Root Nodules Of Leguminous Plants Research Articles

Detection of Rhizobium leguminosarum bv. trifolii and Rhizobium leguminosarum bv. phaseoli isolated from root nodules of leguminous plants by multiplex-PCR

The genetic diversity of 4 R. leguminosarum bv. trifolii and R. leguminosarum bv. Phaseoli that was extracted from root nodes of Trifolium spp and Phaseolus vulgaris L cultivars circulated in Mosul soils were investigated. The bacteria strains under study show similar tolerance levels against some external factors. In the current study, using classical diagnosis methods, similarity and distance indices and DNA polymorphism were examined with the randomly amplified polymorphic DNA. The isolates were distributed through genetic diversification into two main classes, including two main branches, Rl2 and the other sub-group was divided into Rl3, and the neighboring group involved Rl1 and Rl4. It is noted that individual plants and abiotic aspects were less affected than the Genetic factor on bacterial diversity. On the other hand, in multiplex-PCR reaction, Our outcomes revealed specific amplified for R. leguminosarum bv. trifolii with amplification products 419 bp and R. leguminosarum bv. Phaseoli with amplification products 362 bp. Keywords: R. leguminosarum bv. phaseoli, R. leguminosarum bv. trifolii , nodules, multiplex-PCR, RAPD-PCR .

Effects of halotolerant rhizobacteria on rice seedlings under salinity stress

Avirulent halotolerant plant growth-promoting rhizobacteria (HPGPR) located on the roots' periphery can reduce abiotic stressors (such as salinity and drought), enhance plant productivity. Salinity poses a significant challenge for growing agricultural products, like rice, in the coastal regions. It is crucial to enhance production because of limited arable land and the high growth rate of the population. This study targeted to identify HPGPR from legume root nodules and assessed their effects on rice plants experiencing salt stress in coastal regions of Bangladesh. Based on the culture morphology, biochemical, salt, pH, and temperature tolerance traits, sixteen bacteria were isolated from the root nodules of leguminous plants (Common bean, Yardlong bean, Dhaincha, and Shameplant). All the bacterial strains can tolerate 3 % salt concentration, and capable to survive at the highest 45 °C temperature and pH 11 (without isolate 1). Three preeminent bacteria, Agrobacterium tumefaciens (B1), Bacillus subtilis (B2), and Lysinibacillus fusiformis (B3) were specified through morpho-biochemical and molecular (16S rRNA gene sequence) exploration for inoculation. To assess the plant growth-promoting activities, germination tests are applied where bacterial inoculation increased germination in saline and non-saline conditions. Control group (C) showed 89.47 % and bacterial treated groups (C + B1, C + B2, and C + B3) 95 %, 90 %, and 75 % germination after 2 days of inoculation. In (1 % NaCl) saline condition control group revealed 40 % whereas three groups with bacteria showed 60 %, 40 %, and 70 % germination after 3 days, which increased 70 %, 90 %, 85 %, and 95 % respectively after 4 days of inoculation. The HPGPR significantly improved plant development metrics such as root length, shoot length, fresh and arid biomass yield, chlorophyll content, etc. Our results suggest that the salt-resistant bacteria (Halotolerant) have a great potential role in recuperating plant growth and would be cost-effective as a bio-inoculant in saline conditions to be used as a prospective bio-fertilizer for rice production. These findings indicate that the HPGPR has a substantially promising function in reviving plant development in an eco-friendly manner.

Toxicity of Herbicide, Paraquat Dichloride and Insecticide, Lambda-cyhalothrin on Phosphate Solubilizing Bacteria, Pantoa dispersa in Aquatic Ecosystems

