No full-text available
To read the full-text of this research,
you can request a copy directly from the authors.
A Fair-Weather Friend: the Impact of Environmental Factors on Rhizobium laguerreae Nodulation Efficiency
ResearchGate has not been able to resolve any citations for this publication.
Despite global warming, the influence of heat on symbiotic nodules is scarcely studied. In this study, the effects of heat stress on the functioning of nodules formed by Rhizobium leguminosarum bv. viciae strain 3841 on pea (Pisum sativum) line SGE were analyzed. The influence of elevated temperature was analyzed at histological, ultrastructural, and transcriptional levels. As a result, an unusual apical pattern of nodule senescence was revealed. After five days of exposure, a senescence zone with degraded symbiotic structures was formed in place of the distal nitrogen fixation zone. There was downregulation of various genes, including those associated with the assimilation of fixed nitrogen and leghemoglobin. After nine days, the complete destruction of the nodules was demonstrated. It was shown that nodule recovery was possible after exposure to elevated temperature for 3 days but not after 5 days (which coincides with heat wave duration). At the same time, the exposure of plants to optimal temperature during the night leveled the negative effects. Thus, the study of the effects of elevated temperature on symbiotic nodules using a well-studied pea genotype and Rhizobium strain led to the discovery of a novel positional response of the nodule to heat stress.
Symbiotic nitrogen fixation (SNF) by rhizobium, a Gram-negative soil bacterium, is an essential component in the nitrogen cycle and is a sustainable green way to maintain soil fertility without chemical energy consumption. SNF, which results from the processes of nodulation, rhizobial infection, bacteroid differentiation and nitrogen-fixing reaction, requires the expression of various genes from both symbionts with adaptation to the changing environment. To achieve successful nitrogen fixation, rhizobia and their hosts cooperate closely for precise regulation of symbiotic genes, metabolic processes and internal environment homeostasis. Many researches have progressed to reveal the ample information about regulatory aspects of SNF during recent decades, but the major bottlenecks regarding improvement of nitrogen-fixing efficiency has proven to be complex. In this mini-review, we summarize recent advances that have contributed to understanding the rhizobial regulatory aspects that determine SNF efficiency, focusing on the coordinated regulatory mechanism of symbiotic genes, oxygen, carbon metabolism, amino acid metabolism, combined nitrogen, non-coding RNAs and internal environment homeostasis. Unraveling regulatory determinants of SNF in the nitrogen-fixing protagonist rhizobium is expected to promote an improvement of nitrogen-fixing efficiency in crop production.
Genomic evidence indicates that the Rhizobium leguminosarum species complex comprises multiple distinct species, perhaps 18 or more. Of the five earliest genospecies (gs) to be described, only two have formal names: R. leguminosarum sensu stricto (gsE) and Rhizobium ruizarguesonis (gsC). Here, we provide formal descriptions and names for the other three genospecies, based on the publicly available genome sequences for multiple strains of each species: Rhizobium brockwellii sp. nov. (gsA, 37 strains, type strain CC275e T =LMG 6122 T = ICMP 2163 T =NZP 561 T = PDDCC 2163 T =HAMBI 13 T ), Rhizobium johnstonii sp. nov. (gsB, 54 strains, type strain 3841 T = LMG 32736 T =DSM 114642 T ) and Rhizobium beringeri sp. nov. (gsD, 8 strains, type strain SM51 T = LMG 32895 T = DSM 115206 T ). Each species forms a well-supported clade in a phylogeny based on 120 concatenated core genes. All strains have average nucleotide identity (ANI) above 96 % with the relevant type strain and below 96 % with all other type strains. Each species is characterised by a number of genes that are absent or rare in other species.
Citation: Wekesa, C.; Kiprotich, K.; Okoth, P.; Asudi, G.O.; Muoma, J.O.; Furch, A.C.U.; Oelmüller, R. Molecular Characterization of Indigenous Rhizobia from Kenyan Soils Nodulating with Common Beans. Int. J. Mol. Sci. 2023, 24, 9509. Abstract: Kenya is the seventh most prominent producer of common beans globally and the second leading producer in East Africa. However, the annual national productivity is low due to insufficient quantities of vital nutrients and nitrogen in the soils. Rhizobia are symbiotic bacteria that fix nitrogen through their interaction with leguminous plants. Nevertheless, inoculating beans with commercial rhizobia inoculants results in sparse nodulation and low nitrogen supply to the host plants because these strains are poorly adapted to the local soils. Several studies describe native rhizobia with much better symbiotic capabilities than commercial strains, but only a few have conducted field studies. This study aimed to test the competence of new rhizobia strains that we isolated from Western Kenya soils and for which the symbiotic efficiency was successfully determined in greenhouse experiments. Furthermore, we present and analyze the whole-genome sequence for a promising candidate for agricultural application, which has high nitrogen fixation features and promotes common bean yields in field studies. Plants inoculated with the rhizobial isolate S3 or with a consortium of local isolates (COMB), including S3, produced a significantly higher number of seeds and seed dry weight when compared to uninoculated control plants at two study sites. The performance of plants inoculated with commercial isolate CIAT899 was not significantly different from uninoculated plants (p > 0.05), indicating tight competition from native rhizobia for nodule occupancy. Pangenome analysis and the overall genome-related indices showed that S3 is a member of R. phaseoli. However, synteny analysis revealed significant differences in the gene order, orientation, and copy numbers between S3 and the reference R. phaseoli. Isolate S3 is phylogenomically similar to R. phaseoli. However, it has undergone significant genome rearrangements (global mutagenesis) to adapt to harsh conditions in Kenyan soils. Its high nitrogen fixation ability shows optimal adaptation to Kenyan soils, and the strain can potentially replace nitrogenous fertilizer application. We recommend that extensive fieldwork in other parts of the country over a period of five years be performed on S3 to check on how the yield changes with varying whether conditions.
