Root transcriptome analysis of Arabidopsis thaliana exposed to beneficial Bacillus subtilis FB17 rhizobacteria revealed genes for bacterial recruitment and plant defense independent of malate efflux

Our previous work has demonstrated that Arabidopsis thaliana can actively recruit beneficial rhizobacteria Bacillus subtilis strain FB17 (hereafter FB17) through an unknown shoot-to-root long-distance signaling pathway post a foliar bacterial pathogen attack. However, it is still not well understood which genetic targets FB17 affects in plants. Microarray analysis of A. thaliana roots treated with FB17 post 24 h of treatment showed 168 and 129 genes that were up- and down-regulated, respectively, compared with the untreated control roots. Those up-regulated include auxin-regulated genes as well as genes involved in metabolism, stress response, and plant defense. In addition, other defense-related genes, as well as cell-wall modification genes were also down-regulated with FB17 colonization. Expression patterns of 20 selected genes were analyzed by semi-quantitative RT-PCR, validating the microarray results. A. thaliana insertion mutants were used against FB17 to further study the functional response of the differentially expressed genes. Five mutants for the up-regulated genes were tested for FB17 colonization, three (at3g28360, at3g20190 and at1g21240) mutants showed decreased FB17 colonization on the roots while increased FB17 titers was seen with three mutants of the down-regulated genes (at3g27980, at4g19690 and at5g56320). Further, these mutants for up-regulated genes and down-regulated genes were foliar infected with Pseudomonas syringae pv. tomato (hereafter PstDC3000) and analyzed for Aluminum activated malate transporter (ALMT1) expression which showed that ALMT1 may be the key regulator for root FB17 colonization. Our microarray showed that under natural condition, FB17 triggers plant responses in a manner similar to known plant growth-promoting rhizobacteria and to some extent also suppresses defense-related genes expression in roots, enabling stable colonization. The possible implication of this study opens up a new dialogin terms of how beneficial microbes regulate plant genetic response for mutualistic associations.     

ACC Deaminase-Containing Bacillus subtilis Reduces Stress Ethylene-Induced Damage and Improves Mycorrhizal Colonization and Rhizobial Nodulation in Trigonella foenum-graecum Under Drought Stress

This study was aimed at protecting Trigonella plants by reducing stress ethylene levels through ACC (1-aminocyclopropane-1-carboxylic acid) deaminase-containing Bacillus subtilis (LDR2) and promoting plant growth through improved colonization of beneficial microbes like Ensifer meliloti (Em) and Rhizophagus irregularis (Ri) under drought stress. A plant growth-promoting rhizobacterium strain possessing high levels of ACC deaminase characterized as B. subtilis was selected. Application of this strain considerably protected Trigonella plants under severe drought stress conditions; this protection was correlated with reduced levels of ACC (responsible for generation of stress ethylene). The experiment consisted of eight inoculation treatments with different combinations of ACC deaminase-containing rhizobacteria LDR2, Ri, and Em under three water regimes. The tripartite combination of LDR2 + Ri + Em acted synergistically to induce protective mechanisms against decreased soil water availability in Trigonella plants and improved plant weight by 56 % with lower ACC concentration (39 % less than stressed noninoculated plants) under severe drought conditions. Drought-induced changes in biochemical markers like reduced chlorophyll concentration, increased proline content, and higher lipid peroxidation were monitored and clearly indicated the protective effects of LDR2 under drought stress. Under drought conditions, apart from alleviating ethylene-induced damage, LDR2 enhanced nodulation and arbuscular mycorrhizal fungi colonization in the plants resulting in improved nutrient uptake and plant growth.     

