Are drought-resistance promoting bacteria cross-compatible with different plant models?

The association between plant and plant growth promoting bacteria (PGPB) contributes to the successful thriving of plants in extreme environments featured by water shortage. We have recently shown that, with respect to the non-cultivated desert soil, the rhizosphere of pepper plants cultivated under desert farming hosts PGPB communities that are endowed with a large portfolio of PGP traits. Pepper plants exposed to bacterial isolates from plants cultivated under desert farming exhibited a higher tolerance to water shortage, compared with untreated control. This promotion was mediated by a larger root system (up to 40%), stimulated by the bacteria, that enhanced plant ability to uptake water from dry soil. We provide initial evidence that the nature of the interaction can have a limited level of specificity and that PGPB isolates may determine resistance to water stress in plants others than the one of the original isolation. It is apparent that, in relation to plant resistance to water stress, a feature of primary evolutionary importance for all plants, a cross-compatibility between PGPB and different plant models exists at least on a short-term.     

Genetics of phosphate solubilization and its potential applications for improving plant growth-promoting bacteria

Plant growth-promoting bacteria (PGPB) are soil and rhizosphere bacteria that can benefit plant growth by different mechanisms. The ability of some microorganisms to convert insoluble phosphorus (P) to an accessible form, like orthophosphate, is an important trait in a PGPB for increasing plant yields. In this mini-review, the isolation and characterization of genes involved in mineralization of organic P sources (by the action of enzymes acid phosphatases and phytases), as well as mineral phosphate solubilization, is reviewed. Preliminary results achieved in the engineering of bacterial strains for improving capacity for phosphate solubilization are presented, and application of this knowledge to improving agricultural inoculants is discussed.     

An improved method of microencapsulation and its evaluation to protect Lactobacillus spp. in simulated gastric conditions

An improved method of microencapsulation was developed to increase the efficacy of capsules in protecting the encapsulated bacteria under simulated gastric conditions. Lactobacillus acidophilus CSCC 2400 was encapsulated in calcium alginate and tested for its survival in simulated gastric conditions. The effects of different capsule sizes (200, 450, 1000 microm), different sodium alginate concentrations (0.75%, 1%, 1.5%, 1.8% and 2% w/v) and different concentrations of calcium chloride (0.1, 0.2, 1.0 M) on the viability of encapsulated bacteria were investigated. The viability of the cells in the microcapsules increased with an increase in alginate capsule size and gel concentration. There was no significant difference (p>0.05) in the viability of encapsulated cells when the concentration of calcium chloride was increased. Increase in cell load during encapsulation increased the number of bacterial survivors at the end of 3-h incubation in simulated gastric conditions. Hardening the capsule in calcium chloride solution for a longer time (8 h) had no impact on increasing the viability of encapsulated bacteria in a simulated gastric environment. The release of encapsulated cells at different phosphate buffer concentrations was also studied. When encapsulated L. acidophilus CSCC 2400 and L. acidophilus CSCC 2409 were subjected to low pH (pH 2) and high bile concentration (1.0% bile) under optimal encapsulation conditions (1.8% (w/v) alginate, 10(9) CFU/ml, 30 min hardening in 0.1 M CaCl(2) and capsule size 450 microm), there was a significant increase (p<0.05) in viable cell counts, compared to the free cells under similar conditions. Thus the encapsulation method described in this study may be effectively used to protect the lactobacillus from adverse gastric conditions.     

Water flow induced transport of Pseudomonas fluorescens cells through soil columns as affected by inoculant treatment

Abstract Water flow induced transport of Pseudomonas fluorescens cells through soil columns was measured as affected by the inoculant treatment. Bacterial cells were introduced into the topsoil of columns, either encapsulated in alginate beads of different types or mixed with bentonite clay in concentrations ranging from 0.5 to 5.0% (w/v). Survival of bacterial cells was improved with the use of alginate or bentonite. Transport, as determined by destructive sampling of the columns, was reduced with the use of alginate encapsulation. Drying of the beads had no influence on transport. The presence of bentonite in the topsoil, either pre-mixed through the soil, or applied as a slurry together with the bacteria, also reduced transport, except when 0.5% was pre-mixed through the soil. P. fluorescens cells encapsulated in alginate beads prepared with water and supplemented with skim milk powder and bentonite showed the best survival during the time of the experiment and the most reduced transport compared to the control. Therefore, cells encapsulated in this way are suitable, due to their optimal survival and reduced spread, for use in a field experiment with genetically manipulated bacteria.     

