Komatsuna, Amaranth, and buckwheat, i.e., 1,830, 1,220, and 1,030 µg per pot, respectively (five plants were grown in each pot). Therefore, inoculation of Komatsuna and Amaranth with the strains tested here could be effective in enhancing Cs accumulation. The decrease of Cs transfer under uniform- and bottom-Cs treatments would suggest that countermeasures aiming at decreasing the transfer of Cs could rely on ploughing practices.

Phenogenetic profile and agronomic contribution of Azospirillum argentinense Az39T, a reference strain for the South American inoculant industry.

Azospirillum sp. is a plant growth-promoting rhizobacteria largely recognized for its potential to increase the yield of different important crops. In this work, we present a thorough genomic and phenotypic analysis of A. argentinense Az39T to provide new insights into the beneficial mechanisms of this microorganism. Phenotypic analyses revealed the following in vitro abilities: growth at 20-38 °C (optimum, 28 °C), pH 6.0-8.0 (optimum, pH 6.8), and in the presence of 1% (w/v) NaCl; production of variable amounts of PHB as intracellular granules; nitrogen fixation under microaerophilic conditions; IAA synthesis in the presence of L-tryptophan. Through biochemical (API 20NE) and carbon utilization profiling (Biolog) assays, we proved that A. argentinense Az39T is able to use 15 substrates and metabolize 19 different carbon substrates. Lipid composition indicated a predominance of medium and long-chain saturated fatty acids. A total of 6 replicons classified as one main chromosome, three chromids, and two plasmids, according to their tRNA and core essential genes contents, were identified. Az39T genome includes genes associated with multiple plant growth-promoting (PGP) traits such as nitrogen fixation and production of auxins, cytokinin, abscisic acid, ethylene, and polyamines. In addition, Az39T genome harbor genetic elements associated with physiological features that facilitate its survival in the soil and competence for rhizospheric colonization; this includes motility, secretion system, and quorum sensing genetic determinants. A metadata analysis of Az39T agronomic performance in the pampas region, Argentina, demonstrated significant grain yield increases in wheat and maize, proving its potential to provide better growth conditions for dryland cereals. In conclusion, our data provide a detailed insight into the metabolic profile of A. argentinense Az39T, the strain most widely used to formulate non-legume inoculants in Argentina, and allow a better understanding of the mechanisms behind its field performance.

Tracking maize colonization and growth promotion by Azospirillum reveals strain-specific behavior and the influence of inoculation method.

Inoculation of Azospirillum in maize has become a standard practice in Latin America. However, information on the behavior and population survival of the Azospirillum post-inoculation is scarce, making standardization difficult and generating variations in inoculation efficiency across assays. In this study, we tracked the colonization of three agriculturally relevant Azospirillum strains (Ab-V5, Az39, and the ammonium excreting HM053) after different inoculation methods in maize crops by qPCR. Besides, we assessed their ability to promote maize growth by measuring biometric parameters after conducting a greenhouse essay over 42 days. Inoculated plants exhibited Azospirillum population ranging from 103 to 107 cells plant-1 throughout the experiment. While all strains efficiently colonized roots, only A. argentinense Az39 demonstrated bidirectional translocation between roots and shoots, which characterizes a systemic behavior. Optimal inoculation methods for plant growth promotion varied among strains: soil inoculation promoted the best maize growth for the Ab-V5 and Az39 strains, while seed inoculation proved most effective for HM053. The findings of this study demonstrate that the inoculation method affects the behavior of Azospirillum strains and their effectiveness in promoting maize growth, thereby guiding practices to enhance crop yield.

Comparison of prominent Azospirillum strains in Azospirillum-Pseudomonas-Glomus consortia for promotion of maize growth.

