Rhizobium Leguminosarum Research Articles

Impact of Two Strains of Rhizobium leguminosarum on the Adaptation to Terminal Water Deficit of Two Cultivars Vicia faba.

Drought stress has become one of the most uncontrolled and unpredictable constraints on crop production. The purpose of this study was to evaluate the impacts of two different Rhizobium leguminosarum strains on terminal drought tolerance induction in two faba bean genotypes cultivated in Algeria, Aquadulce and Maltais. To this end, we measured physiological parameters—osmoprotectants accumulation, oxidative stress markers and enzyme activities—to assess the effect of R. leguminosarum inoculation on V. faba under terminal water deficiency conditions in greenhouse trials. Upregulation of anti-oxidative mechanisms and production of compatible solutes were found differentially activated according to Rhizobium strain. Drought stress resilience of the Maltais variety was improved using the local Rhizobium strain OL13 compared to the common strain 3841. Symbiosis with OL13 strain leads in particular to a much better production of proline and soluble sugar in nodules but also in roots and leaves of Maltais plant. Even if additional work is still necessary to decipher the mechanism by which a Rhizobium strain can affect the accumulation of osmoprotectants or cellular redox status in all the plants, inoculation with selected Rhizobium could be a promising strategy for improving water stress management in the forthcoming era of climate change.

Phytohormone biosynthetic potential of different Rhizobium species

Plants may not have optimum growth hormone production potential under suboptimal growth and environmental conditions. The exogenous application of microbes is a potential and economical source of plant hormones. A laboratory trial was performed to evaluate the hormone biosynthesis potential of several soil microbes. Rhizobium sp. (Rhizobium trifolii, Rhizobium etli, Rhizobium meliloti, Rhizobium ciceri, Azorhizobium caulinodans, Bradyrhizobium japonicum, Rhizobium vulgaris, and Rhizobium phaseoli) were isolated from root nodules of various legumes and were biochemically characterized. Absorption spectroscopy (535, 254 and 665 nm λ) was used to quantify indole acetic acid (IAA), gibberellin and cytokinin production of different Rhizobium species. B3 isolate of Rhizobium trifolii had yielded (14.54 and 21.37 µg mL-1) IAA equivalents without and with L-tryptophan (L-TRP) supplement, that was statistically at par with same species while significantly greater than other Rhizobium isolates. Gibberellin production was found statistically non-significant in all isolates. Rhizobium phaseoli (Vp1) yielded 1.68 µg mL-1 cytokinin that was at par with the same species isolates and with Rhizobium trifolii but was critically greater in amount than other species. It was concluded that Rhizobium plays a vital role in plant growth and development can produce auxin and gibberellins, but some could produce cytokinin. Keywords: Phytohormone, Biosynthesis, IAA, PGPR, Auxin, Gibberellins, Cytokinin

Fields with no recent legume cultivation have sufficient nitrogen-fixing rhizobia for crops of faba bean (Vicia faba L.)

Purpose(1) To assess the biological N fixation (BNF) potential of varieties of faba bean (Vicia faba L.) cropped with or without compost in an experimental field-scale rotation with no recent history of legumes, (2) to enumerate soil populations of Rhizobium leguminosarum sv. viciae (Rlv), and to genetically characterize the nodulating Rlv strains, (3) compare BNF with other sites in Britain.MethodsBNF was evaluated from 2012 to 2015 using 15 N natural abundance. Treatments were either PK fertilizer or compost. Soil rhizobial populations were determined using qPCR, the symbiotic rhizobia genotyped (16 S rRNA, nodA and nodD genes), and their BNF capacity assessed ex situ. The reliance of legumes on BNF at other British sites was estimated in a single season, and their nodulating symbionts examined.ResultsFaba bean obtained most of its N through BNF (>80%) regardless of variety or year. N-accumulation by cvs Babylon and Boxer increased with compost treatment in 2014/2015. Rhizobial populations were c. 105-106Rlv cells g−1 soil regardless of field or treatment. 157 Rlv microsymbionts grouped into two large nodAD clades; one mainly from V. faba, and the other from various legumes. All isolates nodulated, and some performed better than commercial inoculant strains.ConclusionsFaba bean can provide most of its nitrogen through BNF and leave economically valuable residual N for subsequent crops. Recent legume cropping in northern Europe is not essential for effective nodulation: rhizobia may persist in a range of farmland locations. Nevertheless, there is the potential to apply elite rhizobial strains as inoculants in some soils.

