Genomics and taxonomy of the glyphosate-degrading, copper-tolerant rhizospheric bacterium Achromobacter insolitus LCu2.

A rhizosphere strain, Achromobacter insolitus LCu2, was isolated from alfalfa (Medicago sativa L.) roots. It was able to degrade of 50% glyphosate as the sole phosphorus source, and was found resistant to 10 mM copper (II) chloride, and 5 mM glyphosate-copper complexes. Inoculation of alfalfa seedlings and potato microplants with strain LCu2 promoted plant growth by 30-50%. In inoculated plants, the toxicity of the glyphosate-copper complexes to alfalfa seedlings was decreased, as compared with the noninoculated controls. The genome of A. insolitus LCu2 consisted of one circular chromosome (6,428,890 bp) and encoded 5843 protein genes and 76 RNA genes. Polyphasic taxonomic analysis showed that A. insolitus LCu2 was closely related to A. insolitus DSM23807T on the basis of the average nucleotide identity of the genomes of 22 type strains and the multilocus sequence analysis. Genome analysis revealed genes putatively responsible for (1) plant growth promotion (osmolyte, siderophore, and 1-aminocyclopropane-1-carboxylate deaminase biosynthesis and auxin metabolism); (2) degradation of organophosphonates (glyphosate oxidoreductase and multiple phn clusters responsible for the transport, regulation and C-P lyase cleavage of phosphonates); and (3) tolerance to copper and other heavy metals, effected by the CopAB-CueO system, responsible for the oxidation of copper (I) in the periplasm, and by the efflux Cus system. The putative catabolic pathways involved in the breakdown of phosphonates are predicted. A. insolitus LCu2 is promising in the production of crops and the remediation of soils contaminated with organophosphonates and heavy metals.

Rhizobial Microsymbionts of Kamchatka Oxytropis Species Possess Genes of the Type III and VI Secretion Systems, Which Can Affect the Development of Symbiosis.

A collection of rhizobial strains isolated from root nodules of the narrowly endemic legume species Oxytropis erecta, O. anadyrensis, O. kamtschatica, and O. pumilio originating from the Kamchatka Peninsula (Russian Federation) was obtained. Analysis of the 16S ribosomal RNA gene sequence showed a significant diversity of isolates belonging to families Rhizobiaceae (genus Rhizobium), Phyllobacteriaceae (genera Mesorhizobium, Phyllobacterium), and Bradyrhizobiaceae (genera Bosea, Tardiphaga). A plant nodulation assay showed that only strains belonging to genus Mesorhizobium could form nitrogen-fixing nodules on Oxytropis plants. The strains M. loti 582 and M. huakuii 583, in addition to symbiotic clusters, possessed genes of the type III and type VI secretion systems (T3SS and T6SS, respectively), which can influence the host specificity of strains. These strains formed nodules of two types (elongated and rounded) on O. kamtschatica roots. We suggest this phenomenon may result from Nod factor-dependent and -independent nodulation strategies. The obtained strains are of interest for further study of the T3SS and T6SS gene function and their role in the development of rhizobium-legume symbiosis. The prospects of using rhizobia having both gene systems related to symbiotic and nonsymbiotic nodulation strategies to enhance the efficiency of plant-microbe interactions by expanding the host specificity and increasing nodulation efficiency are discussed.

Bosea caraganae sp. nov. a new species of slow-growing bacteria isolated from root nodules of the relict species Caragana jubata (Pall.) Poir. originating from Mongolia.