Triazole fungicides are widely used in agricultural production for plant protection, including pea (Pisum sativum L.). The use of fungicides can negatively affect the legume-Rhizobium symbiosis. In this study, the effects of triazole fungicides Vintage and Titul Duo on nodule formation and, in particular, on nodule morphology, were studied. Both fungicides at the highest concentration decreased the nodule number and dry weight of the roots 20 days after inoculation. Transmission electron microscopy revealed the following ultrastructural changes in nodules: modifications in the cell walls (their clearing and thinning), thickening of the infection thread walls with the formation of outgrowths, accumulation of poly-β-hydroxybutyrates in bacteroids, expansion of the peribacteroid space, and fusion of symbiosomes. Fungicides Vintage and Titul Duo negatively affect the composition of cell walls, leading to a decrease in the activity of synthesis of cellulose microfibrils and an increase in the number of matrix polysaccharides of cell walls. The results obtained coincide well with the data of transcriptomic analysis, which revealed an increase in the expression levels of genes that control cell wall modification and defense reactions. The data obtained indicate the need for further research on the effects of pesticides on the legume-Rhizobium symbiosis in order to optimize their use.
Our current food production systems are unsustainable, driven in part through the application of chemically fixed nitrogen. We need alternatives to empower farmers to maximise their productivity sustainably. Therefore, we explore the potential for transferring the root nodule symbiosis from legumes to other crops. Studies over the last decades have shown that preexisting developmental and signal transduction processes were recruited during the evolution of legume nodulation. This allows us to utilise these preexisting processes to engineer nitrogen fixation in target crops. Here, we highlight our understanding of legume nodulation and future research directions that might help to overcome the barrier of achieving self-fertilising crops.
In this study, the roles of glutathione (GSH), homoglutathione (hGSH), and their ratio in symbiotic nodule development and functioning, as well as in defense responses accompanying ineffective nodulation in pea (Pisum sativum) were investigated. The expression of genes involved in (h)GSH biosynthesis, thiol content, and localization of the reduced form of GSH were analyzed in nodules of wild-type pea plants and mutants sym33-3 (weak allele, “locked” infection threads, occasional bacterial release, and defense reactions) and sym33-2 (strong allele, “locked” infection threads, defense reactions), and sym40-1 (abnormal bacteroids, oxidative stress, early senescence, and defense reactions). The effects of (h)GSH depletion and GSH treatment on nodule number and development were also examined. The GSH:hGSH ratio was found to be higher in nodules than in uninoculated roots in all genotypes analyzed, with the highest value being detected in wild-type nodules. Moreover, it was demonstrated, that a hGSHS-to-GSHS switch in gene expression in nodule tissue occurs only after bacterial release and leads to an increase in the GSH:hGSH ratio. Ineffective nodules showed variable GSH:hGSH ratios that correlated with the stage of nodule development. Changes in the levels of both thiols led to the activation of defense responses in nodules. The application of a (h)GSH biosynthesis inhibitor disrupted the nitrogen fixation zone in wild-type nodules, affected symbiosome formation in sym40-1 mutant nodules, and meristem functioning and infection thread growth in sym33-3 mutant nodules. An increase in the levels of both thiols following GSH treatment promoted both infection and extension of defense responses in sym33-3 nodules, whereas a similar increase in sym40-1 nodules led to the formation of infected cells resembling wild-type nitrogen-fixing cells and the disappearance of an early senescence zone in the base of the nodule. Meanwhile, an increase in hGSH levels in sym40-1 nodules resulting from GSH treatment manifested as a restriction of infection similar to that seen in untreated sym33-3 nodules. These findings indicated that a certain level of thiols is required for proper symbiotic nitrogen fixation and that changes in thiol content or the GSH:hGSH ratio are associated with different abnormalities and defense responses.
A conservative characteristic of manganese superoxide dismutase is the rapid formation of product inhibition at high temperatures. At lower temperatures, the enzyme is less inhibited and undergoes more catalytic fast cycles before being product-inhibited. The temperature-dependent kinetics could be rationalized by the temperature-dependent coordination in the conserved center of manganese superoxide dismutase. As temperature decreases, a water molecule (WAT2) approaches or even coordinates Mn as the sixth ligand to interfere with O2•--Mn coordination and reduce product inhibition, so the dismutation should mainly proceed in the fast outer-sphere pathway at low temperatures. Cold-activation is an adaptive response to low temperature rather than a passive adaptation to excess superoxide levels since the cold-activated dismutase activity significantly exceeds the amount of superoxide in the cell or mitochondria. Physiologically speaking, cold activation of manganese superoxide dismutase mediates cold stress signaling and transduces temperature (physical signal) degree into H2O2 fluxes (chemical signal), which in turn may act as a second messenger to induce a series of physiological responses such as cold shock.