Aim: The study was aimed to determine the toxicity of paraquat dichloride and lambda-cyhalothrin on phosphate solubilizing bacteria, Pantoa dispersa in aquatic ecosystems.
 Study Design: This study employs randomized block design, statistical analysis of the data and interpretation.
 Place and Duration of the Study: Soil sample was collected from the root nodules of leguminous plants in a sterile polythene bag from the Elele, in Etche L.G.A, Rivers State. The fresh water sample was collected from Bane town in Khana L.G.A, brackish water sample was collected from Choba river in Obio/Akpor L.G.A while the marine water was collected from Bonny River of Bonny L.G.A., all of Rivers State, Nigeria. The samples were collected aseptically and transported in an ice-pack immediately to the Rivers State University, Microbiology laboratory for analysis. The study lasted for three months.
 Methodology: The bacterium, Pantoa dispersa was isolated and identified based on conventional and molecular characterization from water and soil samples. Different concentrations (3.13%, 6.25, 12.50, 25.00%, 50.00% and 75.00%) and the control (0%) of the herbicide (paraquat dichloride) and insecticide, Lambda-cyhalothrin were prepared using fresh, brackish and marine water samples and 10ml of the test organism, Pantoa dispersa was introduced and the survival count was determined at 0, 4hr, 8hr, 12hr and 24hr. The Mean Lethal Concentration (LC50) of the insecticide and herbicide on Pantoa dispersa in the three aquatic ecosystem was determined.
 Results: The LC50 of the herbicide (Paraquat dichloride) was recorded as 15.8% in brackish water, 17.37% in fresh water and 27.44% in marine water. While the LC50 of the insecticide, Lambda-cyhalothrin to Pantoa dispersa was 26.84% in fresh water, 27.26% in brackish water and 32.33% in marine water.
 Conclusion: From the study, the herbicide, Paraquat dichloride was more toxic in the three aquatic ecosystems compared to the insecticide, Lambda-cyhalothrin. The use of these agrochemicals should be monitored as they result in the mortality of beneficial soil bacteria like Pantoa dispersa which is phosphate solubilizing bacteria in aquatic ecosystems.

Single cell‐type transcriptome profiling reveals genes that promote nitrogen fixation in the infected and uninfected cells of legume nodules

Single cell‐type transcriptome profiling reveals genes that promote nitrogen fixation in the infected and uninfected cells of legume nodules

A Systematic Review on Isolation, Identification, and Characterization of Agrobacterium tumefaciens from Leguminous Plants

Agrobacterium tumefaciens causes crown gall disease in plants by transferring their T-DNA which contains some oncogenic genes. At the site of the wound, these bacteria become accumulates and then transfer their T-DNA into host plant cells, which interact with host plant genomic DNA and cause tumor. With the help of knowledge, how the A.tumefaciens transfer their T-DNA to plant cells. And with the knowledge and use of the latest molecular biology techniques, A.tumefaciens is used as a tool for the transformation of our gene of interest into the host plant cells to make genetically modified plants. This review provides a working systematic review of the isolation, identification, and characterization of A.tumefaciens from the leguminous plants. This review also tells us how to transfer the T-DNA into a host plant cell and how to isolate the Agrobacterium tumefaciens from the root nodules of leguminous plants, first of all, collect the root nodules of leguminous plants then wash them properly and streak the isolates of root nodules on LB agar plates. then identify and characterize the Agrobacterium from the isolates because Agrobacterium and rhizobium stay together. For characterization, biochemical tests i.e gram staining and KOH tests performed to confirm the isolated bacteria is gram-negative. Some pathogenic tests such as carrot disk bioassay and potato disc confirm whether isolates caused a tumor or not.

Isolation and Diagnosis of Rhizobium Bacteria Isolated from The Root Nodules of Leguminous Plants and Studying Their Plasmid Content.