Two Gram-stain-negative strains, RCAM04680T and RCAM04685, were isolated from root nodules of the relict legume Caragana jubata (Pall.) Poir. originating from the south-western shore of Lake Khuvsgul (Mongolia). The 16S rRNA gene (rrs) sequencing data showed that these novel isolates belong to the genus Bosea and are phylogenetically closest to the type strains Bosea lathyri LMG 26379T, Bosea vaviloviae LMG 28367T, Bosea massiliensis LMG 26221T and Bosea lupini LMG 26383T (the rrs-similarity levels were 98.7-98.8 %). The recA gene of strain RCAM04680T showed the highest sequence similarity to the type strain B. lupini LMG 26383T (95.4 %), while its atpD gene was closest to that of B. lathyri LMG 26379T (94.4 %). The ITS, dnaK and gyrB sequences of this isolate were most similar to the B. vaviloviae LMG 28367T (86.8 % for ITS, 90.4 % for the other genes). The most abundant fatty acid was C18 : 1ω7c (40.8 %). The whole genomes of strains RCAM04680T and RCAM04685 were identical (100 % average nucleotide identity). The highest average nucleotide identity value (82.8 %) was found between the genome of strain RCAM04680T and B. vaviloviae LMG 28367T. The common nodABC genes required for legume nodulation were absent in both strains; however, some other symbiotic nol, nod, nif and fix genes were detected. Based on the genetic study, as well as analyses of the whole-cell fatty acid compositions and phenotypic properties, a new species, Boseacaraganae sp. nov. (type strain RCAM04680T (=LMG 31125T), is proposed.

Taxonomically Different Co-Microsymbionts of a Relict Legume, Oxytropis popoviana, Have Complementary Sets of Symbiotic Genes and Together Increase the Efficiency of Plant Nodulation.

Ten rhizobial strains were isolated from root nodules of a relict legume Oxytropis popoviana Peschkova. For identification of the isolates, sequencing of rrs, the internal transcribed spacer region, and housekeeping genes recA, glnII, and rpoB was used. Nine fast-growing isolates were Mesorhizobium-related; eight strains were identified as M. japonicum and one isolate belonged to M. kowhaii. The only slow-growing isolate was identified as a Bradyrhizobium sp. Two strains, M. japonicum Opo-242 and Bradyrhizobium sp. strain Opo-243, were isolated from the same nodule. Symbiotic genes of these isolates were searched throughout the whole-genome sequences. The common nodABC genes and other symbiotic genes required for plant nodulation and nitrogen fixation were present in the isolate Opo-242. Strain Opo-243 did not contain the principal nod, nif, and fix genes; however, five genes (nodP, nodQ, nifL, nolK, and noeL) affecting the specificity of plant-rhizobia interactions but absent in isolate Opo-242 were detected. Strain Opo-243 could not induce nodules but significantly accelerated the root nodule formation after coinoculation with isolate Opo-242. Thus, we demonstrated that taxonomically different strains of the archaic symbiotic system can be co-microsymbionts infecting the same nodule and promoting the nodulation process due to complementary sets of symbiotic genes.

Phyllobacterium zundukense sp. nov., a novel species of rhizobia isolated from root nodules of the legume species Oxytropis triphylla (Pall.) Pers.

Gram-negative strains Tri-36, Tri-38, Tri-48T and Tri-53 were isolated from root nodules of the relict legume Oxytropis triphylla (Pall.) Pers. originating from Zunduk Cape (Baikal Lake region, Russia). 16S rRNA gene sequencing showed that the novel isolates were phylogenetically closest to the type strains Phyllobacterium sophorae LMG 27899T, Phyllobacterium brassicacearum LMG 22836T, Phyllobacterium endophyticum LMG 26470T and Phyllobacterium bourgognense LMG 22837T while similarity levels between the isolates and the most closely related strain P. endophyticum LMG 26470T were 98.8-99.5 %. The recA and glnII genes of the isolates showed highest sequence similarities with P. sophorae LMG 27899T (95.4 and 89.5 %, respectively) and P. brassicacearum LMG 22836T (91.4 and 85.1 %, respectively). Comparative analysis of phenotypic properties between the novel isolates and the closest reference strains P. sophorae LMG 27899T, P. brassicacearum LMG 22836T and P. endophyticum LMG 26470T was performed using a microassay system. Average nucleotide identities between the whole genome sequences of the isolates Tri-38 and Tri-48T and P. sophorae LMG 27899T, P. brassicacearum LMG 22836T and P. endophyticum LMG 26470T ranged from 79.23 % for P. endophyticum LMG 26470T to 85.74 % for P. sophorae LMG 27899T. The common nodABC genes required for legume nodulation were absent from strains Tri-38 and Tri-48T, although some other symbiotic nod and fix genes were detected. On the basis of genotypic and phenotypic analysis, a novel species, Phyllobacterium zundukense sp. nov. (type strain Tri-48T=LMG 30371T=RCAM 03910T), is proposed.