Bacillus subtilis: A plant-growth promoting rhizobacterium that also impacts biotic stress

Plants encounter many biotic agents, such as viruses, bacteria, nematodes, weeds, and arachnids. These entities induce biotic stress in their hosts by disrupting normal metabolism, and as a result, limit plant growth and/or are the cause of plant mortality. Some biotic agents, however, interact symbiotically or synergistically with their host plants. Some microbes can be beneficial to plants and perform the same role as chemical fertilizers and pesticides, acting as a biofertilizer and/or biopesticide. Plant growth promoting rhizobacteria (PGPR) can significantly enhance plant growth and represent a mutually helpful plant-microbe interaction. Bacillus species are a major type of rhizobacteria that can form spores that can survive in the soil for long period of time under harsh environmental conditions. Plant growth is enhanced by PGPR through the induction of systemic resistance, antibiosis, and competitive omission. Thus, the application of microbes can be used to induce systemic resistance in plants against biotic agents and enhance environmental stress tolerance. Bacillus subtilis exhibits both a direct and indirect biocontrol mechanism to suppress disease caused by pathogens. The direct mechanism includes the synthesis of many secondary metabolites, hormones, cell-wall-degrading enzymes, and antioxidants that assist the plant in its defense against pathogen attack. The indirect mechanism includes the stimulation of plant growth and the induction of acquired systemic resistance. Bacillus subtilis can also solubilize soil P, enhance nitrogen fixation, and produce siderophores that promote its growth and suppresses the growth of pathogens. Bacillus subtilis enhances stress tolerance in their plant hosts by inducing the expression of stress-response genes, phytohormones, and stress-related metabolites. The present review discusses the activity of B. subtilis in the rhizosphere, its role as a root colonizer, its biocontrol potential, the associated mechanisms of biocontrol and the ability of B. subtilis to increase crop productivity under conditions of biotic and abiotic stress.     

Multiple strategies of plant colonization by beneficial endophytic Enterobacter sp. SA187

Although many endophytic plant growth-promoting rhizobacteria have been identified, relatively little is still known about the mechanisms by which they enter plants and promote plant growth. The beneficial endophyte Enterobacter sp. SA187 was shown to maintain the productivity of crops in extreme agricultural conditions. Here we present that roots of its natural host (Indigofera argentea), alfalfa, tomato, wheat, barley and Arabidopsis are all efficiently colonized by SA187. Detailed analysis of the colonization process in Arabidopsis showed that colonization already starts during seed germination, where seed-coat mucilage supports SA187 proliferation. The meristematic zone of growing roots attracts SA187, allowing epiphytic colonization in the elongation zone. Unlike primary roots, lateral roots are significantly less epiphytically colonized by SA187. Root endophytic colonization was found to occur by passive entry of SA187 at lateral-root bases. However, SA187 also actively penetrates the root epidermis by enzymatic disruption of plant cell wall material. In contrast to roots, endophytic colonization of shoots occurs via stomata, whereby SA187 can actively re-open stomata similarly to pathogenic bacteria. In summary, several entry strategies were identified that allow SA187 to establish itself as a beneficial endophyte in several plant species, supporting its use as a plant growth-promoting bacterium in agriculture systems.     

The Effect of Biocontrol Bacteria on Rhizosphere Bacterial Communities Analyzed by Plating and PCR-DGGE

Bacillus subtilis B579, which was isolated from rhizosphere of cucumber, exhibited an excellent biocontrol activity on soil-born pathogens under greenhouse conditions. It could colonize in rhizosphere of cucumber with large number of populations after inoculated in plant growth season. To reveal the effect of high level colonization of B. subtilis B579 on rhizobacteria community structure, cultivation-based analysis coupled with denaturing gradient gel electrophoresis (DGGE) analysis were used to profile the changes of rhizobacteria community structure sampling at 1 week interval. Cultivation-based and DGGE fingerprinting analysis showed significant plant-dependent and seasonal shifts in rhizobacteria populations. Only minimal and transient effects were observed at 4–9 weeks after sowing in samples of B579 treatment, without the pathogen inoculation and showed the best plant growth potential. Sequencing of dominant bands excised from the gel revealed that Streptomyces sp. was the dominate species in soils before and after sowing. Burkholderia sp. was the dominate species in bulk soil, while Bacillus sp. was dominated in rhizosphere within the growth season. Arthrobacter ramosus and Nocardioides sp. were identified as the specific species in samples treated by B579 at the maturity and flowering stages of cucumber.     