Transport and survival of alginate-encapsulated and free lux-lac marked Pseudomonas aeruginosa UG2Lr cells in soil

Transport and survival of alginate-encapsulated and unencapsulated Pseudomonas aeruginosa UG2Lr through soil microcosms was examined. Bacterial cells encapsulated in alginate beads or mixed with soil were introduced into soil microcosms. Microbial cell survival and cell transport were monitored by destructive sampling and selective plating of the microcosms over a 9-week period. Survival rates were greatest when using encapsulated P. aeruginosa UG2Lr cells. Water flow increased microbial cell dispersal from the site of inoculation. After 3 weeks, encapsulated and free cells showed similar distribution patterns. However, after 9 weeks microbial cell distribution was more extensive throughout the soil in the encapsulated treatments under all conditions. Therefore, alginate encapsulation is a suitable method to enhance survival and distribution of microbial inocula in the soil environment.     

盐胁迫环境下植物促生菌的作用机制研究进展

盐胁迫是限制干旱和半干旱地区作物生产的主要非生物胁迫之一,严重影响作物的生长发育,植物促生菌(Plant growth-promoting bacteria,PGPB)可有效减轻植物的盐胁迫损伤,合理施用PGPB是盐胁迫下促进作物生长的重要途径。本文从盐胁迫环境下PGPB在调节植物激素内稳态、促进养分吸收和诱导植物产生系统耐受性等方面的作用阐述了PGPB提高植物耐盐性、减轻植物胁迫损伤的作用机制。讨论了能够在植物根际稳定定殖并在盐生环境下稳定保持PGP活性的功能菌株对未来农业的可持续发展的重要意义,同时,对该研究方向的重难点和未来的发展趋势作出展望。     

Plant regeneration from encapsulated nodal segments of Dalbergia sissoo Roxb., a timber-yielding leguminous tree species

One of the alternative methods adopted in recent years is to use biotechnological approaches for improving the tree species. The synthetic seeds offer several advantages, e.g., easy handling, storability, reduced size of propagules, and transportability. Germplasm can be effectively stored in the form of synthetic seeds. A protocol has been developed for plant regeneration from encapsulated nodal segments of Dalbergia sissoo Roxb. Nodal segments collected from basal sprouts of mature trees were encapsulated in calcium alginate beads. Inability of nodal segments entrapped in calcium alginate beads to form root was a major problem. To avoid this problem, an appropriate root induction treatment was given to nodal segments for 10 days, prior to encapsulation to allow formation of root primordia. For synthetic seeds production and subsequent conversion into plantlet, nodal segments with root primordia were encapsulated using sodium alginate and calcium chloride as gelling matrix. The best gel complexation was achieved using 3% sodium alginate and 75 mmol/L CaCl2 2H2O. Maximum percentage response (85%) for conversion of encapsulated nodal segments into plantlets was achieved on 1/2-MS medium without plant growth regulators, after 25 days of culture. The frequency of conversion of encapsulated nodal segments into plantlets affected by the concentration of sodium alginate, and the presence or absence of 1/2-MS nutrients in calcium alginate beads. Plantlets with well developed roots and shoots were transferred to pots containing autoclaved mixture of peat moss and soil (1:1). Plants were also established in pots. The conversion of encapsulated nodal segments into plantlets also occurred when calcium alginate beads having entrapped nodal segments were directly sown in autoclaved peat moss moistened with 1/2-MS0 medium. Out of 60 encapsulated nodal segments, in each experiments, stored at 4 degrees C for 30 days, 44 plants developed under in vitro conditions, and 27 on peat moss moistened with 1/2-MS0.     