Garden pea (Pisum sativum L.) is a globally important legume crop. Like other legumes, it forms beneficial symbiotic interactions with the soil bacteria rhizobia, gaining the ability to fix atmospheric nitrogen. In pea nodules, the meristem is long-lasting and results in the formation of several histological zones that implicate a notable differentiation of infected host cells. However, the fine transcriptional changes that accompany differentiation are still unknown. In this study, using laser microdissection followed by RNA-seq analysis, we performed transcriptomic profiling in the early infection zone, late infection zone, and nitrogen fixation zone of 11-day-old nodules of pea wild-type line SGE. As a result, a list of functional groups of differentially expressed genes (DEGs) in different nodule histological zones and a list of genes with the most prominent expression changes during nodule development were obtained. Their analyses demonstrated that the highest amount of DEGs was associated with the nitrogen fixation zone. Among well-known genes controlling nodule development, we revealed genes that can be novel players throughout nodule formation. The characterized genes in pea were compared with those previously described in other legumes and their possible functions in nodule development are discussed.
Biological nitrogen fixation by Rhizobium-legume symbioses represents an environmentally friendly and inexpensive alternative to the use of chemical nitrogen fertilizers in legume crops. Rhizobial inoculants, applied frequently as biofertilizers, play an important role in sustainable agriculture. However, inoculants often fail to compete for nodule occupancy against native rhizobia with inferior nitrogen-fixing abilities, resulting in low yields. Strains with excellent performance under controlled conditions are typically selected as inoculants, but the rates of nodule occupancy compared to native strains are rarely investigated. Lack of persistence in the field after agricultural cycles, usually due to the transfer of symbiotic genes from the inoculant strain to naturalized populations, also limits the suitability of commercial inoculants. When rhizobial inoculants are based on native strains with a high nitrogen fixation ability, they often have superior performance in the field due to their genetic adaptations to the local environment. Therefore, knowledge from laboratory studies assessing competition and understanding how diverse strains of rhizobia behave, together with assays done under field conditions, may allow us to exploit the effectiveness of native populations selected as elite strains and to breed specific host cultivar-rhizobial strain combinations. Here, we review current knowledge at the molecular level on competition for nodulation and the advances in molecular tools for assessing competitiveness. We then describe ongoing approaches for inoculant development based on native strains and emphasize future perspectives and applications using a multidisciplinary approach to ensure optimal performance of both symbiotic partners.
Rhizobia are ecologically important, facultative plant-symbiotic microbes. In nature, there is a large variability in the association of rhizobial strains and host plants of the same species. Here, we evaluated whether plant and rhizobial genotypes influence the initial transcriptional response of rhizobium following perception of a host plant. RNA sequencing of the model rhizobium Sinorhizobium meliloti exposed to root exudates or luteolin (an inducer of nod genes, involved in the early steps of symbiotic interaction) was performed on a combination of three S. meliloti strains and three alfalfa varieties as host plants. The response to root exudates involved hundreds of changes in the rhizobium transcriptome. Of the differentially expressed genes, 35% were influenced by the strain genotype, 16% were influenced by the plant genotype, and 29% were influenced by strain-by-host plant genotype interactions. We also examined the response of a hybrid S. meliloti strain in which the symbiotic megaplasmid (∼20% of the genome) was mobilized between two of the above-mentioned strains. Dozens of genes were upregulated in the hybrid strain, indicative of nonadditive variation in the transcriptome. In conclusion, this study demonstrated that transcriptional responses of rhizobia upon perception of legumes are influenced by the genotypes of both symbiotic partners and their interaction, suggesting a wide spectrum of genetic determinants involved in the phenotypic variation of plant-rhizobium symbiosis.
IMPORTANCE A sustainable way for meeting the need of an increased global food demand should be based on a holobiont perspective, viewing crop plants as intimately associated with their microbiome, which helps improve plant nutrition, tolerance to pests, and adverse climate conditions. However, the genetic repertoire needed for efficient association with plants by the microbial symbionts is still poorly understood. The rhizobia are an exemplary model of facultative plant symbiotic microbes. Here, we evaluated whether genotype-by-genotype interactions could be identified in the initial transcriptional response of rhizobium perception of a host plant. We performed an RNA sequencing study to analyze the transcriptomes of different rhizobial strains elicited by root exudates of three alfalfa varieties as a proxy of an early step of the symbiotic interaction. The results indicated strain- and plant variety-dependent variability in the observed transcriptional changes, providing fundamentally novel insights into the genetic basis of rhizobium-plant interactions. Our results provide genetic insights and perspective to aid in the exploitation of natural rhizobium variation for improvement of legume growth in agricultural ecosystems.
Pisum sativum L. (pea) is one of the most cultivated grain legumes in European countries due to the high protein content of its seeds. Nevertheless, the rhizobial microsymbionts of this legume have been scarcely studied in these countries. In this work, we analyzed the rhizobial strains nodulating the pea in a region from Northwestern Spain, where this legume is widely cultivated. The isolated strains were genetically diverse, and the phylogenetic analysis of core and symbiotic genes showed that these strains belong to different clusters related to R. laguerreae sv. viciae. Representative strains of these clusters were able to produce cellulose and cellulases, which are two key molecules in the legume infection process. They formed biofilms and produced acyl-homoserine lactones (AHLs), which are involved in the quorum sensing regulation process. They also exhibited several plant growth promotion mechanisms, including phosphate solubilization, siderophore, and indole acetic acid production and symbiotic atmospheric nitrogen fixation. All strains showed high symbiotic efficiency on pea plants, indicating that strains of R. laguerreae sv. viciae are promising candidates for the biofertilization of this legume worldwide.