The study included thirty-six isolates of rhizobia bacteria were isolated from the nodules located on the roots of nine types of leguminous family plants that were planted in four areas of the city of Mosul for the winter agricultural season for the year 2020-2019, where they studied the phenotypic and agricultural characteristics of the isolated bacteria in addition to a study of resistance and sensitivity to the isolates of rhizobia bacteria The study included ten antibiotics, and resistance ratios differed between isolates groups, where the highest resistance rate for residual bacteria isolates was 100% for Nystatin and Amoxicillin, and the lowest resistance was for Tetracycline and Streptomycin, as it reached 22.2%. As for the rest of the antibiotics, there is a difference between that. As for heavy metal salts, all the isolates of rhizobia bacteria under study were resistant to both CdCl2 chloride and CoCl2 cobalt by 100%, while their resistance to nickel chloride NiCl2 was 77.7% and the lowest resistance to heavy metal was mercury chloride HgCl2, reaching 33.3%. The plasmid DNA content was described for the studied isolates, as the results showed that there are two types of plasmid DNA bundles, the first type close to large-scale gel drilling called Mega plasmid represents symbiotic plasmids that carry the genes of contract formation and nitrogen fixation and the second type moves far from the gel drilling and they are sizes small equal representing non-symbiotic plasmids.

Extracellular Synthesis and Characterization of Silver Nanoparticles from Alkaliphilic Pseudomonas sp.

Bio-nanotechnology offers eco-friendly processes for the synthesis of stable nanoparticles (NPs). We hypothesized that microorganisms isolated from the root nodules of leguminous plants would biosynthesize silver (Ag) bio-nanoparticles. Clover root nodules enriched with nutrient broth (NB) produced four distinct colonies on NA plates. Microbial colonies were purified by repeated streaking and designated as SS6, SS7, SS8, and SS9 for identification using 16S rRNA sequencing. Four species of Pseudomonas were identified with a similarity score of over 99% using the EZ Taxon search engine, and tested for extracellular biosynthesis of AgNPs. Microorganism Pseudomonas taiwanensis-SS8 with alkaliphilic growth characteristics reduced the AgNO3 solution into AgNPs in the shortest time period. AgNPs were characterized using UV-Vis spectrophotometry and electron and transmission electron microscopy. A number of physical (i.e., temperature and time) and chemical (i.e., pH and growth media) parameters were optimized. An efficient polydispersal biosynthesis of AgNPs at pH 8-9 after 48 hrs in NB growth medium was observed. In addition, the AgNPs showed antimicrobial properties against 16 commonly occurring pathogenic microorganisms.

Antimicrobial Activities of Cysteine-rich Peptides Specific to Bacteriocytes of the Pea Aphid Acyrthosiphon pisum.

Aphids have a mutualistic relationship with the bacterial endosymbiont Buchnera aphidicola. We previously reported seven cysteine-rich peptides in the pea aphid Acyrthosiphon pisum and named them Bacteriocyte-specific Cysteine-Rich (BCR) peptides; these peptides are exclusively expressed in bacteriocytes, special aphid cells that harbor symbionts. Similar symbiotic organ-specific cysteine-rich peptides identified in the root nodules of leguminous plants are named Nodule-specific Cysteine-Rich (NCR) peptides. NCR peptides target rhizobia in the nodules and are essential for symbiotic nitrogen fixation. A BacA (membrane protein) mutant of Sinorhizobium is sensitive to NCR peptides and is unable to establish symbiosis. Based on the structural and expressional similarities between BCR peptides and NCR peptides, we hypothesized that aphid BCR peptides exhibit antimicrobial activity, similar to some NCR peptides. We herein synthesized BCR peptides and investigated their antimicrobial activities and effects on the bacterial membrane of Escherichia coli. The peptides BCR1, BCR3, BCR5, and BCR8 exhibited antimicrobial activities with increased membrane permeability. An sbmA mutant of E. coli, a homolog of bacA of S. meliloti, was more sensitive to BCR peptides than the wild type. Our results suggest that BCR peptides have properties that may be required to control the endosymbiont, similar to NCR peptides in legumes.

What is the true nitrogenase reaction? A guided approach.