About the action of metabolites of plant growth-promoting rhizobacteria Bacillus subtilis on plant salt tolerance (I)

To find out the mode of plant tolerance enhancement against salinity by plant growth-promoting rhizobacteria Bacillus subtilis, metabolites of strains FZB24 and FZB41 were studied in a test system with tomatoes under the influence of high salinity. The culture filtrate (CF) from the fermentative transitional phase, containing the whole range of produced metabolites by B. subtilis, showed to a certain extent tolerance-increasing action at dilution of 0.1% in the test plants with the parameters length, fresh mass and dry mass of shoots and roots as well as leaf area after 7-day treatment and subsequent plant cultivation under high salt stress. Afterwards, the CF was fractionated with adsorber resin and high performance liquid chromatography, and these fractions, as well as fractions from a CF after 19-h fermentation, were also tested with axenic-cultivated tomato seedlings. Fractions with different proteins and peptides, produced by B. subtilis, showed partly activities depending on concentration with regard to plant growth stimulation, including tolerance enhancement against salt stress. Subsequently, also an extract from B. subtilis culture with special concentrated peptides was examined in the axenic plant test system and showed similar activity depending on concentration. The observed effect of the bacterial metabolites is discussed as one part of the mechanism for plant growth stimulation and at the same time salt tolerance, increasing action of the rhizobacterium by its root colonization and interaction with the plant metabolism.     

Diversification of Bacillus subtilis during experimental evolution on Arabidopsis thaliana and the complementarity in root colonization of evolved subpopulations

The soil bacterium Bacillus subtilis is known to suppress pathogens as well as promote plant growth. However, in order to fully exploit the potential as natural fertilizer, we need a better understanding of the interactions between B. subtilis and plants. Here, B. subtilis was examined for root colonization through experimental evolution on Arabidopsis thaliana. The populations evolved rapidly, improved in root colonization and diversified into three distinct morphotypes. In order to better understand the adaptation that had taken place, single evolved isolates from the final transfer were randomly selected for further characterization, revealing changes in growth and pellicle formation in medium supplemented with plant polysaccharides. Intriguingly, certain evolved isolates showed improved root colonization only on the plant species they evolved on, but not on another plant species, namely tomato, suggesting A. thaliana specific adaption paths. Finally, the mix performed better than the sum of its constituents in monoculture, which was demonstrated to be caused by complementarity effects. Our results suggest that genetic diversification occurs in an ecological relevant setting on plant roots and proves to be a stable strategy for root colonization.     

A Novel Interaction between Plant-Beneficial Rhizobacteria and Roots: Colonization Induces Corn Resistance against the Root Herbivore Diabrotica speciosa

A number of soil-borne microorganisms, such as mycorrhizal fungi and rhizobacteria, establish mutualistic interactions with plants, which can indirectly affect other organisms. Knowledge of the plant-mediated effects of mutualistic microorganisms is limited to aboveground insects, whereas there is little understanding of what role beneficial soil bacteria may play in plant defense against root herbivory. Here, we establish that colonization by the beneficial rhizobacterium Azospirillum brasilense affects the host selection and performance of the insect Diabrotica speciosa. Root larvae preferentially orient toward the roots of non-inoculated plants versus inoculated roots and gain less weight when feeding on inoculated plants. As inoculation by A. brasilense induces higher emissions of (E)-β-caryophyllene compared with non-inoculated plants, it is plausible that the non-preference of D. speciosa for inoculated plants is related to this sesquiterpene, which is well known to mediate belowground insect-plant interactions. To the best of our knowledge, this is the first study showing that a beneficial rhizobacterium inoculant indirectly alters belowground plant-insect interactions. The role of A. brasilense as part of an integrative pest management (IPM) program for the protection of corn against the South American corn rootworm, D. speciosa, is considered.     

