Plant-growth promoting effect of newly isolated rhizobacteria varies between two Arabidopsis ecotypes

Various rhizobacteria are known for their beneficial effects on plants, i. e. promotion of growth and induction of systemic resistance against pathogens. These bacteria are categorized as plant growth promoting rhizobacteria (PGPR) and are associated with plant roots. Knowledge of the underlying mechanisms of plant growth promotion in vivo is still very limited, but interference of bacteria with plant hormone metabolism is suggested to play a major role. To obtain new growth promoting bacteria, we started a quest for rhizobacteria that are naturally associated to Arabidopsis thaliana. A suite of native root-associated bacteria were isolated from surface-sterilized roots of the Arabidopsis ecotype Gol-1 derived from a field site near Golm (Berlin area, Germany). We found several Pseudomonas and a Microbacterium species and tested these for growth promotion effects on the Arabidopsis ecotypes Gol-1 and Col-0, and for growth-promotion associated traits, such as auxin production, ACC deaminase activity and phosphate solubilization capacity. We showed that two of the bacteria strains promote plant growth with respect to rosette diameter, stalk length and accelerate development and that the effects were greater when bacteria were applied to Col-0 compared with Gol-1. Furthermore, the capability of promoting growth was not explained by the tested metabolic properties of the bacteria, suggesting that further bacterial traits are required. The natural variation of growth effects, combined with the extensive transgenic approaches available for the model plant Arabidopsis, will build a valuable tool to augment our understanding of the molecular mechanisms involved in the natural Arabidopsis - PGPR association.     

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.     

Stimulation of nitrogen fixation and induction of flavonoid-like compounds by rhizobacteria

Bacteria belonging to fluorescent Pseudomonas and to the spore-forming Bacillus groups, predominantly found in the rhizosphere and rhizoplane of healthy chickpea plants, were studied in order to determine their effect on effective strains of Rhizobium sp. 'Cicer' (Ca181 and Ca313) and their plant growth-promoting ability under aseptic and soil conditions. Co-inoculation of the rhizobacteria with effective Rhizobium strains of chickpea resulted in a significant increase in nodule weight, root and shoot biomass and total plant nitrogen when grown either in sterilized chillum jars or under pot culture conditions. The Rhizobium stimulatory Pseudomonas sp. 'CRP55b' showed maximum increase in all the symbiotic parameters. On co-inoculation with 'Ca181' and 'Ca313', Pseudomonas sp. 'CRP55b' and 'CRS68' resulted in significant increases in nodule weight, root and shoot biomass and total plant nitrogen. The nodule stimulating rhizobacteria enhanced levels of flavonoid-like compounds in roots on seed bacterization. Also, ethyl acetate extracts of culture supernatant fluids when applied to seeds resulted in enhancement of flavonoids in roots, suggesting that the rhizobacteria have a direct influence on root flavonoids which might be an additional factor in nodule promotion by these bacteria. These rhizobacteria also produced fluorescent compounds with absorption maxima at 252 nm, similar to those of plant flavonoids but having a different Rf value. Whether such fluorescent compounds act as signal molecules for induction of plant flavonoids in plant roots requires further study.     

<Emphasis Type="Italic">Bacillus megaterium</Emphasis> strain XTBG34 promotes plant growth by producing 2-pentylfuran

Several chemical changes in soil are associated with plant growth-promoting rhizobacteria. An endosporeforming bacterium, strain XTBG34, was isolated from a Xishuangbanna Tropical Botanical Garden soil sample and identified as Bacillus megaterium. The strain’s volatiles had remarkable plant growth promotion activity in Arabidopsis thaliana plants; after 15 days treatment, the fresh weight of plants inoculated with XTBG34 was almost 2-fold compared with those inoculated with DH5α. Head space volatile compounds produced by XTBG34, trapped with headspace solid phase microextraction and identified by gas chromatography-mass spectrometry, included aldehydes, alkanes, ketones and aroma components. Of the 11 compounds assayed for plant growth promotion activity in divided Petri plates, only 2-pentylfuran increased plant growth. We have therefore identified a new plant growth promotion volatile of B. megaterium XTBG34, which deserves further study in the mechanisms of interaction between plant growth-promoting rhizobacteria and plants.     

