Influence of Enhanced Synthesis of Exopolysaccharides in Rhizobium ruizarguesonis and Overproduction of Plant Receptor to these Compounds on Colonizing Activity of Rhizobia in Legume and Non-Legume Plants and Plant Resistance to Phytopathogenic Fungi.

Rhizobial exopolysaccharides (EPS) may provide stabilization of membranes against external factors, as well as improved surface adhesion, but their role in interaction with legume and non-legume plants is still far from understanding. In this work, the transcriptional regulator RosR of Rhizobium ruizarguesonis, which regulates the synthesis of EPS, was overproduced in a pHC60 plasmid and expressed in the RCAM 1026 strain. This resulted in an improved production of EPS by this recombinant strain. Comparative analysis of the inoculation of pea Pisum sativum plants with R. ruizarguesonis pHC60-rosR and strain carrying the empty plasmid revealed an essential increase in the number of nodules, root length and biomass in plants inoculated with this EPS-overproducing strain. It demonstrates that the enhanced EPS synthesis by rhizobia may stimulate plant root colonization and subsequent nodule formation in pea plants. The influence of enhanced EPS synthesis in rhizobia on colonizing activity was also estimated in non-legume plant tomato Solanum lycopersicum. Our findings shown the increased colonization of the root surface and stimulation of the shoot biomass of inoculated plants. Inoculation of pea and tomato with EPS-overproducing rhizobial strain essentially increased plant resistance to phytopathogenic fungi Fusarium culmorum and F. oxysporum in both legume and non-legume plants, demonstrating a significant biocontrol effect of this recombinant strain. Furthermore, we have identified the PsLYK10 gene that encodes a putative EPS receptor in P. sativum, although no significant effect of PsLYK10 overexpression on nodulation in legume (pea P. sativum) and colonization of roots of non-legume plants by rhizobia was found compared to enhanced production of EPS by rhizobia.

Strain Streptomyces sp. P-56 Produces Nonactin and Possesses Insecticidal, Acaricidal, Antimicrobial and Plant Growth-Promoting Traits.

Streptomycetes produce a huge variety of bioactive metabolites, including antibiotics, enzyme inhibitors, pesticides and herbicides, which offer promise for applications in agriculture as plant protection and plant growth-promoting products. The aim of this report was to characterize the biological activities of strain Streptomyces sp. P-56, previously isolated from soil as an insecticidal bacterium. The metabolic complex was obtained from liquid culture of Streptomyces sp. P-56 as dried ethanol extract (DEE) and possessed insecticidal activity against vetch aphid (Medoura viciae Buckt.), cotton aphid (Aphis gossypii Glov.), green peach aphid (Myzus persicae Sulz.), pea aphid (Acyrthosiphon pisum Harr.) and crescent-marked lily aphid (Neomyzus circumflexus Buckt.), as well as two-spotted spider mite (Tetranychus urticae). Insecticidal activity was associated with production of nonactin, which was purified and identified using HPLC-MS and crystallographic techniques. Strain Streptomyces sp. P-56 also showed antibacterial and antifungal activity against various phytopathogenic bacteria and fungi (mostly for Clavibacfer michiganense, Alternaria solani and Sclerotinia libertiana), and possessed a set of plant growth-promoting traits, such as auxin production, ACC deaminase and phosphate solubilization. The possibilities for using this strain as a biopesticide producer and/or biocontrol and a plant growth-promoting microorganism are discussed.

Draft Genome Sequence of the Bacterium Cupriavidus sp. Strain D39, Inhabiting the Rhizosphere of Pea Plants (Pisum sativum L.).

We report the draft genome sequence of Cupriavidus sp. strain D39, associated with the roots of pea plants. The genome is characterized by a GC content of 63.62% and a total length of 7.7 Mbp and contains several putative genes associated with resistance to metals and plant growth promotion.

Aluminum-Immobilizing Rhizobacteria Modulate Root Exudation and Nutrient Uptake and Increase Aluminum Tolerance of Pea Mutant E107 (brz).

It is well known that plant-growth-promoting rhizobacteria (PGPRs) increase the tolerance of plants to abiotic stresses; however, the counteraction of Al toxicity has received little attention. The effects of specially selected Al-tolerant and Al-immobilizing microorganisms were investigated using pea cultivar Sparkle and its Al-sensitive mutant E107 (brz). The strain Cupriavidus sp. D39 was the most-efficient in the growth promotion of hydroponically grown peas treated with 80 µM AlCl3, increasing the plant biomass of Sparkle by 20% and of E107 (brz) by two-times. This strain immobilized Al in the nutrient solution and decreased its concentration in E107 (brz) roots. The mutant showed upregulated exudation of organic acids, amino acids, and sugars in the absence or presence of Al as compared with Sparkle, and in most cases, the Al treatment stimulated exudation. Bacteria utilized root exudates and more actively colonized the root surface of E107 (brz). The exudation of tryptophan and the production of IAA by Cupriavidus sp. D39 in the root zone of the Al-treated mutant were observed. Aluminum disturbed the concentrations of nutrients in plants, but inoculation with Cupriavidus sp. D39 partially restored such negative effects. Thus, the E107 (brz) mutant is a useful tool for studying the mechanisms of plant-microbe interactions, and PGPR plays an important role in protecting plants against Al toxicity.