Azospirillum are prominent plant growth-promoting rhizobacteria (PGPR) extensively used as phytostimulatory crop inoculants, but only few studies are dealing with Azospirillum-containing mixed inocula involving more than two microorganisms. We compared here three prominent Azospirillum strains as part of three-component consortia including also the PGPR Pseudomonas fluorescens F113 and a mycorrhizal inoculant mix composed of three Glomus strains. Inoculant colonization of maize was assessed by quantitative PCR, transcription of auxin synthesis gene ipdC (involved in phytostimulation) in Azospirillum by RT-PCR, and effects on maize by secondary metabolic profiling and shoot biomass measurements. Results showed that phytostimulation by all the three-component consortia was comparable, despite contrasted survival of the Azospirillum strains and different secondary metabolic responses of maize to inoculation. Unexpectedly, the presence of Azospirillum in the inoculum resulted in lower phytostimulation in comparison with the Pseudomonas-Glomus two-component consortium, but this effect was transient. Azospirillum's ipdC gene was transcribed in all treatments, especially with three-component consortia, but not with all plants and samplings. Inoculation had no negative impact on the prevalence of mycorrhizal taxa in roots. In conclusion, this study brought new insights in the functioning of microbial consortia and showed that Azospirillum-Pseudomonas-Glomus three-component inoculants may be useful in environmental biotechnology for maize growth promotion.

Assessment of compost as a bio-fertilizer for the growth of paddy.

The vegetable wastes were converted into compost by a stepwise degradation and its characteristics were studied and analysed at each stage. The temperature increased from 290C to 60 degrees C on 60th day and reached 33 degrees C on 90th day. Shift of pH from 7.6 to 7.3 on 60th day caused a shift of microflora from 12.01 x 10(7) to 11.13 x 10(8) CFU ml(-1) on 30th day and 63.2 x 10(6) on 60th day and 36.75 x 10(6) on 90th day. Shift of microflora caused high decomposition of the waste into compost which were used for enriching the soil as manures. The other characteristics such as moisture, ash content and C:N ratio established the short period required for preparing a complete compost of good quality. The study showed the efficiency of these organisms as plant growth promoting rhizobacteria. Combinations of microorganisms with compost act as a good biofertilizer which improves the fertility of soil and increases plant growth. Better results were produced by organisms in combinations like Azospirillum, Rhizobium and Azotobacter. The least growth in shoot length (64 cm) total fresh weight (151g) and total dry weight (3.994 g) were observed in paddy grown in soil and Bacillus combination, but microbial mixture of compost and soil gave high paddy growth efficiency. The present study concludes that the rhizospheric organisms play well as plant growth promoting agents and gave a better yield and growth of plants in combination with the compost.

Population dynamics of free living, nitrogen fixing bacteria Azospirillum in Manakkudi mangrove ecosystem, India.

Seasonal variations of population dynamics of free living nitrogen fixing bacteria, Azospirillum in relation to chemical parameters in Manakkudi mangrove ecosystem was assessed in root and rhizosphere soil samples of mangroves and mangrove associated plants. In rhizosphere soil and root samples, the counts of Azospirillum were recorded maximum in Acrostichum aureum as 8.63 +/- 0.92 x 10(4) and 115.48 +/- 17.36 x 10(4) CFU g(-1), respectively. The counts of Azospirillum in non-rhizosphere soil varied from 0.01 +/- 0.001 x 10(4) to 5.77 +/- 0.92 x 10(4) CFU g(-1) and found maximum in February and minimum in March and September. Azospirillum counts in water samples were found maximum (2.24 x 10(4) CFU l(-1)) in February. During seasonal variations maximum counts of Azospirillum were recorded during southwest monsoon season in Avicennia officinalis (1.40 x 10(4) CFU g(-1)) followed by Rhizophora mucronata (1.07 x 10(4) CFU g(-1)). The average maximum population density of Azospirillum counts was found during non monsoon season (9.73 x 10(4) CFU g(-1)) and the average maximum population density of Azospirillum counts was found with the mangrove associated root samples (13.73 x 10(4) CFU g(-1)). Of the selected isolates Azospirillum lipoferum (60%) was found to be predominant followed by Azospirillum brasilense (25%), Azospirillum irakense (5%), Azospirillum halopraeferens (5%) and Azospirillum amazonense (5%). Of the isolated species, A. halopraeferens exhibited better growth at 35 g l(-1) NaCl. The level of Fe, Cu, Zn and Mn were varied from 0.91 to 15.93 ppm. The level of Mn (12.13 ppm) was found maximum during non-monsoon of rhizosphere soil sample. Highest rainfall (192.80 mm) and atmospheric temperature (25.10 degrees C) were recorded during south west monsoon and non monsoon seasons. The increased population density was greatly influenced by the pH (r = +0.686). The present finding provides enough information on the nitrogen flow through biological process in Manakkudi mangrove ecosystem which can be useful for the effective implementation of mangrove management plan.