Microvirga ossetica sp. nov., a species of rhizobia isolated from root nodules of the legume species Vicia alpestris Steven.

Gram-stain-negative strains V5/3MT, V5/5K, V5/5M and V5/13 were isolated from root nodules of Vicia alpestris plants growing in the North Ossetia region (Caucasus). Sequencing of the partial 16S rRNA gene (rrs) and four housekeeping genes (dnaK, gyrB, recA and rpoB) showed that the isolates from V. alpestris were most closely related to the species Microvirga zambiensis (order Rhizobiales, family Methylobacteriaceae) which was described for the single isolate from root nodule of Listia angolensis growing in Zambia. Sequence similarities between the Microvirga-related isolates and M. zambiensis WSM3693T ranged from 98.5 to 98.7 % for rrs and from 79.7 to 95.8 % for housekeeping genes. Cellular fatty acids of the isolates V5/3MT, V5/5K, V5/5M and V5/13 included important amounts of C18 : 1ω7c (54.0-67.2 %), C16 : 0 (6.0-7.8 %), C19 : 0 cyclo ω8c (3.1-10.2 %), summed feature 2 (comprising one or more of iso-C16 : 1 I, C14 : 0 3-OH and unknown ECL 10.938, 5.8-22.5 %) and summed feature 3 (comprising C16 : 1ω7c and/or iso-C15 : 02-OH, 2.9-4.0 %). DNA-DNA hybridization between the isolate V5/3MT and M. zambiensis WSM3693T revealed DNA-DNA relatedness of 35.3 %. Analysis of morphological and physiological features of the novel isolates demonstrated their unique phenotypic profile in comparison with reference strains from closely related species of the genus Microvirga. On the basis of genotypic and phenotypic analysis, a novel species named Microvirga ossetica sp. nov. is proposed. The type strain is V5/3MT (=LMG 29787T=RCAM 02728T). Three additional strains of the species are V5/5K, V5/5M and V5/13.

Aluminum exclusion from root zone and maintenance of nutrient uptake are principal mechanisms of Al tolerance in Pisum sativum L.

Our study aimed to evaluate intraspecific variability of pea (Pisum sativum L.) in Al tolerance and to reveal mechanisms underlying genotypic differences in this trait. At the first stage, 106 pea genotypes were screened for Al tolerance using root re-elongation assay based on staining with eriochrome cyanine R. The root re-elongation zone varied from 0.5 mm to 14 mm and relationships between Al tolerance and provenance or phenotypic traits of genotypes were found. Tolerance index (TI), calculated as a biomass ratio of Al-treated and non-treated contrasting genotypes grown in hydroponics for 10 days, varied from 30% to 92% for roots and from 38% to 90% for shoots. TI did not correlate with root or shoot Al content, but correlated positively with increasing pH and negatively with residual Al concentration in nutrient solution in the end of experiments. Root exudation of organic acid anions (mostly acetate, citrate, lactate, pyroglutamate, pyruvate and succinate) significantly increased in several Al-treated genotypes, but did not correlate with TI. Al-treatment decreased Ca, Co, Cu, K, Mg, Mn, Mo, Ni, S and Zn contents in roots and/or shoots, whereas contents of several elements (P, B, Fe and Mo in roots and B and Fe in shoots) increased, suggesting that Al toxicity induced substantial disturbances in uptake and translocation of nutrients. Nutritional disturbances were more pronounced in Al sensitive genotypes. In conclusion, pea has a high intraspecific variability in Al tolerance and this trait is associated with provenance and phenotypic properties of plants. Transformation of Al to unavailable (insoluble) forms in the root zone and the ability to maintain nutrient uptake are considered to be important mechanisms of Al tolerance in this plant species.

The cadmium-tolerant pea (Pisum sativum L.) mutant SGECdt is more sensitive to mercury: assessing plant water relations.