In Russia, tetramethylthiuram disulfide (TMTD) is a fungicide widely used in the cultivation of legumes, including the pea (Pisum sativum). Application of TMTD can negatively affect nodulation; nevertheless, its effect on the histological and ultrastructural organization of nodules has not previously been investigated. In this study, the effect of TMTD at three concentrations (0.4, 4, and 8 g/kg) on nodule development in three pea genotypes (laboratory lines Sprint-2 and SGE, and cultivar ‘Finale’) was examined. In SGE, TMTD at 0.4 g/kg reduced the nodule number and shoot and root fresh weights. Treatment with TMTD at 8 g/kg changed the nodule color from pink to green, indicative of nodule senescence. Light and transmission electron microscopy analyses revealed negative effects of TMTD on nodule structure in each genotype. ‘Finale’ was the most sensitive cultivar to TMTD and Sprint-2 was the most tolerant. The negative effects of TMTD on nodules included the appearance of a senescence zone, starch accumulation, swelling of cell walls accompanied by a loss of electron density, thickening of the infection thread walls, symbiosome fusion, and bacteroid degradation. These results demonstrate how TMTD adversely affects nodules in the pea and will be useful for developing strategies to optimize fungicide use on legume crops.
Drought stress is a major limitation in enhancing agricultural productivity to fulfill the food demand for the world's population. Fertigation of plants with a variety of bio-chemicals is being used to create drought resistance in wheat; however, the previous work has been limited in addressing these issues in plants at different growth stages. Therefore, a greenhouse study was conducted to ameliorate the drought stress in two wheat varieties (Chakwal-50 and Faisalabad-2008) by foliar application of 24-epibrassinolide (EBL). It was evident that drought stress had a negative effect on the growth, photosynthesis, and yield of wheat plants. EBL significantly enhanced the plant growth both under optimal and drought conditions. EBL improved the plant height, spike length, and the dry weights of roots, shoots, and grains as compared to control. Furthermore, the foliar application of EBL positively enhanced the osmolyte accumulation, increased the amounts of photosynthetic pigments, and improved the gas exchange parameters. The EBL minimized the oxidative stress by reducing elec-trolyte leakage, malondialdehyde, and hydrogen peroxide contents whereas it enhanced the activities of antioxidant enzymes, such as catalase, superoxide dis-mutase, and peroxidase under drought stress. The EBL significantly improved the level of stress hormones, such as abscisic acid, indol acetic acid, and cytokinin under drought stress. The growth response of Chakwal-50 was higher than that of Faisalabad-2008 when exposed to EBL under drought stress. Overall, the EBL plays a major role in the enhancement of growth, biomass, yield, and decrease in oxidative damage in wheat under drought conditions, however; field investigations with different doses of EBL are needed before any further recommendation.
In central Europe, soybean cultivation is gaining increasing importance to reduce protein imports from overseas and make cropping systems more sustainable. In the field, despite the inoculation of soybean with commercial rhizobia, its nodulation is low. In many parts of Europe, limited information is currently available on the genetic diversity of rhizobia and, thus, biological resources for selecting high nitrogen-fixing rhizobia are inadequate. These resources are urgently needed to improve soybean production in central Europe. The objective of the present study was to identify strains that have the potential to increase nitrogen fixation by and the yield of soybean in German soils. We isolated and characterized 77 soybean rhizobia from 18 different sampling sites. Based on a multilocus sequence analysis (MLSA), 71% of isolates were identified as Bradyrhizobium and 29% as Rhizobium. A comparative analysis of the nodD and nifH genes showed no significant differences, which indicated that the soybean rhizobia symbiotic genes in the present study belong to only one type. One isolate, GMF14 which was tolerant of a low temperature (4°C), exhibited higher nitrogen fixation in root nodules and a greater plant biomass than USDA 110 under cold conditions. These results strongly suggest that some indigenous rhizobia enhance biological nitrogen fixation and soybean yield due to their adaption to local conditions.
Fabeae legumes such as pea and faba bean form symbiotic nodules with a large diversity of soil Rhizobium leguminosarum symbiovar viciae (Rlv) bacteria. However, bacteria competitive to form root nodules (CFN) are generally not the most efficient to fix dinitrogen, resulting in a decrease in legume crop yields. Here, we investigate differential selection by host plants on the diversity of Rlv.
A large collection of Rlv was collected by nodule trapping with pea and faba bean from soils at five European sites. Representative genomes were sequenced. In parallel, diversity and abundance of Rlv were estimated directly in these soils using metabarcoding. The CFN of isolates was measured with both legume hosts. Pea/faba bean CFN were associated to Rlv genomic regions.
Variations of bacterial pea and/or faba bean CFN explained the differential abundance of Rlv genotypes in pea and faba bean nodules. No evidence was found for genetic association between CFN and variations in the core genome, but variations in specific regions of the nod locus, as well as in other plasmid loci, were associated with differences in CFN.
These findings shed light on the genetic control of CFN in Rlv and emphasise the importance of host plants in controlling Rhizobium diversity.
Background. A comparative analysis out of the structural organization of the symbiotic nodules of the pea initial line SGE and the mutant line SGECdt, characterized by increased tolerance to cadmium and increased its accumulation, was carried out.
Materials and methods.Nodules of initial line SGE and mutant SGECdt were analyzed using light and transmission electron microscopy.