Post translational modifications of Trifolitoxin: a blue fluorescent peptide antibiotic

Trifolitoxin (TFX, C41H63N15O15S) is a selective, ribosomally-synthesized, post-translationally modified, peptide antibiotic, produced by Rhizobium leguminosarum bv. trifolii T24. TFX specifically inhibits α-proteobacteria, including the plant symbiont Rhizobium spp., the plant pathogen Agrobacterium spp. and the animal pathogen Brucella abortus. TFX-producing strains prevent legume root nodulation by TFX-sensitive rhizobia. TFX has been isolated as a pair of geometric isomers, TFX1 and TFX2, which are derived from the biologically inactive primary amino acid sequence: Asp-Ile-Gly-Gly-Ser-Arg-Gln-Gly-Cys-Val-Ala. Gly-Cys is present as a thiazoline ring and the Arg-Gln-Gly sequence is extensively modified to a UV absorbing, blue fluorescent chromophore. The chromophore consists of a conjugated, 5-membered heterocyclic ring and side chain of modified glutamine.

Genome-Scale Metabolic Modelling of Lifestyle Changes in Rhizobium leguminosarum.

ABSTRACTBiological nitrogen fixation in rhizobium-legume symbioses is of major importance for sustainable agricultural practices. To establish a mutualistic relationship with their plant host, rhizobia transition from free-living bacteria in soil to growth down infection threads inside plant roots and finally differentiate into nitrogen-fixing bacteroids. We reconstructed a genome-scale metabolic model for Rhizobium leguminosarum and integrated the model with transcriptome, proteome, metabolome, and gene essentiality data to investigate nutrient uptake and metabolic fluxes characteristic of these different lifestyles. Synthesis of leucine, polyphosphate, and AICAR is predicted to be important in the rhizosphere, while myo-inositol catabolism is active in undifferentiated nodule bacteria in agreement with experimental evidence. The model indicates that bacteroids utilize xylose and glycolate in addition to dicarboxylates, which could explain previously described gene expression patterns. Histidine is predicted to be actively synthesized in bacteroids, consistent with transcriptome and proteome data for several rhizobial species. These results provide the basis for targeted experimental investigation of metabolic processes specific to the different stages of the rhizobium-legume symbioses.IMPORTANCE Rhizobia are soil bacteria that induce nodule formation on plant roots and differentiate into nitrogen-fixing bacteroids. A detailed understanding of this complex symbiosis is essential for advancing ongoing efforts to engineer novel symbioses with cereal crops for sustainable agriculture. Here, we reconstruct and validate a genome-scale metabolic model for Rhizobium leguminosarum bv. viciae 3841. By integrating the model with various experimental data sets specific to different stages of symbiosis formation, we elucidate the metabolic characteristics of rhizosphere bacteria, undifferentiated bacteria inside root nodules, and nitrogen-fixing bacteroids. Our model predicts metabolic flux patterns for these three distinct lifestyles, thus providing a framework for the interpretation of genome-scale experimental data sets and identifying targets for future experimental studies.