Root Exudate-Induced Alterations in Bacillus cereus Cell Wall Contribute to Root Colonization and Plant Growth Promotion

The outcome of an interaction between plant growth promoting rhizobacteria and plants may depend on the chemical composition of root exudates (REs). We report the colonization of tobacco, and not groundnut, roots by a non-rhizospheric Bacillus cereus (MTCC 430). There was a differential alteration in the cell wall components of B. cereus in response to the REs from tobacco and groundnut. Attenuated total reflectance infrared spectroscopy revealed a split in amide I region of B. cereus cells exposed to tobacco-root exudates (TRE), compared to those exposed to groundnut-root exudates (GRE). In addition, changes in exopolysaccharides and lipid-packing were observed in B. cereus grown in TRE-amended minimal media that were not detectable in GRE-amended media. Cell-wall proteome analyses revealed upregulation of oxidative stress-related alkyl hydroperoxide reductase, and DNA-protecting protein chain (Dlp-2), in response to GRE and TRE, respectively. Metabolism-related enzymes like 2-amino-3-ketobutyrate coenzyme A ligase and 2-methylcitrate dehydratase and a 60 kDa chaperonin were up-regulated in response to TRE and GRE. In response to B. cereus, the plant roots altered their exudate-chemodiversity with respect to carbohydrates, organic acids, alkanes, and polyols. TRE-induced changes in surface components of B. cereus may contribute to successful root colonization and subsequent plant growth promotion.     

A new bioorganic fertilizer can effectively control banana wilt by strong colonization with Bacillus subtilis N11

Fusarium wilt is one of the most serious diseases caused by a soil-borne pathogen affecting banana production. The goal of this study was to evaluate the capability of a novel bio-organic fertilizer (BIO2) that integrated the biocontrol agent Bacillus subtilis N11, and mature composts to control Fusarium wilt of banana in pot experiments. The results showed that the application of the BIO2 significantly decreased the incidence rate of Fusarium wilt compared to the control. To determine the antagonistic mechanism of the strain, we also studied the colonization of the natural biocontrol agent on banana roots using a GFP marker. The studies were performed in a hydroponic culture system, a sand system and a natural soil system. The results indicated that the bacteria colonized predominantly by forming biofilms along the elongation and differentiation zones of the roots. The fact that similar observations were obtained in all three systems suggests that colonization by N11 can be studied in a defined system. The population of B. subtilis N11 in the rhizosphere and on banana roots was also monitored. We speculate that the colonization pattern of B.subtilis N11 can be linked to the mechanism of protection of plants from fungal infection.     

Plant growth promoting rhizobacteria improve growth and essential oil yield in Origanum majorana L.

Effects of root colonization by plant growth promoting rhizobacteria (PGPR) on biomass, and qualitative and quantitative composition of essential oils, were determined in the aromatic crop Origanum majorana L. (sweet marjoram). PGPR strains evaluated were Pseudomonas fluorescens, Bacillus subtilis, Sinorhizobium meliloti, and Bradyrhizobium sp. Only P. fluorescens and Bradyrhizobium sp. showed significant increases in shoot length, shoot weight, number of leaf, number of node, and root dry weight, in comparison to control plants or plants treated with other PGPR. Essential oil yield was also significantly increased relative to non-inoculated plants, without alteration of oil composition. P. fluorescens has clear commercial potential for economic cultivation of O. majorana.     

Accelerated Growth Rate and Increased Drought Stress Resilience of the Model Grass Brachypodium distachyon Colonized by Bacillus subtilis B26