Results. The non-treated nodules of SGE and SGECdt were characterized by a similar histological and ultrastructural organization. In the nodules of SGE exposed to 100 M CdCl2 in infected cells, the following abnormalities were observed: expansion of the peribacteroid space, destruction of the symbiosome membrane, fusion of symbiosomes and, as a result, the formation of symbiosomes containing several bacteroids. In the nodules of SGECdt, infected cells did not undergo pronounced changes. In the nodules of SGE exposed to 1 mM CdCl2, at the base of the nodule, senescent infected cells with completely destroyed cytoplasm and degrading bacteroids appeared. Also there were present cells in which the contents of symbiosomes were lysing, and only the ghosts of the bacteroids remained in them. In SGECdt, in some infected cells, abnormalities were manifested in an increase in the peribacteroid space, partial destruction of symbiosome membranes, fusion of symbiosomes, and release of bacteroids into the vacuole.
Conclusions. The tolerance of pea nodules to cadmium can be significantly increased due to a single recessive cdt mutation.
Biological nitrogen fixation (BNF) in legumes plays a critical role in improving soil fertility. Despite this vital role, there is limited information on the genetic diversity and BNF of bacteria nodulating common bean (Phaseolus vulgaris L.). This study evaluated the genetic diversity and symbiotic nitrogen fixation of bacteria nodulating common bean in soils of Western Kenya. The genetic diversity was determined using 16S rRNA gene partial sequences while BNF was estimated in a greenhouse experiment. The sequences of the native isolates were closely affiliated with members from the genera Pantoea, Klebsiella, Rhizobium, Enterobacter and Bacillus. These results show that apart from rhizobia, there are non-rhizobial strains in the nodules of common bean. The symbiotic efficiency (SE) of native isolates varied and exhibited comparable or superior BNF compared to the local commercial inoculants (CIAT 899 and Strain 446). Isolates (MMUST 003 [KP027691], MMUST 004 [KP027687], MMUST 005 [KP027688], KSM 001 [KP027682], KSM 002 [KP027680], KSM 003 [KP027683] and KSM 005 [KP027685]) recorded equal or significantly higher SE (p < 0.05) compared to N supplemented treatments. The results demonstrate the presence of genetic diversity of native bacteria nodulating bean that are effective in N fixation. These elite bacterial strains should be exploited as candidates for the development of Phaseolus vulgaris inoculants.
In this study we analyzed and compared the organization of the tubulin cytoskeleton in nodules of Medicago truncatula and Pisum sativum .
We combined antibody labeling and green fluorescent protein tagging with laser confocal microscopy to observe microtubules (MTs) in nodules of both wild‐type (WT) plants and symbiotic plant mutants blocked at different steps of nodule development.
The 3D MT organization of each histological nodule zone in both M. truncatula and P. sativum is correlated to specific developmental processes. Endoplasmic MTs appear to support infection thread growth, infection droplet formation and bacterial release into the host cytoplasm in nodules of both species. No differences in the organization of the MT cytoskeleton between WT and bacterial release mutants were apparent, suggesting both that the phenotype is not linked to a defect in MT organization and that the growth of hypertrophied infection threads is supported by MTs. Strikingly, bacterial release coincides with a change in the organization of cortical MTs from parallel arrays into an irregular, crisscross arrangement. After release, the organization of endoplasmic MTs is linked to the distribution of symbiosomes.
The 3D MT organization of each nodule histological zone in M. truncatula and P. sativum was analyzed and linked to specific developmental processes.
Starch content and activities of some enzymes of starch metabolism were determined in wild-type, N2-fixing (fix+) nodules and in two non-N2-fixing (fix−) nodules induced by Bradyrhizobium japonicum mutant strains, T5-95 and T8-1, on soybean (Glycine max L.) roots. The T5-95 nodules are similar to wild type in ultrastructure, but the T8-1 nodules are different in that the bacteroids are not released from the infection thread. After initial accumulation to relatively high concentration, starch was depleted during nitrogen fixation in fix+ nodules. However, in fix− nodules, the accumulated starch was not metabolized. The activity of starch-bound starch synthase (EC 2.4.1.21) declined in fix+ nodules but remained high in fix− nodules. The activity of α-amylase (EC 3.2.1.1) was only slightly higher than wild type in T5-95 but was four times higher than wild type in T8-1 nodules. The activity of starch phosphorylase (EC 2.4.1.1) increased in all nodule types from 14 to 21 days postinfection. A positive correlation was observed between the capacity of nodules to fix N2 and their capacity to degrade starch. Collectively, these results support the concept that starch accumulated during early stages of nodule development is metabolized to supply energy for nitrogen fixation and to meet the metabolic demands of bacteroids. Key words: nitrogen fixation, starch content, effective and ineffective nodules, starch synthase, starch phosphorylase, α-amylase.
Screening of Rhizobium leguminosarum bv. phaseoli strains showed some that were able to nodulate common beans (Phaseolus vulgaris L.) at high temperatures (35 and 38°C/8 h/day). The nodulation ability was not related to the capability to grow or produce
melanin-like pigment in culture media at high temperatures. However, nodules formed at high temperatures were ineffective
and plants did not accumulate N in shoots. Two thermal shocks of 40°C/8 h/day at flowering time drastically decreased nitrogenase
activity and nodule relative efficiency of plants otherwise grown at 28°C. Recovery of nitrogenase activity began only after
seven days, when new nodules formed; total incorporation of N in tops did not recover for 2 weeks. Non-inoculated beans receiving
mineral N were not affected by the thermal shock, and when growing continuously at 35 or 38°C had total N accumulated in shoots
reduced by only 18%.