Organic acids, sugars, and L-tryptophane in exudates of vegetables growing on stonewool and their effects on activities of rhizosphere bacteria

The influence of stonewool substrate on the exudation of the major soluble carbon nutrients and of the auxin precursor tryptophane for Pseudomonas biocontrol agents was studied. To this end, the composition of the organic acids and sugars, as well that of tryptophane, of axenically collected exudates of seed, seedlings, and roots of tomato, cucumber, and sweet pepper was determined. The major results were as follows. i) The total amount of organic acid is much higher than that of total sugar. ii) Exudation of both organic acids and sugars increases during plant growth. iii) Citric, succinic, and malic acids represent the major organic acids, whereas fructose and glucose are the major sugars. iv) Compared with glass beads as a neutral substrate, stonewool substantially stimulates exudation of organic acids and sugars. v) It appeared that enhanced root-tip-colonizing bacteria isolated previously from the rhizosphere of tomato and cucumber grow much better in minimal medium with citrate as the sole carbon source than other, randomly selected rhizobacteria do. This indicates that the procedure which selects for excellent root-tip colonizers enriches for strains which utilize the major exudate carbon source citrate. vi) The content of L-tryptophane, the direct precursor of auxin, is approximately 60-fold higher in seedling exudates of tomato and sweet pepper than in cucumber seedling exudates, indicating a higher possibility of plant growth stimulation after inoculation with auxin-producing rhizobacteria for tomato and sweet pepper crops than for cucumber. However, the biocontrol strain Pseudomonas fluorescens WCS365, which is able to convert tryptophane into auxin, did not stimulate growth of these three crops. In contrast, this strain did stimulate growth of roots of radish, a plant which exudes nine times more tryptophane than tomato does.     

Sucrose triggers a novel signaling cascade promoting Bacillus subtilis rhizosphere colonization

Beneficial rhizobacteria promote plant growth and protect plants against phytopathogens. Effective colonization on plant roots is critical for the rhizobacteria to exert beneficial activities. How bacteria migrate swiftly in the soil of semisolid or solid nature remains unclear. Here we report that sucrose, a disaccharide ubiquitously deployed by photosynthetic plants for fixed carbon transport and storage, and abundantly secreted from plant roots, promotes solid surface motility (SSM) and root colonization by Bacillus subtilis through a previously uncharacterized mechanism. Sucrose induces robust SSM by triggering a signaling cascade, first through extracellular synthesis of polymeric levan, which in turn stimulates strong production of surfactin and hyper-flagellation of the cells. B. subtilis poorly colonizes the roots of Arabidopsis thaliana mutants deficient in root-exudation of sucrose, while exogenously added sucrose selectively shapes the rhizomicrobiome associated with the tomato plant roots, promoting specifically bacilli and pseudomonad. We propose that sucrose activates a signaling cascade to trigger SSM and promote rhizosphere colonization by B. subtilis. Our findings also suggest a practicable approach to boost prevalence of beneficial Bacillus species in plant protection.Subject terms: Soil microbiology, Bacteriology     

Response of spring rape (Brassica napus var. oleifera L.) to inoculation with plant growth promoting rhizobacteria containing 1-aminocyclopropane-1-carboxylate deaminase depends on nutrient status of the plant

Responses of rape (Brassica napus var. oleifera L.) to inoculation with plant growth promoting rhizobacteria, Pseudomonas putida Am2, Pseudomonas putida Bm3, Alcaligenes xylosoxidans Cm4, and Pseudomonas sp. Dp2, containing 1-aminocyclopropane-l-carboxylate (ACC) deaminase were studied using growth pouch and soil cultures. In growth pouch culture, the bacteria significantly increased root elongation of phosphorus-sufficient seedlings, whereas root elongation of phosphorus-deficient seedlings was not affected or was even inhibited by the bacteria. Bacterial stimulation of root elongation of phosphorus-sufficient seedlings was eliminated in the presence of a high ammonia concentration (1 mM) in the nutrient solution. Bacterial effects on root elongation of potassium-deficient and potassium-sufficient seedlings were similar. The bacteria also decreased inorganic phosphate content in shoots of potassium- and phosphorus-sufficient seedlings, reduced ethylene production by phosphorus-sufficient seedlings, and inhibited development of root hairs. The effects of treatment with Ag+, a chemical inhibitor of plant ethylene production, on root elongation, ethylene evolution, and root hair formation were similar to bacterial treatments. The number of bacteria on the roots of phosphorus-deficient seedlings was not limited by phosphorus deficiency. In pot experiments with soil culture, inoculation of seeds with bacteria and treatment with aminoethoxyvinylglycine, an inhibitor of ethylene biosynthesis in plants, increased root and (or) shoot biomass of rape plants. Stimulation of plant growth caused by the bacteria was often associated with a decrease in the content of nutrients, such as P, K, S, Mo, and Ba, in shoots, depending on the strain used. The results obtained show that the growth-promoting effects of ACC-utilizing rhizobacteria depend significantly on the nutrient status of the plant.     