Mixed planting with a leguminous plant outperforms bacteria in promoting growth of a metal remediating plant through histidine synthesis

The effectiveness of plant growth promoting bacteria (PGPB) in improving metal phytoremediation is still limited by stunted plant growth under high soil metal concentrations. Meanwhile, mixed planting with leguminous plants is known to improve yield in nutrient deficient soils but the use of a metal tolerant legume to enhance metal tolerance of a phytoremediator has not been explored. We compared the use of Pseudomonas brassicacearum, Rhizobium leguminosarum, and the metal tolerant leguminous plant Vicia sativa to promote the growth of Brassica juncea in soil contaminated with 400 mg Zn kg^–1, and used synchrotron based microfocus X-ray absorption spectroscopy to probe Zn speciation in plant roots. B. juncea grew better when planted with V. sativa than when inoculated with PGPB. By combining PGPB with mixed planting, B. juncea recovered full growth while also achieving soil remediation efficiency of >75%, the maximum ever demonstrated for B. juncea. μXANES analysis of V. sativa suggested possible root exudation of the Zn chelates histidine and cysteine were responsible for reducing Zn toxicity. We propose the exploration of a legume-assisted-phytoremediation system as a more effective alternative to PGPB for Zn bioremediation.     

Enhanced Cd extraction of oilseed rape (Brassica napus) by plant growth-promoting bacteria isolated from Cd hyperaccumulator Sedum alfredii Hance

Four plant growth-promoting bacteria (PGPB) were used as study materials, among them two heavy metal-tolerant rhizosphere strains SrN1 (Arthrobacter sp.) and SrN9 (Bacillus altitudinis) were isolated from rhizosphere soil, while two endophytic strains SaN1 (Bacillus megaterium) and SaMR12 (Sphingomonas) were identified from roots of the cadmium (Cd)/zinc (Zn) hyperaccumulator Sedum alfredii Hance. A pot experiment was carried out to investigate the effects of these PGPB on plant growth and Cd accumulation of oilseed rape (Brassica napus) plants grown on aged Cd-spiked soil. The results showed that the four PGPB significantly boosted oilseed rape shoot biomass production, improved soil and plant analyzer development (SPAD) value, enhanced Cd uptake of plant and Cd translocation to the leaves. By fluorescent in situ hybridization (FISH) and green fluorescent protein (GFP), we demonstrated the studied S. alfredii endophytic bacterium SaMR12 were able to colonize successfully in the B. napus roots. However, all four PGPB could increase seed Cd accumulation. Due to its potential to enhance Cd uptake by the plant and to restrict Cd accumulation in the seeds, SaMR12 was selected as the most promising microbial partner of B. napus when setting up a plant–microbe fortified remediation system.     

Plant growth-promoting bacteria as inoculants in agricultural soils

Plant-microbe interactions in the rhizosphere are the determinants of plant health, productivity and soil fertility. Plant growth-promoting bacteria (PGPB) are bacteria that can enhance plant growth and protect plants from disease and abiotic stresses through a wide variety of mechanisms; those that establish close associations with plants, such as the endophytes, could be more successful in plant growth promotion. Several important bacterial characteristics, such as biological nitrogen fixation, phosphate solubilization, ACC deaminase activity, and production of siderophores and phytohormones, can be assessed as plant growth promotion (PGP) traits. Bacterial inoculants can contribute to increase agronomic efficiency by reducing production costs and environmental pollution, once the use of chemical fertilizers can be reduced or eliminated if the inoculants are efficient. For bacterial inoculants to obtain success in improving plant growth and productivity, several processes involved can influence the efficiency of inoculation, as for example the exudation by plant roots, the bacterial colonization in the roots, and soil health. This review presents an overview of the importance of soil-plant-microbe interactions to the development of efficient inoculants, once PGPB are extensively studied microorganisms, representing a very diverse group of easily accessible beneficial bacteria.     

Enhancing cell survival of atrazine degrading Rhodococcus erythropolis NI86/21 cells encapsulated in alginate beads