Immobilization mechanism of Cd2+/HCrO4-/CrO42- ions and carboxin on montmorillonite modified with Rhizobium leguminosarum bv. trifolii exopolysaccharide

The modification of clay minerals by exopolysaccharides (EPSs) may significantly increase their adsorption capacity. Therefore, this study focused on the impact of the EPS synthesised by the soil bacterium Rhizobium leguminosarum bv. trifolii on the adsorptive features of montmorillonite relative to two heavy metal ions (cadmium/Cd(II) and chromium/Cr(VI)) and a pesticide (carboxin). The characterization of montmorillonite was carried out using various methods: X-ray diffraction, X-ray photoelectron spectroscopy, Fourier-transform infrared spectroscopy, scanning electron microscopy. The adsorption/desorption data were modelled using theoretical equations (Langmuir-Freundlich, Redlich-Peterson, etc.). The obtained results showed that EPS promoted the accumulation of heavy metals on the clay mineral and, simultaneously, contributed to a lower degree of their desorption. This resulted from complexation between the biopolymer and the Cd(II)/Cr(VI) ions. After montmorillonite modification with 100 mg/L EPS, the maximum noted growth in Cd(II) adsorption was 26.10%, whereas it was 20.30% for Cr(VI). The adsorbed amounts of Cd(II) and Cr(VI) were then 24.24 and 14.45 mg/g, respectively. In the case of carboxin, the EPS effect on its adsorption/desorption rates was opposite – its adsorption level decreasing by 10.80%, was 0.27 mg/g. Thus, the presence of EPS-producing bacteria could reduce the bioavailability of the heavy metals, but not of the selected pesticide.

Tea plant-legume intercropping simultaneously improves soil fertility and tea quality by changing bacillus species composition.

The tea plant is an economically important crop in China, but long-term monoculture and substantial chemical nitrogen fertilizer input cause soil acidification, which in turn affects the nutrient supply and tea quality. Intercropping has drawn more attention in tea gardens because this pattern is expected to improve soil fertility and tea quality and change the soil microbial community composition. However, the roles of some key microorganisms in rhizosphere soils have not been well characterized. Here, a “soybean in summer and smooth vetch in winter” strategy was used in a tea garden to investigate the effects of intercropped legumes on soil fertility, tea quality, and potential changes in beneficial bacteria such as Bacillus. Our data showed that when soybeans were turned into the soil, the intercropping system exhibited higher soil organic matter (SOM), total nitrogen (TN), tea quality indices, and expression of the Camellia sinensis glutamine synthetase gene (CsGS). Notably, intercropping significantly affected the bacterial communities, decreasing the relative abundance of Bacillus but increasing its absolute abundance. Bacillus amyloliquefaciens BM1 was isolated from intercropped soil and showed outstanding plant growth-promoting (PGP) properties when co-inoculated with rhizobia. In winter, intercropping with smooth vetch had a beneficial effect on soil properties and tea quality. Co-inoculation with strain BM1 and Rhizobium leguminosarum Vic5 on smooth vetch (Vicia villosa) produced huge improvements in SOM, TN, and tea leaf quality, which were accompanied by the highest level of amino acids and the lowest levels of polyphenols and caffeine (p < 0.05). Our findings demonstrate that intercropping with some legumes in the tea garden is a strategy that increases SOM, TN, and tea quality, and the optional use of some PGP Bacillus species produces an amplification effect.

Differential Response of Rhizobium Leguminosarum Inoculation for Inducing Water Deficit Tolerance in Triticum Durum

Limited soil water availability is a major threat to agricultural productivity because it inhibits plant growth and yields. Soil microorganisms having the ability to alleviate abiotic stress and promoting plant development would be highly valuable tools in sustainable agriculture. To this purpose, two Rhizobium leguminosarum strains were tested for their potential to induce tolerance against water deficits in Triticum durum grown under two hydric conditions in greenhouse experiments. For this, physiological and biochemical parameters were measured as criteria to assess the effect of R. leguminosarum inoculation on T. durum under water deficiency. In the presence of rhizobia, wheat plants were able to withstand water stress more effectively than un-inoculated plant, as indicated by the recorded increases in relative water content of inoculated plants compared to un-inoculated plants, higher accumulation of osmoprotectant (soluble sugar), lower MDA and H2O2 accumulation, and increased in antioxidant responses (peroxidase). Under water-limited condition, co-inoculation of selected rhizobia was better than single application for one of studied genotype, which let us hypothesize that this variation in responsiveness indicates a possible genotype effect. As a result, rhizobia from various legumes have a huge potential for enhancing the water deficiency stress tolerance of cereals.