Heavy metals have multiple effects on plant growth and physiology, including perturbation of plant water status. These effects were assessed by exposing the unique Cd-tolerant and Cd-accumulating pea (Pisum sativum L.) mutant SGECd(t) and its wild-type (WT) line SGE to either cadmium (1, 4 µM CdCl2) or mercury (0.5, 1, 2 µM HgCl2) in hydroponic culture for 12 days. When exposed to Cd, SGECd(t) accumulated more Cd in roots, xylem sap, and shoot, and had considerably more biomass than WT plants. WT plants lost circa 0.2 MPa turgor when grown in 4 µM CdCl2, despite massive decreases in whole-plant transpiration rate and stomatal conductance. In contrast, root Hg accumulation was similar in both genotypes, but WT plants accumulated more Hg in leaves and had a higher stomatal conductance, and root and shoot biomass compared with SGECd(t). Shoot excision resulted in greater root-pressure induced xylem exudation of SGECd(t) in the absence of Cd or Hg and following Cd exposure, whereas the opposite response or no genotypic differences occurred following Hg exposure. Exposing plants that had not been treated with metal to 50 µM CdCl2 for 1h increased root xylem exudation of WT, whereas 50 µM HgCl2 inhibited and eliminated genotypic differences in root xylem exudation, suggesting differences between WT and SGECd(t) plants in aquaporin function. Thus, root water transport might be involved in mechanisms of increased tolerance and accumulation of Cd in the SGECd(t) mutant. However, the lack of cross-tolerance to Cd and Hg stress in the mutant indicates metal-specific mechanisms related to plant adaptation.

Extra-slow-growing Tardiphaga strains isolated from nodules of Vavilovia formosa (Stev.) Fed.

Eleven extra-slow-growing strains were isolated from nodules of the relict legume Vavilovia formosa growing in North Ossetia (Caucasus) and Armenia. All isolates formed a single rrs cluster together with the type strain Tardiphaga robiniae LMG 26467(T), while the sequencing of the 16S-23S rDNA intergenic region (ITS) and housekeeping genes glnII, atpD, dnaK, gyrB, recA and rpoB divided them into three groups. North Ossetian isolates (in contrast to the Armenian ones) were clustered separately from the type strain LMG 26467(T). However, all isolates were classified as T. robiniae because the DNA-DNA relatedness between them and the type strain LMG 26467(T) was 69.6% minimum. Two symbiosis-related genes (nodM and nodT) were amplified in all isolated Tardiphaga strains. It was shown that the nodM gene phylogeny is similar to that of ITS and housekeeping genes. The presence of the other symbiosis-related genes in described Tardiphaga strains, which is recently described genus of rhizobia, as well as their ability to form nodules on any plants are under investigation.

Bosea vaviloviae sp. nov., a new species of slow-growing rhizobia isolated from nodules of the relict species Vavilovia formosa (Stev.) Fed.

The Gram-negative, rod-shaped slow-growing strains Vaf-17, Vaf-18(T) and Vaf-43 were isolated from the nodules of Vavilovia formosa plants growing in the hard-to-reach mountainous region of the North Ossetian State Natural Reserve (north Caucasus, Russian Federation). The sequencing of 16S rDNA (rrs), ITS region and five housekeeping genes (atpD, dnaK, recA, gyrB and rpoB) showed that the isolated strains were most closely related to the species Bosea lathyri (class Alphaproteobacteria, family Bradyrhizobiaceae) which was described for isolates from root nodules of Lathyrus latifolius. However the sequence similarity between the isolated strains and the type strain B. lathyri LMG 26379(T) for the ITS region was 90 % and for the housekeeping genes it was ranged from 92 to 95 %. All phylogenetic trees, except for the rrs-dendrogram showed that the isolates from V. formosa formed well-separated clusters within the Bosea group. Differences in phenotypic properties of the B. lathyri type strain and the isolates from V. formosa were studied using the microassay system GENIII MicroPlate BioLog. Whole-cell fatty acid analysis showed that the strains Vaf-17, Vaf-18(T) and Vaf-43 had notable amounts of C16:0 (4.8-6.0 %), C16:0 3-OH (6.4-6.6 %), C16:1 ω5c (8.8-9.0 %), C17:0 cyclo (13.5-13.9 %), C18:1 ω7c (43.4-45.4 %), C19:0 cyclo ω8c (10.5-12.6 %) and Summed Feature (SF) 3 (6.4-8.0 %). The DNA-DNA relatedness between the strains Vaf-18(T) and B. lathyri LMG 26379(T) was 24.0 %. On the basis of genotypic and phenotypic analysis a new species Bosea vaviloviae sp. nov. (type strain RCAM 02129(T) = LMG 28367(T) = Vaf-18(T)) is proposed.