Only diazotrophic bacteria, called Rizhobia, living as symbionts in the root nodules of leguminous plants and certain free-living prokaryotic cells can fix atmospheric N2 . In these microorganisms, nitrogen fixation is carried out by the nitrogenase protein complex. However, the reduction of nitrogen to ammonia has an extremely high activation energy due to the stable (unreactive) N≡N triple bond. The structural and functional features of the nitrogenase protein complex, based on the stepwise transfer of eight electrons from reduced ferredoxin to the nitrogenase, coupled to the hydrolysis of 16 ATP molecules, to fix one N2 molecule into two NH3 molecules, is well understood. Yet, a number of different nitrogenase-catalyzed reactions are present in biochemistry textbooks, which might cause misinterpretation. In this article, we show that when trying to balance the reaction catalyzed by the nitrogenase protein complex, it is important to show explicitly the 16 H(+) released by the hydrolysis of the 16 ATP molecules needed to fix the atmospheric N2.

A term�kenys�get jav�t� bakt�riumos talajolt�s t�rt�neti �ttekint�se

The first bacteria capable of biological nitrogen-fixation and for this reason for the replacement of inorganic fertilizers was found in the root-nodules of leguminous plants more than 100 years ago. The other nitrogen-fixing bacteria, the associative azospirillums, on the other hand do not create visible systems, alterations in the root system. They were therefore found and reported only about 40 years ago. The isolated and identified bacteria are well-used in the agriculture since then, but still we can find unknown elements behind of their functioning. Nowadays second and third generations of microbial inoculums are applied. The soil-plant-microbe systems are so multifactorial arrangements, than there are several biotic and abiotic factors influencing of their real functioning. We should learn more how those systems are really working. The beneficial effects of those microbial inoculums will be discussed in this review with a historical background.

Physiological and molecular identification of polyhydroxybutyrates (PHB) producing micro-organisms isolated from root nodules of leguminous plants

Polyhydroxybutyrates (PHBs) are synthesized and deposited as cytoplasmic inclusions in various bacteria like Rhizobium sp., Rhodobacter sphaeroides, etc. This work was focused on isolating a PHB-producing bacterium capable of growing in conventional media used for industrial biopolymer production, its physiological and molecular identification and characterization. Thus, a bacterial isolation process was carried out from samples of root nodules of various leguminous plants. Synthetic plastics pose a number of serious problems. Therefore, there is an urgent need to have a biodegradable alternative to these synthetic polymers. Rhizobium spp. is able to produce PHB which can be used as biodegradable plastic. Although, the cost of production is high because of high cost of media ingredients and chemicals used for their extraction from the cell. In the present study, attempts have been made to isolate and characterize the Rhizobiumisolates capable of producing PHB and to analyze the effect of cheap carbon and nitrogen sources on their growth and PHB production. Key words: Polyhydroxybutyrate, biodegradable plastic, Rhizobium.

Ectopic expression of miR160 results in auxin hypersensitivity, cytokinin hyposensitivity, and inhibition of symbiotic nodule development in soybean.

Symbiotic root nodules in leguminous plants result from interaction between the plant and nitrogen-fixing rhizobia bacteria. There are two major types of legume nodules, determinate and indeterminate. Determinate nodules do not have a persistent meristem, while indeterminate nodules have a persistent meristem. Auxin is thought to play a role in the development of both these types of nodules. However, inhibition of rootward auxin transport at the site of nodule initiation is crucial for the development of indeterminate nodules but not determinate nodules. Using the synthetic auxin-responsive DR5 promoter in soybean (Glycine max), we show that there is relatively low auxin activity during determinate nodule initiation and that it is restricted to the nodule periphery subsequently during development. To examine if and what role auxin plays in determinate nodule development, we generated soybean composite plants with altered sensitivity to auxin. We overexpressed microRNA393 to silence the auxin receptor gene family, and these roots were hyposensitive to auxin. These roots nodulated normally, suggesting that only minimal/reduced auxin signaling is required for determinate nodule development. We overexpressed microRNA160 to silence a set of repressor auxin response factor transcription factors, and these roots were hypersensitive to auxin. These roots were not impaired in epidermal responses to rhizobia but had significantly reduced nodule primordium formation, suggesting that auxin hypersensitivity inhibits nodule development. These roots were also hyposensitive to cytokinin and had attenuated expression of key nodulation-associated transcription factors known to be regulated by cytokinin. We propose a regulatory feedback loop involving auxin and cytokinin during nodulation.