The Three Sisters of Agriculture: An Active Learning Activity on Symbiotic Nitrogen Fixation

This active learning exercise introduces students to the plant microbiome and the contributions that bacteria make to plant growth and food production. The Three Sisters are an ancient indigenous practice in which three plant crops and the associated microbiome contribute to each other&rsquo;s growth. In this symbiotic relationship, the associated bacteria (<em>Rhizobium leguminosarum</em> biovar <em>phaseoli</em>) inhabit root nodules in bean plants, converting atmospheric nitrogen to ammonia through the process of nitrogen fixation. The plant takes up the ammonia as its nitrogen source, and provides the bacteria with organic carbon. The Lesson contains a pre-class reading, a 50-minute class session, and an after-class reading. In the first round of small group work during the class, students discuss and report back on the symbiosis between the three plants and between the bacteria and the plants, and the contributions to sustainable agriculture. In the second round of discussion and reporting, students discuss nitrogen fixation, emphasizing the <em>nod</em> genes for polypeptides involved in forming the root nodules and the <em>nif</em> genes that encode the nitrogenase enzyme complex that carries out nitrogen fixation. The after class reading provides students with an example of enhancing plant growth by adding nitrogen-fixing bacteria externally to beans. Altogether, this exercise provides students with a real life scenario relevant to sustainable agriculture. <em>Primary Image:</em>&nbsp;Nitrogen fixation in bean root nodules. Nodule formation starts with the secretion of flavonoids from bean roots, leading to the induction of nodulation factors in the bacteria. After root curling and nodule formation, the enzyme nitrogenase catalyzes the reaction from atmospheric nitrogen to plant accessible ammonia.

A fluorometric assay to estimate pea leaf weevil (Coleoptera: Curculionidae) larval feeding damage using leghaemoglobin in root nodules of faba bean (Fabaceae)

Abstract The pea leaf weevil, Sitona lineatus Linnaeus (Coleoptera: Curculionidae), is an invasive pest of field pea, Pisum sativum Linnaeus, and faba bean, Vicia faba Linnaeus (Fabaceae). Larvae feed on leguminous root nodules and associated Rhizobium leguminosarum Frank (Rhizobiacea) nitrogen-fixing bacteria. Larval feeding causes economic damage, but the current method to assess nodule damage is laborious. Leghaemoglobin, the oxygen-carrying molecule in root nodules, is degraded as larvae feed. Measurement of leghaemoglobin could be an alternative method to estimate larval damage. Here, we developed a fluorometric assay to measure the variation in haem fluorescence, which relates to leghaemoglobin content, from nodulated roots. Roots were collected from caged faba bean plants with or without weevil infestation. Faba bean yield and haem fluorescence were inversely correlated with the percent damaged nodules. A plant growth score was positively correlated with haem fluorescence. This method can be used to assess nodule damage in pulse crops from pea leaf weevil and potentially from other biotic stresses, and it may have wider application to studies of nitrogen fixation.

NoiD, a DedA membrane protein required for homeostasis maintaining of Rhizobium leguminosarum biovar viciae during symbiosis with Pisum sativum

NoiD, a DedA membrane protein required for homeostasis maintaining of Rhizobium leguminosarum biovar viciae during symbiosis with Pisum sativum

Distribution of Heterotrophic and Nitrogen fixing Rhizobacteria in Irrigation Sites of Lake Alau Borno State Northeastern Nigeria