Biological N(2) fixation represents the major source of N input in agricultural soils including those in arid regions. The major N(2)-fixing systems are the symbiotic systems, which can play a significant role in improving the fertility and productivity of low-N soils. The Rhizobium-legume symbioses have received most attention and have been examined extensively. The behavior of some N(2)-fixing systems under severe environmental conditions such as salt stress, drought stress, acidity, alkalinity, nutrient deficiency, fertilizers, heavy metals, and pesticides is reviewed. These major stress factors suppress the growth and symbiotic characteristics of most rhizobia; however, several strains, distributed among various species of rhizobia, are tolerant to stress effects. Some strains of rhizobia form effective (N(2)-fixing) symbioses with their host legumes under salt, heat, and acid stresses, and can sometimes do so under the effect of heavy metals. Reclamation and improvement of the fertility of arid lands by application of organic (manure and sewage sludge) and inorganic (synthetic) fertilizers are expensive and can be a source of pollution. The Rhizobium-legume (herb or tree) symbiosis is suggested to be the ideal solution to the improvement of soil fertility and the rehabilitation of arid lands and is an important direction for future research.
Rhizobium laguerreae is regarded as a promising candidate for biofertilization of legume plants worldwide through its high efficiency in symbiosis. In this paper, we report high-quality sequences of six R. laguerreae strains with total genome completeness from 93.5% to 97.5%.
In this work, we used MALDI-TOF MS for identification of strains nodulating Pisum sativum L. (peas)
in soils from Valladolid, Salamanca and León where the 30 % of the total Spanish production of peas is located. The results obtained showed a correct identification, with score values higher than 2.0, of strains isolated in the soil from León as Rhizobium leguminosarum and those isolated in soils from Valladolid and Salamanca as Rhizobium laguerreae. These results confirmed that MALDI-TOF MS is a reliable technique for identification at species level of fast-growing rhizobia. They also showed that this methodology is able to differentiate between sister species, not distinguishable on the basis of 16S rRNA gene analysis, as occurs in the case of R. leguminosarum and R. laguerreae.
Trifolium subterraneum plants grown at 30°C root temperature nodulated 3 days after inoculation with Rhizobium trifolii and nitrogenase activity (determined by acetylene-reduction) was detected 3 days later. While all four strains of R. trifolii examined formed effective nodules at 22°C, at 30°C only the nodules formed by strain TAI showed a rapid increase in nitrogenase activity. Light and electron microscopy of the nodules formed by one temperature-sensitive strain (NA30) showed the following abnormalities: multiple branching and distortion of the infection threads; failure of many bacteria to develop into characteristic bacteroids; continued division of these bacteria to give atypical multiple occupancy of the membrane envelopes; release of polysaccharide from ruptured infection threads into the host cytoplasm; rapid degeneration of the membrane envelopes and their contents. While the development of the membrane envelopes appeared to be under host control, the effect of the higher temperature on bacteroid development was highly strain specific.
Upon transfer of nodulated plants from 22 to 30°C, bacteroid tissue in nodules formed by the temperature-sensitive strain NA30 broke down rapidly but nodules formed by strain TAI were less severely affected.
The nodulation and growth of cowpea plants (Vigna sinensis Endl. ex Hassk. var. Poona), grown in the CERES phytotron glasshouses, were examined. The plants were grown under six controlled temperature regimes (21, 24, 27, 30, 33, or 36°C day temperature) with ammonium nitrate (NH4N03) additions of 0, 10, 30, and 90 mg nitrogen per pot of six plants; and were grown either under natural daylight or under natural daylight reduced in intensity by one-third by mesh screens. Primary root nodulation was significantly affected by temperature, NH4NO3 level, and light intensity, with an optimum temperature of 24°C. Secondary root nodulation was also affected by light intensity and temperature, but the nodulation pattern (with an optimum temperature of 33°) was almost the inverse of the primary root pattern. Temperature significantly influenced the fresh weight of nodules per plant and the nodule size, with a lesser effect of the NH4NO3 level. Plant dry weight production (tops and roots) was governed by the temperature, NH4NO3 level, and light intensity, the maximum total dry weight being produced at 27°C. Temperature, light intensity, and NH4NO3 level all influenced the plant combined- nitrogen uptake. The nodule leghaemoglobin concentration was significantly reduced at the two extreme temperatures 21° and 36°C. Temperature also markedly affected the nodule structure – particularly the distribution of starch within the nodule.
Several new varieties of marrowfats, dun peas and maple peas were bred by Dr R. J. Mansholt, Westpolder, Ir C. Koopman and the Plant Breeding Station C.B. (Dir. Ir C. Koopman), Hoofddorp, over a period of years.The parentage of the varieties figures in the diagrams 1–3, while the varietal range of all the agricultural peas grown in the Netherlands during the years 1934–1954 were copied from the Descriptive List of Varieties of Field Crops (Table 1).The cultivation of round blue peas in the Netherlands is based on varieties of domestic plant breeders and this is also the case with the growing of marrowfats, dun peas and maple peas. The Netherlands List of Varieties mentions only one yellow pea which is, moreover, exclusively cultivated for export.In de loop der jaren zijn tal van rassen van schokkers, capucijners en rozijnerwten gekweekt door Dr R. J. Mansholt te Westpolder, Ir C. Koopman en het Veredelingsbedrijf C.B. (Dir.: Ir C. Koopman) te Hoofddorp. Gebr. Kool te Andijk en het Proefstation voor de Groenteteelt in de Vollegrond te Alkmaar legden zich toe op de veredeling van de rozijnerwten.De afstamming van de rassen is aangegeven in de figuren 1–3, terwijl de rassenstatistiek van alle in Nederland verbouwde landbouwerwten over de jaren 1934–1954 aan de Beschrijvende rassenlijst voor landbouwgewassen werd ontleend (tabel 1). Evenals de teelt van groene erwten in Nederland gebaseerd is op rassen van eigen kwekers, is dit ook het geval met de teelt van schokkers, capucijners en rozijnerwten. De Nederlandse rassenlijst vermeldt slechts 1 gele erwt, die bovendien uitsluitend voor export wordt geteeld.