AIMS: To develop a method to produce beads with encapsulated Rhodococcus erythropolis NI86/21 with high cell density, extended shelf life, ease of handling and good atrazine degradation capabilities in both liquid and in agricultural soil. METHODS AND RESULTS: Our findings show that the supplementary recovery step in nutrient broth media shortly after cell encapsulation facilitates cell survival in both wet and dry beads upon extended storage at 4 degrees C. Air drying has little or no impact on encapsulated R. erythropolis cell's ability to degrade atrazine in liquid or soil. Bead storage for periods extending up to 12 months at 4 degrees C did not affect the capacity of R. erythropolis encapsulated cells to degrade atrazine in either BMN or nonsterile soil extracts. Bentonite-amended beads formulated with 1% skim milk and exposed to the supplementary growth step, outperformed all other bead formats. These beads provided adequate numbers of vigorous R. erythropolis cells in either liquid or soil media to degrade atrazine. CONCLUSIONS: Supplementary growth in nutrient broth media immediately following cell encapsulation greatly enhances R. erythropolis cells survival in both wet and dry beads upon extended storage at 4 degrees C. Wet and dried beads have similar capacity for atrazine degradation, and their usefulness and appeal in agronomic practise will only be known after bioassay evaluation and successful demonstration at field scale using incurred residues. SIGNIFICANCE AND IMPACT OF THE STUDY: R. erythropolis NI86/21 encapsulated cells have the potential to reduce residual atrazine in soil, thereby minimizing the likelihood of off-site transport to ground or river water and reduce the loss of crops because of phytotoxicity of residual herbicide. Owing to their ease of handling, storage and possible compatibilities with pre-existing mechanical equipment, dried bead formats are ideally suited for agricultural and remediational applications.     

In vitro analyses are not reliable predictors of the plant growth promotion capability of bacteria; a Pseudomonas fluorescens strain that promotes the growth and yield of wheat

Aims: In this study, we set out to identify bacteria that can be used to promote the growth of cereals, while concurrently investigating the merits of using a range of such tests to preselect bacteria for glasshouse studies. Methods and Results: A panel of 15 strains isolated from the rhizosphere and phyllosphere of cereals was tested for the ability to improve the germination of wheat seeds and for production of a range of factors associated with plant growth promotion. In parallel, all bacteria were tested for their ability to improve biomass and grain yield when applied as a soil amendment in glasshouse trials. Conclusions: There was no significant correlation between growth promotion potential in the glasshouse and the results of either the phenotypic or the germination tests. Glasshouse tests identified that only one strain, Pseudomonas fluorescens strain MKB37, gave a significant increase in head weight and grain yield. Significance and Impact of the Study: While this study has identified a candidate for further field tests, it has also highlighted the fact that the modes of action for plant growth‐promoting bacteria (PGPB) are still not fully understood, and that there is no efficient and effective screening method for identifying PGPB by laboratory tests.     

Inoculants of plant growth-promoting bacteria for use in agriculture

An assessment of the current state of bacterial inoculants for contemporary agriculture in developed and developing countries is critically evaluated from the point of view of their actual status and future use. Special emphasis is given to two new concepts of inoculation, as yet unavailable commercially: (i) synthetic inoculants under development for plant-growth-promoting bacteria (PGPB) (Bashan and Holguin, 1998), and (ii) inoculation by groups of associated bacteria.This review contains: A brief historical overview of bacterial inoculants; the rationale for plant inoculation with emphasis on developing countries and semiarid agriculture, and the concept and application of mixed inoculant; discussion of microbial formulation including optimization of carrier-compound characteristics, types of existing carriers for inoculants, traditional formulations, future trends in formulations using unconventional materials, encapsulated synthetic formulations, macro and micro formulations of alginate, encapsulation of beneficial bacteria using other materials, regulation and contamination of commercial inoculants, and examples of modern commercial bacterial inoculants; and a consideration of time constraints and application methods for bacterial inoculants, commercial production, marketing, and the prospects of inoculants in modern agriculture.     

Impact of Alginate Composition: From Bead Mechanical Properties to Encapsulated HepG2/C3A Cell Activities for In Vivo Implantation

Recently, interest has focused on hepatocytes’ implantation to provide end stage liver failure patients with a temporary support until spontaneous recovery or a suitable donor becomes available. To avoid cell damage and use of an immunosuppressive treatment, hepatic cells could be implanted after encapsulation in a porous biomaterial of bead or capsule shape. The aim of this study was to compare the production and the physical properties of the beads, together with some hepatic cell functions, resulting from the use of different material combinations for cell microencapsulation: alginate alone or combined with type I collagen with or without poly-L-lysine and alginate coatings. Collagen and poly-L-lysine increased the bead mechanical resistance but lowered the mass transfer kinetics of vitamin B12. Proliferation of encapsulated HepG2/C3A cells was shown to be improved in alginate-collagen beads. Finally, when the beads were subcutaneously implanted in mice, the inflammatory response was reduced in the case of alginate mixed with collagen. This in vitro and in vivo study clearly outlines, based on a systematic comparison, the necessity of compromising between material physical properties (mechanical stability and porosity) and cell behavior (viability, proliferation, functionalities) to define optima hepatic cell microencapsulation conditions before implantation.     