Plant growth-promoting bacteria (PGB) induce positive effects in plants, for instance, increased growth and reduced abiotic stresses susceptibility. The mechanisms by which these bacteria impact the host plant are numerous, diverse and often specific. Here, we studied the agronomical, molecular and biochemical effects of the endophytic PGB Bacillus subtilis B26 on the full life cycle of Brachypodium distachyon Bd21, an established model species for functional genomics in cereal crops and temperate grasses. Inoculation of Brachypodium with B. subtilis strain B26 increased root and shoot weights, accelerated growth rate and seed yield as compared to control plants. B. subtilis strain B26 efficiently colonized the plant and was recovered from roots, stems and blades as well as seeds of Brachypodium, indicating that the bacterium is able to migrate, spread systemically inside the plant, establish itself in the aerial plant tissues and organs, and is vertically transmitted to seeds. The presence of B. subtilis strain B26 in the seed led to systemic colonization of the next generation of Brachypodium plants. Inoculated Brachypodium seedlings and mature plants exposed to acute and chronic drought stress minimized the phenotypic effect of drought compared to plants not harbouring the bacterium. Protection from the inhibitory effects of drought by the bacterium was linked to upregulation of the drought-response genes, DREB2B-like, DHN3-like and LEA-14-A-like and modulation of the DNA methylation genes, MET1B-like, CMT3-like and DRM2-like, that regulate the process. Additionally, total soluble sugars and starch contents increased in stressed inoculated plants, a biochemical indication of drought tolerance. In conclusion, we show a single inoculation of Brachypodium with a PGB affected the whole growth cycle of the plant, accelerating its growth rates, shortening its vegetative period, and alleviating drought stress effects. These effects are relevant to grasses and cereal crops.     

Sustained growth promotion in arabidopsis with long-term exposure to the beneficial soil bacterium Bacillus subtilis (GB03)

Volatile emissions from the commercial growth promoting soil bacterium Bacillus subtilis (GB03) are effective in augmenting short-term growth, photosynthetic capacity and salt tolerance in Petri-dish grown arabidopsis seedlings. In contrast, the impact sustained GB03 volatile exposure on plant growth and development has yet to be examined. here is provided physical and physiological data establishing that bacterial volatiles induce long-term growth promotion, elevated photosynthetic capacity and iron accumulation, as well as delayed albeit higher seed count compared with water-treated control plants. Plants were grown unrestricted in double Magenta boxes containing solid MS media for up to twelve weeks with GB03 volatiles introduced in separate containers within the chamber so that plant bacterial interactions were only by air-borne transmission. These results establish that GB03 volatiles induce sustained beneficial effects on Arabidopsis growth including robust and extended vegetative growth followed by elevated seed set.Key words: Bacillus subtilis (GB03), photosynthetic efficiency, plant growth promoting rhizobacteria (PGPR), seed set, volatile organic compound (VOCs)     

Study of mechanisms for plant growth promotion elicited by rhizobacteria in Arabidopsis thaliana

Plant growth-promoting rhizobacteria (PGPR) colonize plant roots and exert beneficial effects on plant health and development. We are investigating the mechanisms by which PGPR elicit plant growth promotion from the viewpoint of signal transduction pathways within plants. We report here our first study to determine if well-characterized PGPR strains, which previously demonstrated growth promotion of various other plants, also enhance plant growth in Arabidopsis thaliana. Eight different PGPR strains, including Bacillus subtilis GB03, B. amyloliquefaciens IN937a, B. pumilus SE-34, B. pumilus T4, B. pasteurii C9, Paenibacillus polymyxa E681, Pseudomonas fluorescens 89B-61, and Serratia marcescens 90-166, were evaluated for elicitation of growth promotion of wild-type and mutant Arabidopsis in vitro and in vivo. In vitro testing on MS medium indicated that all eight PGPR strains increased foliar fresh weight of Arabidopsis at distances of 2, 4, and 6 cm from the site of bacterial inoculation. Among the eight strains, IN937a and GB03 inhibited growth of Arabidopsis plants when the bacteria were inoculated 2 cm from the plants, while they significantly increased plant growth when inoculated 6 cm from the plants, suggesting that a bacterial metabolite that diffused into the agar accounted for growth promotion with this strain. In vivo, eight PGPR strains promoted foliar fresh weight under greenhouse conditions 4 weeks after sowing. To define signal transduction pathways associated with growth promotion elicited by PGPR, various plant-hormone mutants of Arabidopsis were evaluated in vitro and in vivo. Elicitation of growth promotion by PGPR strains in vitro involved signaling of brassinosteroid, IAA, salicylic acid, and gibberellins. In vivo testing indicated that ethylene signaling was involved in growth promotion. Results suggest that elicitation of growth promotion by PGPR in Arabidopsis is associated with several different signal transduction pathways and that such signaling may be different for plants grown in vitro vs. in vivo.