[Morpho-functional changes of spring wheat plants under interrelation with diasotrophs of Azospirillas].

The morphological and functional changes of photosynthetic apparatus of spring wheat at inoculation by active strains of Aospirillium genus diazotrophs were studied. Inoculation resulted in an increase of mesophyll cell size of 3 spring wheat leaves and chloroplasts in them. The largest number of grains and volume of photomembranes were presented in the chloroplasts of plants inoculated by A. brasilense 77, which coincided with an increase in chlorophyll b content. In the mesophyll cells of plants inoculated by A. brasilense 102 a greater number of chloroplasts, mitochondria and peroxysomes was found as compared to other options. The spring wheat plants inoculated by the strains contained a significant amount of carotenoids.

Genomic insights into the versatility of the plant growth-promoting bacterium Azospirillum amazonense.

BACKGROUND: The species Azospirillum amazonense belongs to a well-known genus of plant growth-promoting bacteria. This bacterium is found in association with several crops of economic importance; however, there is a lack of information on its physiology. In this work, we present a comprehensive analysis of the genomic features of this species. RESULTS: Genes of A. amazonense related to nitrogen/carbon metabolism, energy production, phytohormone production, transport, quorum sensing, antibiotic resistance, chemotaxis/motility and bacteriophytochrome biosynthesis were identified. Noteworthy genes were the nitrogen fixation genes and the nitrilase gene, which could be directly implicated in plant growth promotion, and the carbon fixation genes, which had previously been poorly investigated in this genus. One important finding was that some A. amazonense genes, like the nitrogenase genes and RubisCO genes, were closer phylogenetically to Rhizobiales members than to species of its own order. CONCLUSION: The species A. amazonense presents a versatile repertoire of genes crucial for its plant-associated lifestyle.

Host plant secondary metabolite profiling shows a complex, strain-dependent response of maize to plant growth-promoting rhizobacteria of the genus Azospirillum.

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 10d 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.

Response to saline stress and aquaporin expression in Azospirillum-inoculated barley seedlings.

The ability of two strains of Azospirillum brasilense to mitigate NaCl stress in barley plants was evaluated. Barley seedlings were inoculated and subjected to 200 mM NaCl for 18 days. Several days after NaCl treatment, a significant decline in biomass as well as in height was observed in uninoculated plants. However, smaller reductions in biomass and height were detected in plants inoculated with strain Az39. All the stressed plants showed significantly higher Na(+) but lower K(+) contents in their shoots. The growth rate of uninoculated plants was adversely affected by saline treatment, which was associated with higher putrescine content and lower levels of HvPIP2;1 transcripts in the roots. Azospirillum inoculation triggered the transcription of this gene. Our results suggest that barley plants inoculated with A. brasilense may be better prepared to thrive under saline conditions. To our knowledge, this is the first report showing an effect of Azospirillum inoculation on the expression of PIP2;1, a gene involved in the synthesis of root water channels.

Fluctuation of microbial activities after influent load variations in a full-scale SBR: recovery of the biomass after starvation.