Effects of bioaugmentation strategies in UASB reactors with a methanogenic consortium for removal of phenolic compounds

The removal of phenol, ortho- (o-) and para- (p-)cresol was studied with two series of UASB reactors using unacclimatized granular sludges bioaugmented with a consortium enriched against these substances. The parameters studied were the amount of inoculum added to the sludges and the method of immobilization of the inoculum. Two methods were used, adsorption to the biomass or encapsulation within calcium alginate beads. In the bioaugmentation by adsorption experiment, and with a 10% inoculum, complete phenol removal was obtained after 36 d, while 178 d were required in the control reactor. For p-cresol, 95% removal was obtained in the bioaugmented reactor on day 48 while 60 d were required to achieve 90% removal in the control reactor. For o-cresol, the removals were only marginally better with the bioaugmented reactors. Tests performed with the reactors biomass under non-limiting substrate concentrations showed that the specific activities of the bioaugmented biomasses were larger than the original biomass for phenol, and p-cresol even after 276 of operations, showing that the inoculum bacteria successfully colonized the sludge granules. Immobilization of the inoculum by encapsulation in calcium alginate beads, was performed with 10% of the inoculum. Results showed that the best activities were obtained when the consortium was encapsulated alone and the beads added to the sludges. This reactor presented excellent activity and the highest removal of the various phenolic compounds a few days after start-up. After 90 d, a high-phenolic compounds removal was still observed, demonstrating the effectiveness of the encapsulation technique for the start-up and maintenance of high-removal activities.     

Tuning structural durability of yeast-encapsulating alginate gel beads with interpenetrating networks for sustained bioethanol production

Microorganisms have become key components in many biotechnological processes to produce various chemicals and biofuels. The encapsulation of microbial cells in calcium cross-linked alginate gel beads has been extensively studied due to several advantages over using free cells. However, industrial use of alginate gel beads has been hampered by the low structural stability of the beads. In this study, we demonstrate that the incorporation of interpenetrating covalent cross-links in an ionically cross-linked alginate gel bead significantly enhances the bead's structural durability. The interpenetrating network (IPN) was prepared by first cross-linking alginate chemically modified with methacrylic groups, termed methacrylic alginate (MA), with calcium ions and subsequently conducting a photo cross-linking reaction. The resulting methacrylic alginate gel beads (IPN-MA) exhibited higher stiffness, ultimate strength and ultimate strain and also remained more stable in media either subjected to high shear or supplemented with chelating agents than calcium cross-linked alginate gel beads. Furthermore, yeast cells encapsulated in IPN-MA gel beads remained more metabolically active in ethanol production than those in calcium cross-linked alginate gel beads. Overall, the results of this study will be highly useful in designing encapsulation devices with improved structural durability for a broad array of prokaryotic and eukaryotic cells used in biochemical and industrial processes.     

Shoot endophytic plant growth-promoting bacteria reduce cadmium toxicity and enhance switchgrass (<Emphasis Type="Italic">Panicum virgatum</Emphasis> L.) biomass

The aims of the study were to increase the biomass and to alleviate the deleterious effects of cadmium (Cd) in the switchgrass cultivars (Panicum virgatum L.) Alamo and Cave-in-Rock (CIR) under cadmium (Cd) stress using Cd-tolerant shoot endophytic plant growth-promoting bacteria (PGPB). Four shoot endophytic bacterial strains, viz. Bc09, So23, E02, and Oj24, were isolated from the above-ground parts of plants grown in a Cd-polluted soil and were successfully identified by 16S rRNA gene sequencing as Pseudomonas grimontii, Pantoea vagans, Pseudomonas veronii, and Pseudomonas fluorescens, respectively. These four strains were adapted to high CdCl₂ concentrations as they had higher Cd uptake capacities. In addition, they possessed a huge amount of growth regulatory activities e.g., indole acetic acid production, 1-aminocyclopropane-1-carboxylic acid deaminase (ACCD) activity, and phosphate solubilization. Growth particularly the height and biomass of both cultivars increased significantly in response to PGPB inoculation in the 20 µM CdCl₂ stress. The shoot biomass of the PGPB-inoculated Alamo was higher than the CIR under Cd stress. Interestingly, the level of Cd inside PGPB-inoculated plant tissues and the translocation factors were lower compared with the noninoculated Cd control plants. CIR plants exhibited higher Cd content than Alamo plants. Through confocal microscopy, green fluorescence was observed in roots and leaf tissues 2 days after the inoculation of green fluorescent protein (GFP)-labeled bacteria in Alamo, which confirmed the successful colonization of bacteria inside the plant tissues. These shoot endophytic PGPB and switchgrass interactions are useful for the sustainable biomass production of bioenergy crop in a Cd-contaminated environment.     