Synergy between Rhizobial Co-Microsymbionts Leads to an Increase in the Efficiency of Plant-Microbe Interactions.

Combined inoculation of legumes with rhizobia and plant growth-promoting rhizobacteria or endophytes is a known technique for increasing the efficiency of nitrogen-fixing symbiosis and plant productivity. The aim of this work was to expand knowledge about the synergistic effects between commercial rhizobia of pasture legumes and root nodule bacteria of relict legume species. Pot experiments were performed on common vetch (Vicia sativa L.) and red clover (Trifolium pratense L.) co-inoculated with the participation of the corresponding commercial rhizobial strains (R. leguminosarum bv. viciae RCAM0626 and R. leguminosarum bv. trifolii RCAM1365) and seven strains isolated from nodules of relict legumes inhabiting the Baikal Lake region and the Altai Republic: Oxytropis popoviana, Astragalus chorinensis, O. tragacanthoides and Vicia costata. The inoculation of plants with combinations of strains (commercial strain plus the isolate from relict legume) had a different effect on symbiosis depending on the plant species: the increase in the number of nodules was mainly observed on vetch, whereas increased acetylene reduction activity was evident on clover. It was shown that the relict isolates differ significantly in the set of genes related to different genetic systems that affect plant-microbe interactions. At the same time, they had additional genes that are involved in the formation of symbiosis and determine its effectiveness, but are absent in the used commercial strains: symbiotic genes fix, nif, nod, noe and nol, as well as genes associated with the hormonal status of the plant and the processes of symbiogenesis (acdRS, genes for gibberellins and auxins biosynthesis, genes of T3SS, T4SS and T6SS secretion systems). It can be expected that the accumulation of knowledge about microbial synergy on the example of the joint use of commercial and relict rhizobia will allow in the future the development of methods for the targeted selection of co-microsymbionts to increase the efficiency of agricultural legume-rhizobia systems.

Rhizobacteria Mitigate the Negative Effect of Aluminum on Pea Growth by Immobilizing the Toxicant and Modulating Root Exudation.

High soil acidity is one of the main unfavorable soil factors that inhibit the growth and mineral nutrition of plants. This is largely due to the toxicity of aluminum (Al), the mobility of which increases significantly in acidic soils. Symbiotic microorganisms have a wide range of beneficial properties for plants, protecting them against abiotic stress factors. This report describes the mechanisms of positive effects of plant growth-promoting rhizobacteria Pseudomonas fluorescens SPB2137 on four pea (Pisum sativum L.) genotypes grown in hydroponics and treated with 80 µM AlCl3. In batch culture, the bacteria produced auxins, possessed 1-aminocyclopropane-1-carboxylate (ACC) deaminase activity, alkalized the medium and immobilized Al, forming biofilm-like structures and insoluble phosphates. Inoculation with Ps. fluorescens SPB2137 increased root and/or shoot biomass of Al-treated plants. The bacteria alkalized the nutrient solution and transferred Al from the solution to the residue, which contained phosphorus that was exuded by roots. As a result, the Al concentration in roots decreased, while the amount of precipitated Al correlated negatively with its concentration in the solution, positively with the solution pH and negatively with Al concentration in roots and shoots. Treatment with Al induced root exudation of organic acids, amino acids and sugars. The bacteria modulated root exudation via utilization and/or stimulation processes. The effects of Al and bacteria on plants varied depending on pea genotype, but all the effects had a positive direction and the variability was mostly quantitative. Thus, Ps. fluorescens SPB2137 improved the Al tolerance of pea due to immobilization and exclusion of toxicants from the root zone.

Endophytes from Halotolerant Plants Aimed to Overcome Salinity and Draught.

The aim of our research was to study the endosphere of four halophytic plants: Salicornia europaea L., Salsola australis (R.Br.), Bassia sedoides (Pall.) and Kochia prostrata (L.) Schrad. from arid and saline areas of the Stavropol Territory, Russia. In total, 28 endophyte strains were isolated from the roots and stems of these halophytic plants. Most of the isolates (23 out of 28) were identified as Bacillus sp. while others belonged to the genera Oceanobacillus, Paenibacillus, Pantoea, Alcaligenes and Myroides. Three strains of Bacillus sp. (Se5R, Se1-1R, and Se1-3S), isolated from the S. europaea were capable of growth at 55 °C and in 10% of NaCl. Strains Se1-4S, Kp20-2S, and Bs11-2S Bacillus sp. (isolated from the S. australis, K. prostrata and B. sedoides, respectively) demonstrated strong plant growth promoting activity: 85-265% over control lettuce plants and a high degree of growth suppression (59.1-81.2%) of pathogenic fungi Fusarium oxysporum, Bipolaris sorokiniana and Rhizoctonia solani. Selected strains can be promising candidates for the development of bioinoculants to facilitate salt soil phytoremediation and be beneficial for mitigating the salt stress to the plants growing in salt-affected habitats.