Influence of a Bacillus sp. on physiological activities of two arbuscular mycorrhizal fungi and on plant responses to PEG-induced drought stress

The effects of bacterial inoculation (Bacillus sp.) on the development and physiology of the symbiosis between lettuce and the arbuscular mycorrhizal (AM) fungi Glomus mosseae (Nicol. and Gerd.) Gerd. and Trappe and Glomus intraradices (Schenck and Smith) were investigated. Plant growth, mineral nutrition and gas-exchange values in response to bacterial inoculation after PEG-induced drought stress were also evaluated. In AM plants, inoculation with Bacillus sp. enhanced fungal development and metabolism, measured as succinate dehydrogenase (SDH) and alkaline phosphatase (ALP) activities, more than plant growth. Under non-stressed conditions, G. intraradices colonization increased all plant physiological values to a higher extent when in dual inoculation with the bacterium. Under stress conditions, the bacterium had an important stimulatory effect on G. intraradices development. Under such conditions, the effects of the bacterium on photosynthetic rate, water use efficiency (WUE) and stomatal conductance of lettuce plants differed with the fungus species. Plant-gas exchange was enhanced in G. intraradices- and reduced in G. mosseae-colonized plants when co-inoculated with Bacillus sp. Thus, the effects of each fungus on plant physiology were modulated by the bacterium. Stress was detrimental, particularly in G. intraradices-colonized plants without the bacterium, reducing intra and extraradical mycelium growth and vitality (SDH), as well as plant-gas exchange. Nevertheless, Bacillus sp. inoculation improved all these plant and fungal parameters to the same level as in non-stressed plants. The highest amount of alive and active AM mycelium for both fungi was obtained after co-inoculation with Bacillus sp. These results suggest that selected free-living bacteria and AM fungi should be co-inoculated to optimize the formation and functioning of the AM symbiosis in both normal and adverse environments.     

The autophagy-associated Atg8 gene family operates both under favourable growth conditions and under starvation stresses in Arabidopsis plants

Arabidopsis plants possess a family of nine AtAtg8 gene homologues of the yeast autophagy-associated Apg8/Aut7 gene. To gain insight into how these genes function in plants, first, the expression patterns of five AtAtg8 homologues were analysed in young Arabidopsis plants grown under favourable growth conditions or following exposure to prolonged darkness or sugar starvation. Promoters, plus the entire coding regions (exons and introns) of the AtAtg8 genes, were fused to the beta-glucuronidase reporter gene and transformed into Arabidopsis plants. In all plants, grown under favourable growth conditions, beta-glucuronidase staining was much more significant in roots than in shoots. Different genes showed distinct spatial and temporal expression patterns in roots. In some transgenic plants, beta-glucuronidase staining in leaves was induced by prolonged darkness or sugar starvation. Next, Arabidopsis plants were transformed with chimeric gene-encoding Atg8f protein fused to N-terminal green fluorescent protein and C-terminal haemagglutinin epitope tags. Analysis of these plants showed that, under favourable growth conditions, the Atg8f protein is efficiently processed and is localized to autophagosome-resembling structures, both in the cytosol and in the central vacuole, in a similar manner to its processing and localization under starvation stresses. Moreover, treatment with a cocktail of proteasome inhibitors did not prevent the turnover of this protein, implying that its turnover takes place in the vacuoles, as occurs in yeasts. The results suggest that, in plants, the cellular processes involving the Atg8 genes function efficiently in young, non-senescing tissues, both under favourable growth conditions and under starvation stresses.     

Effectiveness of rhizobacteria containing ACC deaminase for growth promotion of peas (Pisum sativum) under drought conditions

A series of experiments were conducted to assess the effectiveness of rhizobacteria containing 1-aminocyclopropane- 1-carboxylate (ACC) deaminase for growth promotion of peas under drought conditions. Ten rhizobacteria isolated from the rhizosphere of different crops (peas, wheat, and maize) were screened for their growth promoting ability in peas under axenic condition. Three rhizobacterial isolates, Pseudomonas fluorescens biotype G (ACC-5), P. fluorescens (ACC-14), and P. putida biotype A (Q-7), were selected for pot trial on the basis of their source, ACC deaminase activity, root colonization, and growth promoting activity under axenic conditions. Inoculated and uninoculated (control) seeds of pea cultivar 2000 were sown in pots (4 seeds/pot) at different soil moisture levels (25, 50, 75, and 100% of field capacity). Results revealed that decreasing the soil moisture levels from 100 to 25% of field capacity significantly decreased the growth of peas. However, inoculation of peas with rhizobacteria containing ACC deaminase significantly decreased the "drought stress imposed effects" on growth of peas, although with variable efficacy at different moisture levels. At the lowest soil moisture level (25% field capacity), rhizobacterial isolate Pseudomonas fluorescens biotype G (ACC-5) was found to be more promising compared with the other isolates, as it caused maximum increases in fresh weight, dry weight, root length, shoot length, number of leaves per plant, and water use efficiency on fresh and dry weight basis (45, 150, 92, 45, 140, 46, and 147%, respectively) compared with respective uninoculated controls. It is highly likely that rhizobacteria containing ACC deaminase might have decreased the drought-stress induced ethylene in inoculated plants, which resulted in better growth of plants even at low moisture levels. Therefore, inoculation with rhizobacteria containing ACC deaminase could be helpful in eliminating the inhibitory effects of drought stress on the growth of peas.     