1.
Nodule structure was examined in the red clover cultivar S123 and in lines (designated H2) separately bred for high yield withRhizobium trifolii strains RCR 0403 and RCR 5. Plants were grown at 16/11, 22/17 and 27/22C in a 16 h day at 25,000 lux.
2.
The larger yields of bred lines compared with S123 were directly correlated with larger aggregate nodule size, larger infected zones and with more bacteroid tissue. Over all treatments the correlation of plant dry weight with aggregate areas of bacteroid tissue, assessed in median longitudinal section, accounted for 79% of the variance.
3.
Yields were unrelated to the proportion of uninfected cells and vacuoles within the bacteroid zone.
4.
Effects of host type and strain on nodule structure and yield were greatest at the moderate temperature and least and most irregular at high temperature.
5.
Bacteriod tissue degenerated more rapidly in S123 than in the bred lines, more rapidly with RCR 0403 than RCR 5 and more rapidly at the high temperature than at moderate or low temperatures.
6.
Aggregate nodule size irrespective of treatment was correlated with the aggregate sizes of nodule meristem and differentiating tissues. Nodule comtex comprised a larger proportion of the nodule section at 17 days than subsequently. Other than as specified above, the size relationships of the different tissues of the nodule were unaffected by host selection, temperature, bacterial strain or plant age.
7.
At 22/17C over a 17–25 day period the increment in dry matter per mm2 active nodule tissue was similar for H2 and S123 plants and was less for RCR 0403 (3.33 mg) than for RCR 5 (6.87 mg). At 27/22C the effeciency of bacteroid tissue was much less,viz. 0.67 and 1.23 mg per mm2 respectively.
8.
Nodule tissue areas and volumes were closely related so that unit volumes of active bacteriod tissue in general promoted similar dry matter increment in S123 and bred lines,viz. about 50.5 mg mm–3 at 22/17 for RCR 0403 and 80.8 mg mm–3 for RCR 5. The latter was associated with a fixation rate of 0.157 mg N mm–3 active tissue day–1.
In short-season soybean production areas, low soil temperature is the major factor limiting plant growth and yield. The decreases in soybean yield at low temperatures are mainly due to nitrogen limitation. Genistein, the most effective plant-to-bacterium signal in the soybean (Glycine max (L.) Merr.) nitrogen fixation symbiosis, was used to pretreat Bradyrhizobium japonicum. We have previously reported that this increased soybean nodulation and nitrogen fixation in growth chamber studies. Two field experiments were conducted on two adjacent sites in 1994 to determine whether the incubation of B. japonicum with genistein, prior to application as an inoculant, or genistein, without B. japonicum, applied onto seeds in the furrow at the time of planting, increased soybean grain yield and protein yield in short season areas. The results of these experiments indicated that genistein-preincubated bradyrhizobia increased the grain yield and protein yield of AC Bravor, the later maturing of the two cultivars tested. Genistein without B. japonicum, applied onto seeds in the furrow at the time of planting also increased both grain and protein yield by stimulation of native soil B. japonicum. Interactions existed between genistein application and soybean cultivars, and indicated that the cultivar with the greatest yield potential responded more to genistein addition.
The formulation and commercial production of any Rhizobium or Bradyrhizobium legume inoculant requires the integration of physical, chemical and biological parameters leading to both high target organism populations and long term survival of these organisms over time at less than optimum conditions. To achieve this, all raw materials processing and bacteriological needs must be addressed, and quality products developed within narrow budgetary constraints. It is only through an understanding of the needs and limitations of both the products physical and Rhizobium/Bradyrhizobium requirements that an effective and efficient legume inoculant can be commercially produced.
The rhizobia-legume symbiosis is the main source of fixed nitrogen for many agricultural systems. However, it is inhibited by low soil temperature. To date, research on nodulation has involved either qualitative or destructive analyses. The use of computer-based image analysis potentially allows nodules to be followed during the course of development. Seedlings of bean (Phaseolus vulgaris L.), lentil (Lens culinaris Medik.) and pea (Pisum sativum L.) were transplanted into plastic growth pouches suspended in water baths maintained at 10, 15, 20 or 25 8 C. Two days after transplanting, all plants were inoculated with appropriate rhizobial strains. Seven days after inoculation, plant roots were scanned; this was repeated weekly for 7 weeks. Data on nodule length were collected through image analysis. Nodule length was correlated with nodule size and development. There were increases in the precision of estimates of environmental effects through observation of individual nodule development, as opposed to averages for populations of nodules. The effects of root temperature on nodulation and nodule development were observed both in the delayed onset of nodulation and in reduced subsequent nodule growth rate, resulting in effects on final nodule size.