This study was conducted to know the population of rhizobacteria in both irrigation and non-irrigation sites of the dam. The dense population of these organism indirectly promote plant growth and development. Five sites (A, B, C, D and E) were used to collect soil samples randomly and transported to the laboratory for analysis. Total heterotrophic bacterial count was done using nutrient agar (NA) and nitrogen fixing bacteria was counted using Ashbey’s media (AM). The result shows that highest number of total heterotrophic bacteria in site C (46.0×106) cfu/g in irrigation site whereas higher count in non-irrigation site was (13.0×106) site D, the nitrogen fixing bacterial count in irrigation site was higher at site E with (12.0×106) and for the non-irrigation site was higher at site D with (14.0×106) The total heterotrophic bacteria isolated in the soil sample are the species of Bacillus alvei, Bacillus alvei, Bacillus cereus, Bacillus cereus, Pseudomonas putida, Klebsiella aeruginosa and Enterobacter aeruginosa. Likewise, the Nitrogen fixing bacteria isolated are the species of Rhizobium leguminosarum, Klebsiella pneumonia, Bacillus lentus, Azotobacter nigricans, Azotobacter tropicalis, Azotobacter spp, and Azotobacter tropicalis. The long history of agricultural activities in the lake area has directly influenced the diversity of microbial population in the area.

Transferring of Exopolysaccharide (EPS) Gene Cluster from Rhizobium leguminosarum to Pseudomonas fluorescens by Horizontal Gene Transfer Techniques

Transferring of Exopolysaccharide (EPS) Gene Cluster from Rhizobium leguminosarum to Pseudomonas fluorescens by Horizontal Gene Transfer Techniques

Selection and characterization of Spanish Trifolium-nodulating rhizobia for pasture inoculation

Identification of elite nitrogen-fixing rhizobia strains is a continuous and never ending effort, since new legume species can be cultivated in different agro systems or are introduced into new areas. This current study reports on the taxonomic affiliation and symbiotic proficiency of nine strains of Trifolium-nodulating rhizobia isolated from different pasture areas in Spain, as well as three Rhizobium leguminosarum bv. trifolii reference strains, on eleven Trifolium species. Based on 16S rRNA gene sequences the strains belonged to the R. leguminosarum species complex. Additional phylogenetic analyses of the housekeeping genes recA, atpD and rpoB showed the strains were closely related to the species R. leguminosarum, R. laguerreae, R. indicum, R. ruizarguesonis or R. acidisoli. In addition, three strains had no clear affiliation and could represent putative new species, although two of the reference strains were positioned close to R. ruizarguesonis. nodC gene phylogeny allowed the discrimination between strains isolated from annual or perennial Trifolium species and placed all of them in the symbiovar trifolii. Neither geographic origin nor host-plant species could be correlated with the taxonomic affiliation of the strains and a high degree of phenotypic diversity was found among this set of strains. The strong interaction of plant species with the rhizobial strains found for biological nitrogen fixation (BNF) was noteworthy, and allowed the identification of rhizobial strains with a maximum proficiency for certain trefoil species. Several strains showed high BNF potential with a wide range of clover species, which made them valuable strains for inoculant manufacturers and they would be particularly useful for inoculation of seed mixtures in natural or cultivated pastures.

Effects of Meloidogyne incognita, Pseudomonas syringae pv. pisi and Rhizobium leguminosarum inoculated alone, simultaneously, and sequentially, on the growth and biochemical parameters of pea (Pisum sativum) in three soil types

AbstractEffects ofMeloidogyne incognita,Pseudomonas syringaepv.pisiandRhizobium leguminosarumwere studied on growth and biochemical parameters of pea (Pisum sativumL.) in three soil types. Plants grown in 20% fly ash attained higher plant growth, chlorophyll and carotenoid followed by loam soil and 20% sand. Inoculation ofR. leguminosarumresulted in increased plant growth, nodulation, chlorophyll and carotenoid over control. Root nodulation and proline contents were high in plants grown in 20% sand and least in 20% fly ash. Inoculation ofM. incognitaprior toP. syringaepv.pisiresulted in a greater reduction in plant growth, nodulation, chlorophyll and carotenoid content and least whereP. syringaepv.pisiwas inoculated prior toM. incognita. Inoculation of pathogens increased proline contents. Galling and population ofM. incognitawas high in 20% sand followed by loam soil and fly ash amended soil.P. syringaepv.pisiandR. leguminosarumhad adverse effect on galling and nematode population. The principal component analysis identifies interaction of pathogens and showed segregation of various treatments in the plots.