Choice of hydrogen uptake (Hup) status in legume‐rhizobia symbioses

The H2 is an obligate by-product of N-fixation. Recycling of H2 through uptake hydrogenase (Hup) inside the root nodules of leguminous plants is often considered an advantage for plants. However, many of the rhizobium-legume symbioses found in nature, especially those used in agriculture are shown to be Hup−, with the plants releasing H2 produced by nitrogenase activity from root nodules into the surrounding rhizosphere. Recent studies have suggested that, H2 induces plant-growth-promoting rhizobacteria, which may explain the widespread of Hup− symbioses in spite of the low energy efficiency of such associations. Wild legumes grown in Nova Scotia, Canada, were surveyed to determine if any plant-growth characteristics could give an indication of Hup choice in leguminous plants. Out of the plants sampled, two legumes, Securigera varia and Vicia cracca, showed Hup+ associations. Securigera varia exhibited robust root structure as compared with the other plants surveyed. Data from the literature and the results from this study suggested that plants with established root systems are more likely to form the energy-efficient Hup+ symbiotic relationships with rhizobia. Conversely, Hup− associations could be beneficial to leguminous plants due to H2-oxidizing plant-growth-promoting rhizobacteria that allow plants to compete successfully, early in the growing season. However, some nodules from V. cracca tested Hup+, while others were Hup−. This was similar to that observed in Glycine max and Pisum sativum, giving reason to believe that Hup choice might be affected by various internal and environmental factors.

Environmental signals and regulatory pathways that influence exopolysaccharide production in rhizobia.

Rhizobia are Gram-negative bacteria that can exist either as free-living bacteria or as nitrogen-fixing symbionts inside root nodules of leguminous plants. The composition of the rhizobial outer surface, containing a variety of polysaccharides, plays a significant role in the adaptation of these bacteria in both habitats. Among rhizobial polymers, exopolysaccharide (EPS) is indispensable for the invasion of a great majority of host plants which form indeterminate-type nodules. Various functions are ascribed to this heteropolymer, including protection against environmental stress and host defense, attachment to abiotic and biotic surfaces, and in signaling. The synthesis of EPS in rhizobia is a multi-step process regulated by several proteins at both transcriptional and post-transcriptional levels. Also, some environmental factors (carbon source, nitrogen and phosphate starvation, flavonoids) and stress conditions (osmolarity, ionic strength) affect EPS production. This paper discusses the recent data concerning the function of the genes required for EPS synthesis and the regulation of this process by several environmental signals. Up till now, the synthesis of rhizobial EPS has been best studied in two species, Sinorhizobium meliloti and Rhizobium leguminosarum. The latest data indicate that EPS synthesis in rhizobia undergoes very complex hierarchical regulation, in which proteins engaged in quorum sensing and the regulation of motility genes also participate. This finding enables a better understanding of the complex processes occurring in the rhizosphere which are crucial for successful colonization and infection of host plant roots.

Vacuolar organization in the nodule parenchyma is important for the functioning of pea root nodules.

Different models have been proposed to explain the operation of oxygen diffusion barrier in root nodules of leguminous plants. This barrier participates in protection of oxygen-sensitive nitrogenase, the key enzyme in nitrogen fixation, from inactivation. Details concerning structural and biochemical properties of the barrier are still lacking. Here, the properties of pea root nodule cortical cells were examined under normal conditions and after shoot removal. Microscopic observations, including neutral red staining and epifluorescence investigations, showed that the inner and outer nodule parenchyma cells exhibit different patterns of the central vacuole development. In opposition to the inner part, the outer parenchyma cells exhibited vacuolar shrinkage and formed cell wall infoldings. Shoot removal induced vacuolar shrinkage and formation of infoldings in the inner parenchyma and uninfected cells of the symbiotic tissue, as well. It is postulated that cells which possess shrinking vacuoles are sensitive to the external osmotic pressure. The cells can give an additional resistance to oxygen diffusion by release of water to the intercellular spaces.Immunolocalization studies proved higher expression of endo-β-1,4-glucanases within expanding cells of the outer cortex of pea nodules comparing with nodule endodermis or nodule parenchyma, so it is suggested that (1) endo-glucanases are involved in growth related modifications of cell walls and (2) enlarged cells decrease nodule conductance to oxygen.