Isolation and Characterization of 1-Hydroxy-2,6,6-trimethyl-4-oxo-2-cyclohexene-1-acetic Acid, a Metabolite in Bacterial Transformation of Abscisic Acid.

We report the discovery of a new abscisic acid (ABA) metabolite, found in the course of a mass spectrometric study of ABA metabolism by the rhizosphere bacterium Rhodococcus sp. P1Y. Analogue of (+)-ABA, enriched in tritium in the cyclohexene moiety, was fed in bacterial cells, and extracts containing radioactive metabolites were purified and analyzed to determine their structure. We obtained mass spectral fragmentation patterns and nuclear magnetic resonance spectra of a new metabolite of ABA identified as 1-hydroxy-2,6,6-trimethyl-4-oxo-2-cyclohexene-1-acetic acid, which we named rhodococcal acid (RA) and characterized using several other techniques. This metabolite is the second bacterial ABA degradation product in addition to dehydrovomifoliol that we described earlier. Taken together, these data reveal an unknown ABA catabolic pathway that begins with side chain disassembly, as opposed to the conversion of the cyclohexene moiety in plants. The role of ABA-utilizing bacteria in interactions with other microorganisms and plants is also discussed.

Rhizosphere Bacterium Rhodococcus sp. P1Y Metabolizes Abscisic Acid to Form Dehydrovomifoliol.

The phytohormone abscisic acid (ABA) plays an important role in plant growth and in response to abiotic stress factors. At the same time, its accumulation in soil can negatively affect seed germination, inhibit root growth and increase plant sensitivity to pathogens. ABA is an inert compound resistant to spontaneous hydrolysis and its biological transformation is scarcely understood. Recently, the strain Rhodococcus sp. P1Y was described as a rhizosphere bacterium assimilating ABA as a sole carbon source in batch culture and affecting ABA concentrations in plant roots. In this work, the intermediate product of ABA decomposition by this bacterium was isolated and purified by preparative HPLC techniques. Proof that this compound belongs to ABA derivatives was carried out by measuring the molar radioactivity of the conversion products of this phytohormone labeled with tritium. The chemical structure of this compound was determined by instrumental techniques including high-resolution mass spectrometry, NMR spectrometry, FTIR and UV spectroscopies. As a result, the metabolite was identified as (4RS)-4-hydroxy-3,5,5-trimethyl-4-[(E)-3-oxobut-1-enyl]cyclohex-2-en-1-one (dehydrovomifoliol). Based on the data obtained, it was concluded that the pathway of bacterial degradation and assimilation of ABA begins with a gradual shortening of the acyl part of the molecule.

The Role of Symbiotic Microorganisms, Nutrient Uptake and Rhizosphere Bacterial Community in Response of Pea (Pisum sativum L.) Genotypes to Elevated Al Concentrations in Soil.

Aluminium being one of the most abundant elements is very toxic for plants causing inhibition of nutrient uptake and productivity. The aim of this study was to evaluate the potential of microbial consortium consisting of arbuscular mycorrhizal fungus (AMF), rhizobia and PGPR for counteracting negative effects of Al toxicity on four pea genotypes differing in Al tolerance. Pea plants were grown in acid soil supplemented with AlCl3 (pHKCl = 4.5) or neutralized with CaCO3 (pHKCl = 6.2). Inoculation increased shoot and/or seed biomass of plants grown in Al-supplemented soil. Nodule number and biomass were about twice on roots of Al-treated genotypes after inoculation. Inoculation decreased concentrations of water-soluble Al in the rhizosphere of all genotypes grown in Al-supplemented soil by about 30%, improved N2 fixation and uptake of fertilizer 15N and nutrients from soil, and increased concentrations of water-soluble nutrients in the rhizosphere. The structure of rhizospheric microbial communities varied to a greater extent depending on the plant genotype, as compared to soil conditions and inoculation. Thus, this study highlights the important role of symbiotic microorganisms and the plant genotype in complex interactions between the components of the soil-microorganism-plant continuum subjected to Al toxicity.

Herbicidal potential of stagonolide, a new phytotoxic nonenolide from Stagonospora cirsii.

Stagonospora cirsii is a pathogen of Cirsium arvense, causing necrotic lesions on leaves of this noxious weed. The fungus produced toxic metabolites when grown in liquid culture. A new phytotoxin, named stagonolide, was isolated and characterized as (8R,9R)-8-hydroxy-7-oxo-9-propyl-5-nonen-9-olide by spectroscopic methods. Stagonolide was shown to be a nonhost-specific but selective phytotoxin. Leaves of C. arvense were most sensitive and leaves of tomato and pepper (both Solanaceae) were less sensitive to stagonolide, which was assayed at 5 x 10(-3) M, than other plants. Stagonolide assayed at 5 x 10(-6) M was demonstrated to be a strong inhibitor of root growth in seedlings of C. arvense and some other Asteraceae species. Seedlings growth in wheat and radish was much less affected by the toxin, and seedlings of cucumber were insensitive to it.