The auxin-signaling pathway is required for the lateral root response of Arabidopsis to the rhizobacterium Phyllobacterium brassicacearum

Plant root development is highly responsive both to changes in nitrate availability and beneficial microorganisms in the rhizosphere. We previously showed that Phyllobacterium brassicacearum STM196, a plant growth-promoting rhizobacteria strain isolated from rapeseed roots, alleviates the inhibition exerted by high nitrate supply on lateral root growth. Since soil-borne bacteria can produce IAA and since this plant hormone may be implicated in the high nitrate-dependent control of lateral root development, we investigated its role in the root development response of Arabidopsis thaliana to STM196. Inoculation with STM196 resulted in a 50% increase of lateral root growth in Arabidopsis wild-type seedlings. This effect was completely abolished in aux1 and axr1 mutants, altered in IAA transport and signaling, respectively, indicating that these pathways are required. The STM196 strain, however, appeared to be a very low IAA producer when compared with the high-IAA-producing Azospirillum brasilense sp245 strain and its low-IAA-producing ipdc mutant. Consistent with the hypothesis that STM196 does not release significant amounts of IAA to the host roots, inoculation with this strain failed to increase root IAA content. Inoculation with STM196 led to increased expression levels of several IAA biosynthesis genes in shoots, increased Trp concentration in shoots, and increased auxin-dependent GUS staining in the root apices of DR5::GUS transgenic plants. All together, our results suggest that STM196 inoculation triggers changes in IAA distribution and homeostasis independently from IAA release by the bacteria.     

Abnormal accumulation of sugars and phenolics in tobacco roots expressing the Agrobacterium T-6b oncogene and the role of these compounds in 6b-induced growth

The Agrobacterium T-DNA oncogene 6b induces tumors and modifies the growth of transgenic plants by an unknown mechanism. We have investigated changes in roots of tobacco seedlings that express a dexamethasone-inducible T-6b (dex-T-6b) gene. On induction medium with sucrose, intact or isolated dex-T-6b roots accumulated sucrose, glucose, and fructose and changed their growth, contrary to noninduced roots. Root fragments bridging agar blocks with or without sucrose accumulated sugars at the site of sucrose uptake, resulting in local growth. Induced root fragments showed enhanced uptake of 14C-labeled sucrose, glucose, and fructose. When seedlings were placed on sucrose-free induction medium, sugar levels strongly decreased in roots and increased in cotyledons. Collectively, these results demonstrate that 6b stimulates sugar uptake and retention with drastic effects on growth. Apart from sugars, phenolic compounds also have been found to accumulate in 6b tissues and have been proposed earlier to play a role in 6b-induced growth. Induced dex-T-6b roots accumulated high levels of 5-caffeoylquinic acid (or chlorogenic acid [CGA]), but only under conditions where endogenous sugars increased. Inhibition of phenylalanine ammonia-lyase with the competitive inhibitor 2-aminoindan-2-phosphonic acid (AIP) abolished CGA accumulation without modifying sugar accumulation or affecting the 6b phenotype. We conclude that the absorption, retention, and abnormal accumulation of sugars are essential factors in 6b-induced growth changes, whereas phenylpropanoids only marginally contribute to the 6b seedling phenotype.     

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.     

Analysis of the sucrose synthase gene family in Arabidopsis

The properties and expression patterns of the six isoforms of sucrose synthase in Arabidopsis are described, and their functions are explored through analysis of T-DNA insertion mutants. The isoforms have generally similar kinetic properties. Although there is variation in sensitivity to substrate inhibition by fructose this is unlikely to be of major physiological significance. No two isoforms have the same spatial and temporal expression patterns. Some are highly expressed in specific locations, whereas others are more generally expressed. More than one isoform is expressed in all organs examined. Mutant plants lacking individual isoforms have no obvious growth phenotypes, and are not significantly different from wild-type plants in starch, sugar and cellulose content, seed weight or seed composition under the growth conditions employed. Double mutants lacking the pairs of similar isoforms sus2 and sus3, and sus5 and sus6, are also not significantly different in these respects from wild-type plants. These results are surprising in the light of the marked phenotypes observed when individual isoforms are eliminated in crop plants including pea, maize, potato and cotton. A sus1/sus4 double mutant grows normally in well-aerated conditions, but shows marked growth retardation and accumulation of sugars when roots are subjected to hypoxia. The sucrose synthase activity in roots of this mutant is 3% or less of wild-type activity. Thus under well-aerated conditions sucrose mobilization in the root can proceed almost entirely via invertases without obvious detriment to the plant, but under hypoxia there is a specific requirement for sucrose synthase activity.