It's life, Martinus, but not as we know it

It's life, Martinus, but not as we know it

Development of real-time PCR assay for detection and quantification ofSinorhizobium melilotiin soil and plant tissue

Sinorhizobium meliloti is a nitrogen-fixing alpha-proteobacterium present in soil and symbiotically associated with root nodules of leguminous plants. To date, estimation of bacterial titres in soil is achieved by most-probable-number assays based on the number of nodules on the roots of test plants. Here, we report the development of two real-time PCR (qPCR) assays to detect the presence of S. meliloti in soil and plant tissues by targeting, in a species-specific fashion, the chromosomal gene rpoE1 and the pSymA gene nodC. rpoE1 and nodC primer pairs were tested on DNA extracted from soil samples unspiked and spiked with known titres of S. meliloti and from plant root samples nodulated with S. meliloti. Results obtained were well in agreement with viable titres of S. meliloti cells estimated in the same samples. The developed qPCR assays appear to be enough sensitive, precise and species-specific to be used as a complementary tool for S. meliloti titre estimation. These two novel markers offer the possibility of quick and reliable estimation of S. meliloti titres in soil and plant roots contributing new tools to explore S. meliloti biology and ecology including viable but nonculturable fraction.

Involvement of polyamines in the root nodule regulation of soybeans (Glycine max)

In leguminous plants, infection with rhizobia leads to the formation of root nodules that fix atmospheric nitrogen and supply it as ammonium to the host plant cells. The formation of nitrogen-fixing nodules is nutritionally benefi- cial, but excessive nodule production appears to be detrimental to the host legumes because of the resultant over-consumption of photosynthetic products. Therefore, the number of root nodules in leguminous plants is tightly regulated by shoot-root signaling known as a feedback or autoregulation of nodulation. Recently, we found that foliar application of polyamines and an effective inhibitor of polyamine biosynthesis regulates the number of nodules in soybean plants. In this review, we discuss the possible function of polyamines as a systemic regulator of nodule formation.

Expressed nifH Genes of Endophytic Bacteria Detected in Field-Grown Sweet Potatoes (Ipomoea batatas L.)

We examined the nitrogenase reductase (nifH) genes of endophytic diazotrophic bacteria expressed in field-grown sweet potatoes (Ipomoea batatas L.) by reverse transcription (RT)-PCR. Gene fragments corresponding to nifH were amplified from mRNA obtained from the stems and storage roots of field-grown sweet potatoes several months after planting. Sequence analysis revealed that these clones were homologous to the nifH sequences of Bradyrhizobium, Pelomonas, and Bacillus sp. in the DNA database. Investigation of the nifH genes amplified from the genomic DNA extracted from these sweet potatoes also showed high similarity to various α-proteobacteria including Bradyrhizobium, β-proteobacteria, and cyanobacteria. These results suggest that bradyrhizobia colonize and express nifH genes not only in the root nodules of leguminous plants but also in sweet potatoes as diazotrophic endophytes.

Metabolism of Reactive Oxygen Species Is Attenuated in Leghemoglobin-Deficient Nodules of Lotus japonicus

Leghemoglobins together with high rates of respiration are believed to be major sources of reactive oxygen species (ROS) in root nodules of leguminous plants. High capacities of antioxidative systems apparently protect this organ from oxidative damage. Using leghemoglobin-RNA interference (LbRNAi) lines of Lotus japonicus, we found that loss of leghemoglobin results in significantly lower H(2)O(2) levels in nodules. Transcript levels and catalytic activities of ascorbate-glutathione cycle enzymes involved in H(2)O(2) detoxification as well as concentrations of reduced ascorbate were also altered in LbRNAi nodules. Thus, symbiotic leghemoglobins contribute significantly to ROS generation in functional nodules.