Enhancement of drought tolerance in diverse Vicia faba cultivars by inoculation with plant growth-promoting rhizobacteria under newly reclaimed soil conditions

Water deficit has devastating impacts on legume production, particularly with the current abrupt climate changes in arid environments. The application of plant growth-promoting rhizobacteria (PGPR) is an effective approach for producing natural nitrogen and attenuating the detrimental effects of drought stress. This study investigated the influence of inoculation with the PGPR Rhizobium leguminosarum biovar viciae (USDA 2435) and Pseudomonas putida (RA MTCC5279) solely or in combination on the physio-biochemical and agronomic traits of five diverse Vicia faba cultivars under well-watered (100% crop evapotranspiration [ETc]), moderate drought (75% ETc), and severe drought (50% ETc) conditions in newly reclaimed poor-fertility sandy soil. Drought stress substantially reduced the expression of photosynthetic pigments and water relation parameters. In contrast, antioxidant enzyme activities and osmoprotectants were considerably increased in plants under drought stress compared with those in well-watered plants. These adverse effects of drought stress reduced crop water productivity (CWP) and seed yield‐related traits. However, the application of PGPR, particularly a consortium of both strains, improved these parameters and increased seed yield and CWP. The evaluated cultivars displayed varied tolerance to drought stress: Giza-843 and Giza-716 had the highest tolerance under well-watered and moderate drought conditions, whereas Giza-843 and Sakha-4 were more tolerant under severe drought conditions. Thus, co-inoculation of drought-tolerant cultivars with R. leguminosarum and P. putida enhanced their tolerance and increased their yield and CWP under water-deficit stress conditions. This study showed for the first time that the combined use of R. leguminosarum and P. putida is a promising and ecofriendly strategy for increasing drought tolerance in legume crops.

DNA Methylation Patterns Differ between Free-Living Rhizobium leguminosarum RCAM1026 and Bacteroids Formed in Symbiosis with Pea (Pisum sativum L.).

Rhizobium leguminosarum (Rl) is a common name for several genospecies of rhizobia able to form nitrogen-fixing nodules on the roots of pea (Pisum sativum L.) while undergoing terminal differentiation into a symbiotic form called bacteroids. In this work, we used Oxford Nanopore sequencing to analyze the genome methylation states of the free-living and differentiated forms of the Rl strain RCAM1026. The complete genome was assembled; no significant genome rearrangements between the cell forms were observed, but the relative abundances of replicons were different. GANTC, GGCGCC, and GATC methylated motifs were found in the genome, along with genes encoding methyltransferases with matching predicted target motifs. The GGCGCC motif was completely methylated in both states, with two restriction–modification clusters on different replicons enforcing this specific pattern of methylation. Methylation patterns for the GANTC and GATC motifs differed significantly depending on the cell state, which indicates their possible connection to the regulation of symbiotic differentiation. Further investigation into the differences of methylation patterns in the bacterial genomes coupled with gene expression analysis is needed to elucidate the function of bacterial epigenetic regulation in nitrogen-fixing symbiosis.

Effect of plant growth promoting rhizobacteria (PGPR) on the growth of habanero pepper (Capsicum chinense Jacq.)

Objective: To evaluate the effect of plant growth promoting rhizobacteria (PGPR) on the growth and production of habanero pepper (Capsicum chinense Jacq.). Design/Methodology/Approach: Twelve strains of PGPR were evaluated in habanero pepper seeds of the orange variety. The species of PGPR were Rhizobium leguminosarum: (CP Méx 46), Pseudomonas spp: (P fluorescens, C2, A7, A9, A9m, Avm); Azospirillum, (Sp7, Sp 59, UAP 40, UAP154), plus a control treatment, giving a total of 13 treatments. The study variables were seedling emergence (SE), plant height (PH), white fruit incidence (WFI), virotic plants (VP), days to flowering (DF) and fresh fruit yield (FFY). The experimental design was random blocks with four repetitions. Results: An effect on the growth of habanero pepper from PGPRs was found in all the variables studied. Seedling emergence and their height was favored by strains Sp9 (84.16%) and A7 (73.44). The number of white flies decreased with the inoculation of CP Méx 46, while the incidence of virosis decreased in plants inoculated with SP9 (32.00%). The highest yield of fresh fruit was found in plants with the strain AVM with 16636 kg ha-1. Findings/Conclusions: The effect of inoculation with PGPR is in function of the strain used and the study variable, growth stage and development stage of the habanero pepper plant.