Genetic diversity of rhizobia isolated from nodules of the relic species Vavilovia formosa (Stev.) Fed.

Sixteen bacterial strains were isolated from root nodules of Vavilovia formosa plants originated from the North Ossetian State Natural Reserve (Caucasus, Russia). Phylogenetic analysis of these strains was performed using partial 16S rRNA gene and internally transcribed spacer (ITS) sequences. The results showed that the isolates belong to three families of root nodule bacteria. Twelve of them were related to the genus Rhizobium (family Rhizobiaceae) but four strains can be most probably identified as Phyllobacterium-related (family Phyllobacteriaceae), Bosea- and Rhodopseudomonas-related (family Bradyrhizobiaceae). Amplified fragment length polymorphism clustering was congruent with ITS phylogeny but displayed more variability for Rhizobium isolates, which formed a single group at the level of 30 % similarity. We expect that the isolates obtained can belong to new taxa at genus, species or subspecies levels. The results of PCR amplification of the nodulation genes nodC and nodX showed their presence in all Rhizobium isolates and one Rhodopseudomonas-related isolate. The nodC gene sequences of V. formosa isolates were closely related to those of the species Rhizobium leguminosarum bv. viciae but formed separate clusters and did not intermingle with any reference strains. The presence of the nodX gene, which is necessary for nodulation of Afghan peas (Pisum sativum L.) originated from the Middle East, allows the speculation that these wild-type pea cultivars may be the closest existing relatives of V. formosa. Thus, the studies of genetic diversity and symbiotic genes of V. formosa microsymbionts provide the primary information about their phylogeny and contribute to the conservation of this relict leguminous species.

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.

RIAM: A Universal Accessible Protocol for the Isolation of High Purity DNA from Various Soils and Other Humic Substances.

A single universal open protocol RIAM (named after Research Institute for Agricultural Microbiology) for the isolation of high purity DNA from different types of soils and other substrates (high and low in humic, clay content, organic fertilizer, etc.) is proposed. The main features of the RIAM protocol are the absence of the sorption-desorption stage on silica columns, the use of high concentrations of phosphate in buffers, which prevents DNA sorption on minerals, and DNA precipitation using CTAB. The performance of RIAM was compared with a reference commercial kit and showed very good results in relation to the purity and quantity of DNA, as well as the absence of inhibitory activity on PCR. In all cases, the RIAM ensured the isolation of DNA in quantities much greater than the commercial kit without the effect of PCR inhibition up to 50 ng DNA per reaction in a volume of 15 µL. The latter circumstance along with the ability of the protocol to extract low molecular weight DNA fractions makes the method especially suitable for those cases where quantitative assessments, detection of minor components of soil microbiota, and completeness of isolation of all DNA fractions are required.

Water Stress, Cadmium, and Plant Genotype Modulate the Rhizosphere Microbiome of Pisum sativum L.

Drought and heavy metals seriously affect plant growth and the biodiversity of the associated rhizosphere microbiomes, which, in turn, could be involved in the adaptation of plants to these environmental stresses. Rhizosphere soil was collected from a three-factor pot experiment, where pea line SGE and its Cd-tolerant mutant SGECdt were cultivated under both optimal and limited water conditions and treated with a toxic Cd concentration. The taxonomic structure of the prokaryotic rhizosphere microbiome was analyzed with the high-throughput sequencing of 16S rRNA amplicon libraries. A permutation test demonstrated statistically significant effects of Cd and water stress but not of pea genotype on the rhizosphere microbiome structure. Phylogenetic isometric log-ratio data transformation identified the taxonomic balances that were affected by abiotic factors and pea genotypes. A small number of significant (log ratio [-3.0:+3.0]) and phylogenetically deep balances characterized water stress, while a larger number of weak (log ratio [-0.8:+0.8]) phylogenetically lower balances described the influence of the plant genotype. Stress caused by cadmium took on an intermediate position. The main conclusion of the study is that the most powerful factor affecting the rhizosphere microbiome was water stress, and the weakest factor was plant genotype since it demonstrated a very weak transformation of the taxonomic structure of rhizosphere microbiomes in terms of alpha diversity indices, beta diversity, and the log ratio values of taxonomic balances.