Generation of Alginate Microspheres for Biomedical Applications

Alginate-based materials have received considerable attention for biomedical applications because of their hydrophilic nature, biocompatibility, and physical architecture. Applications include cell encapsulation, drug delivery, stem cell culture, and tissue engineering scaffolds. In fact, clinical trials are currently being performed in which islets are encapsulated in PLO coated alginate microbeads as a treatment of type I diabetes. However, large numbers of islets are required for efficacy due to poor survival following transplantation. The ability to locally stimulate microvascular network formation around the encapsulated cells may increase their viability through improved transport of oxygen, glucose and other vital nutrients. Fibroblast growth factor-1 (FGF-1) is a naturally occurring growth factor that is able to stimulate blood vessel formation and improve oxygen levels in ischemic tissues. The efficacy of FGF-1 is enhanced when it is delivered in a sustained fashion rather than a single large-bolus administration. The local long-term release of growth factors from islet encapsulation systems could stimulate the growth of blood vessels directly towards the transplanted cells, potentially improving functional graft outcomes. In this article, we outline procedures for the preparation of alginate microspheres for use in biomedical applications. In addition, we describe a method we developed for generating multilayered alginate microbeads. Cells can be encapsulated in the inner alginate core, and angiogenic proteins in the outer alginate layer. The release of proteins from this outer layer would stimulate the formation of local microvascular networks directly towards the transplanted islets.     

Use of immobilized cells of the strainCorynebacterium sp. for 4-nitrophenol degradation

4-Nitrophenol degrading bacterial strainCorynebacterium sp. 8/3 was isolated from chemically polluted soil. The product of cometabolic transformation of 4-nitrophenol was identified as 4-nitrocatechol., Effect of immobilization (encapsulation in calcium alginate) ofCorynebacterium sp. cells on the process of 4-nitrophenol transformation was investigated. 4-Nitrophenol was converted by encapsulated cells and encapsulation had a protective effect, on 4-nitrophenol degrading bacteria in repeated cycles of incubation. Transformation of 4-nitrophenol to 4-nitrocatechol by encapsulated cells was influenced by pH of medium but was not influenced by concentration of alginate and CaCl₂. The count of viable cells in alginate beads declined approximately by one order of magnitude after 10 d of incubation. Presented at the 4th Mini-Symposium on Biosorption and Microbial Degradation, Prague, Czech Republic, November 26–29, 1996.     

Survival of and wheat-root colonization by alginate encapsulated Herbaspirillum spp

The survival ofHerbaspirillum spp. cells added directly or encapsulated in alginate beads and colonization of wheat roots was evaluated in soil microcosms. Cells entrapped in alginate in the presence of JNFb-broth and introduced into unplanted non-sterile clay loamy and sandy soils survived better than cells added directly to the same soils after 50 d incubation. On amendment by JNFb broth and/or skim milk the entrapped cells survived better than those prepared in water. Encapsulated cells survived better in a heavier textured soil (clay-loamy) than in a lighter (sandy) soil. Wheat plants growing in microcosms inoculated with various bead types from day 0 to day 30 exhibited high levels of histosphere colonization, nitrogenase activity (in situ) measured by acetylene reduction assay, plant dry mass and total N content but no symptoms of mottled stripe disease were observed. Comparable results of growth criteria and nitrogenase activity, but relatively lower bacterial populations, were obtained with wheat grown for 45 d after the inoculant had been introduced into the soil with different bead types.