First report of Serratia marcescens associated with black rot of Citrus sinensis fruit , and evaluation of its biological control measures in Bangladesh

Background: The present study was designed to isolate and identify the phyto-pathogen responsible for black rot of Citrus sinensis, and to determine its biological control measures. Methods: The pathogen was isolated from infected oranges and cultured on Luria-Bertani medium. Gram staining method was used to identify the morphological characteristics of the causal agents of the black rot. Advanced molecular technique was applied to facilitate proper detection of the isolated bacteria. Phylogenetic trees were analyzed using the Neighbor-Joining method. Antimicrobial screening was conducted by disc diffusion method. Antagonistic activity was evaluated by well diffusion method. Results: Gram staining of the causal agent showed rod shaped, small and pink bacteria. Polymerase chain reaction of the 16S ribosomal RNA gene amplified an approximately 1465 bp product. The nucleotide sequences of the isolated bacterial sample 1 (BS1) and bacterial sample 2 (BS2) had 99.34% and 99.45% similarities with the reference of Serratia marcescens sequence in NCBI GenBank. The obtained sequences were deposited in GenBank. Two isolates showed virulence capability on some fresh fruits, which confirmed the stain detection and Koch’s postulates. Allium sativum extract showed the largest (27.33±1.5 mm) diameter of zone of inhibition against BS1, at 30µg/disc concentration. In the antagonistic assay, Rhizobium leguminosarum showed largest (19±1 mm) zone of inhibition against BS1. Conclusions: Findings of the current investigations are constructive for identification of causative pathogens in Citrus sinensis black rot disease and their biological control measures.

EVALUATION OF PATHOGENICITY OF NODULE BACTERIA STRAIN RHIZOBIUM LEGUMINOSARUM G 222

Objective. Investigate pathogenic (non-pathogenic) properties of a new strain of nodule bacteria Rhizobium leguminosarum G 222. Methods. Microbiological (obtaining a suspension of R. leI guminosarum G 222, determination of its titre by opacity optical standard and by seeding on a digest medium; microscopy of stained imprint smears of internal organs of experimental animals and seeding of tissue samples on a digest medium), pathoanatomical (to determine possible invasiveness and dissemination of bacteria in the tissues of internal organs of animals — in the model of outbred white mice after administration of a suspension of live bacterial cells (oral doses from 0.5 × 109 to 5 × 109 CFU in 0.5 cm3 of sterile isotonic sodium chloride solution per mouse and intraperitoneal doses from 1 × 109 to 5 × 109 CFU/0.5 cm3) and statistical. Results. Over 15 days of observations after administration of a suspension of live bacterial cells, no death of experimental animals was registered. The strain did not lead to any changes in the general condition of the mice. No changes in their behaviour were reported. Fifteen days after the start of the study, it was found that this strain is non-infective (non-invasive), does not disseminate and does not reproduce in the body of experimental animals. Oral and intraperitoneal doses of a suspension of live bacterial cells did not result in bacterial invasion of animal internal organs. No retrocultures were registered. Gross examination did not find characteristic changes in the internal organs of the experimental animals. The obtained results indicate the avirulence of the strain for the studied warm-blooded animals (intraperitoneal LD50 &gt; 5 billion cells/mouse, oral LD50 &gt; 5 billion cells/mouse). Conclusion: According to the results regarding the lack of virulence and according to regulations, the new strain R. leguminosarum G 222 belongs to the group of avirulent microorganisms that are not able to invade the internal organs of studied warm-blooded laboratory animals and can be considered nonpathogenic and used as a basis for microbial preparations to increase crop yields.