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.

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.

Microbial Consortium of PGPR, Rhizobia and Arbuscular Mycorrhizal Fungus Makes Pea Mutant SGECdt Comparable with Indian Mustard in Cadmium Tolerance and Accumulation.

Cadmium (Cd) is one of the most widespread and toxic soil pollutants that inhibits plant growth and microbial activity. Polluted soils can be remediated using plants that either accumulate metals (phytoextraction) or convert them to biologically inaccessible forms (phytostabilization). The phytoremediation potential of a symbiotic system comprising the Cd-tolerant pea (Pisum sativum L.) mutant SGECdt and selected Cd-tolerant microorganisms, such as plant growth-promoting rhizobacterium Variovorax paradoxus 5C-2, nodule bacterium Rhizobium leguminosarum bv. viciae RCAM1066, and arbuscular mycorrhizal fungus Glomus sp. 1Fo, was evaluated in comparison with wild-type pea SGE and the Cd-accumulating plant Indian mustard (Brassica juncea L. Czern.) VIR263. Plants were grown in pots in sterilized uncontaminated or Cd-supplemented (15 mg Cd kg-1) soil and inoculated or not with the microbial consortium. Cadmium significantly inhibited growth of uninoculated and particularly inoculated SGE plants, but had no effect on SGECdt and decreased shoot biomass of B. juncea. Inoculation with the microbial consortium more than doubled pea biomass (both genotypes) irrespective of Cd contamination, but had little effect on B. juncea biomass. Cadmium decreased nodule number and acetylene reduction activity of SGE by 5.6 and 10.8 times, whereas this decrease in SGECdt was 2.1 and 2.8 times only, and the frequency of mycorrhizal structures decreased only in SGE roots. Inoculation decreased shoot Cd concentration and increased seed Cd concentration of both pea genotypes, but had little effect on Cd concentration of B. juncea. Inoculation also significantly increased concentration and/or accumulation of nutrients (Ca, Fe, K, Mg, Mn, N, P, S, and Zn) by Cd-treated pea plants, particularly by the SGECdt mutant. Shoot Cd concentration of SGECdt was twice that of SGE, and the inoculated SGECdt had approximately similar Cd accumulation capacity as compared with B. juncea. Thus, plant-microbe systems based on Cd-tolerant micro-symbionts and plant genotypes offer considerable opportunities to increase plant HM tolerance and accumulation.

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.

Rhizosphere bacteria containing 1-aminocyclopropane-1-carboxylate deaminase increase yield of plants grown in drying soil via both local and systemic hormone signalling.

Decreased soil water availability can stimulate production of the plant hormone ethylene and inhibit plant growth. Strategies aimed at decreasing stress ethylene evolution might attenuate its negative effects. An environmentally benign (nonchemical) method of modifying crop ethylene relations - soil inoculation with a natural root-associated bacterium Variovorax paradoxus 5C-2 (containing the enzyme 1-aminocyclopropane-1-carboxylate (ACC) deaminase that degrades the ethylene precursor ACC), was assessed with pea (Pisum sativum) plants grown in drying soil. Inoculation with V. paradoxus 5C-2, but not with a transposome mutant with massively decreased ACC deaminase activity, improved growth, yield and water-use efficiency of droughted peas. Systemic effects of V. paradoxus 5C-2 included an amplified soil drying-induced increase of xylem abscisic acid (ABA) concentration, but an attenuated soil drying-induced increase of xylem ACC concentration. A local bacterial effect was increased nodulation by symbiotic nitrogen-fixing bacteria, which prevented a drought-induced decrease in nodulation and seed nitrogen content. Successfully deploying a single bacterial gene in the rhizosphere increased yield and nutritive value of plants grown in drying soil, via both local and systemic hormone signalling. Such bacteria may provide an easily realized, economic means of sustaining crop yields and using irrigation water more efficiently in dryland agriculture.