Due to variations in the production levels, a full-scale sequencing batch reactor (SBR) for post-treatment of tannery wastewater was exposed to low and high ammonia load periods. In order to study how these changes affected the N-removal capacity, the microbiology of the reactor was studied by a diverse set of techniques including molecular tools, activity tests, and microbial counts in samples taken along 3 years. The recover capacity of the biomass was also studied in a lab-scale reactor operated with intermittent aeration without feeding for 36 days. The results showed that changes in the feeding negatively affected the nitrifying community, but the nitrogen removal efficiencies could be restored after the concentration stress. Species substitution was observed within the nitrifying bacteria, Nitrosomonas europaea and Nitrobacter predominated initially, and after an ammonia overload period, Nitrosomonas nitrosa and Nitrospira became dominant. Some denitrifiers, with nirS related to Alicycliphilus, Azospirillum, and Marinobacter nirS, persisted during long-term reactor operation, but the community fluctuated both in composition and in abundance. This fluctuating community may better resist the continuous changes in the feeding regime. Our results showed that a nitrifying-denitrifying SBR could be operated with low loads or even without feeding during production shut down periods.

Effects of colonization of a bacterial endophyte, Azospirillum sp. B510, on disease resistance in rice.

Agriculturally important grasses contain numerous diazotrophic bacteria, the interactions of which are speculated to have some other benefits to the host plants. In this study, we analyzed the effects of a bacterial endophyte, Azospirillum sp. B510, on disease resistance in host rice plants. Rice plants (Oryza sativa cv. Nipponbare) were inoculated with B510 exhibited enhanced resistance against diseases caused by the virulent rice blast fungus Magnaporthe oryzae and by the virulent bacterial pathogen Xanthomonas oryzae. In the rice plants, neither salicylic acid (SA) accumulation nor expression of pathogenesis-related (PR) genes was induced by interaction with this bacterium, except for slight induction of PBZ1. These results indicate the possibility that strain B510 is able to induce disease resistance in rice by activating a novel type of resistance mechanism independent of SA-mediated defense signaling.

Azospirillum palatum sp. nov., isolated from forest soil in Zhejiang province, China.

A Gram-negative bacterium designated ww 10(T) was isolated by plating dilutions from forest soil in Zhejiang province, China. Strain ww 10(T) was investigated by polyphasic taxonomic study including phenotypic and phylogenetic analysis. Cells of ww 10(T) were Gram-negative, strictly aerobic, motile with peritrichous flagella and rod-shaped. The strain grew optimally at 30-37 masculineC and pH 6.0-8.0. The major fatty acids were C(18:1)omega7C, cyclo-C(19:0) omega8C and C(16:0). The respiratory quinones contained a large amount of Q-10, a moderate component of Q-9 and a minor of Q-8. The G+C content of genomic DNA was about 67.3 mol%. Phylogenetic analysis revealed that strain ww 10(T) belongs to the phyletic cluster of genus Azospirillum and displayed 16S rRNA gene sequence similarity lower than 96.3% to the four closest described species of the genera Azospirillum and Roseomonas. Results of polyphasic taxonomic analysis showed that strain ww 10(T) represents a novel species in the genus Azospirillum, for which the name Azospirillum palatum sp. nov., is proposed. The type strain is ww 10(T) (=LMG 24444(T)=KCTC 13200(T)=CCTCC AB 207189(T)).

Joint immobilization of plant growth-promoting bacteria and green microalgae in alginate beads as an experimental model for studying plant-bacterium interactions.

A simple, quantitative experimental model, offering a convenient and basic approach to studies of plant-bacterium interactions, is proposed. This involves immobilizing a unicellular, freshwater microalga, a Chlorella species, serving as the plant, with a plant growth-promoting bacterium, an Azospirillum species, in small alginate beads to allow close interaction and to avoid external interference from bacterial contaminants.