A study has been made concerning the effect of high temperatures on the symbiosis of Rhizobium leguminosarum and pea plants (Pisum sativum). At 30°C, no nodules were found on the roots of plants growing in nutrient solution after inoculation with the appropriate bacteria. This is in contrast to the ready nodulation at lower temperatures, and to the observation that at the high temperature both partners of the symbiosis, when growing separately, develop normally, provided that they dispose of adequate amounts of combined nitrogen.From the results of experiments recorded in Chapter 3 it was concluded that the inhibition of nodulation by high temperatures occurs only when the sites of nodulation are exposed to such temperatures. Incubation of other parts of the plant at 30 °C (Table 1), or incubating the entire plant during several days before inoculation at that temperature, had no effect.The unfolding of the first true leaf of pea plants was found to be correlated with the onset of the liability of the root system to infection by R.leguminosarum. By blackening root systems with charcoal at different times, it was found that nodulation occurred usually on parts of the roots that were formed from one day before until 3-4 days after inoculation (Figs. 7, 8, 9. Table 2).An inventarisation of morphological characters of pea roots, affected by high temperatures and by the presence of rhizobia, is given in Chapter 4. Numbers of nodules were found to be dependent not only on temperature, but also on unknown factors that could not be controlled (Table 4a). To a lesser extent, the same was true of the nodulation time (Table 4b). For this reason. the main part of the present investigation was restricted to a comparison of nodulation phenomena at two temperatures viz. 22 °C at which nodulation took place readily, and 30°C at which usually no nodules were formed. Root-growth rate and formation of lateral roots were found to be affected by temperature as well as by the presence of rhizobia (Tables 5 and 6, Fig. 10).By transferring pea plants from 22 to 30°C at different times after inoculation, the temperature sensitivity of various stages of nodule formation was ascertained. Nodulation was found to be suppressed when the 22°C-incubation ended at 3 days after inoculation, but a number of nodules were formed when the 22°C-period was terminated at 4 days, demonstrating that between 3 and 4 days after inoculation, a highly temperature-sensitive stage of nodulation was terminated (Fig. 11). Although nodule initiations beyond this stage developed to nodules at 30°C, the size of the latter remained considerably smaller than those of control plants incubated continuously at 22°C (Fig. 14), whereas leghaemoglobin and acetylene-reducing activity did not develop (Table 8). This demonstrates that in addition to the main temperature-sensitive stage, later stages of nodulation are adversely affected by high temperatures.Chapter 6 contains the results of experiments in which it was shown that enhancement of the photosynthetic activity of pea plants (by increasing the light intensity or by extending the illumination period) raised the probability of such plants of being nodulated when growing at moderately high temperatures (Fig. 18). The same effect was obtained by inserting a diurnal period at 22 °C, particularly during the dark period, which decreases the respiratory activity and consequently enhances the carbohydrate content of the plants (Table 9).In Chapter 7 experiments on the first contact between R. leguminosarum and pea roots are recorded. Attachment of the rhizobia to pea roots was found to be adversely affected by high temperatures (Table 14). Once attached to the root, rhizobial growth in the rhizosphere at 30°C, is faster than at 22°C (Table 16).Chapter 8 contains the results of experiments with antibiotics added to R. leguminosarum -pea plant associations at different periods after inoculation to find out at what time the bacteria were no more susceptible to the antibiotics. This was found to be the case at 3-4 days after inoculation (Table 15), i.e. the time at which the main temperature-sensitive period ended (Fig. 11).The main effect of high temperatures during the first 3 days after inoculation is connected with root-hair formation and root-hair deformation (Chapter 9). Pea plants at 22°C form many root hairs but deformations do not occur (Fig. 22a). R. leguminosarum and also other Rhizobium species tested stimulate root-hair development and deformation of root hairs (curling, branching) (Figs. 23a to 27a, Table 17) which precede infection. This depends on the formation by the rhizobia of a dialysable compound (Fig. 42). Pea plants without rhizobia, incubated at 30°C, were found to form no root hairs (Fig. 22b, Table 17). In the presence of R. leguminosarum or a number of other Rhizobium species, root hairs at 30 °C were formed in relatively large numbers, but then they had an abnormal, spheriod shape (Figs. 23b to 27b) and did not give rise to nodule formation.In conclusion, it can be stated that a number of processes are involved in causing the adverse effect of high temperatures on nodulation and nitrogen fixation of the Rhizobium -pea association. Interference with the formation and deformation of root hairs in the presence of an established flora of R. leguminosarum was shown to be responsible for the absence of nodules. Reduced growth of the nodules, absence of leghaemoglobin formation, absence of nitrogenase activity (both characters probably being linked) are further important processes, adversely affected by high temperatures.
Bacteroid differentiation was examined in developing and mature alfalfa nodules elicited by wild-type or Fix- mutant strains of Rhizobium meliloti. Ultrastructural studies of wild-type nodules distinguished five steps in bacteroid differentiation (types 1 to 5), each being restricted to a well-defined histological region of the nodule. Correlative studies between nodule development, bacteroid differentiation, and acetylene reduction showed that nitrogenase activity was always associated with the differentiation of the distal zone III of the nodule. In this region, the invaded cells were filled with heterogeneous type 4 bacteroids, the cytoplasm of which displayed an alternation of areas enriched with ribosomes or with DNA fibrils. Cytological studies of complementary halves of transversally sectioned mature nodules confirmed that type 4 bacteroids were always observed in the half of the nodule expressing nitrogenase activity, while the presence of type 5 bacteroids could never be correlated with acetylene reduction. Bacteria with a transposon Tn5 insertion in pSym fix genes elicited the development of Fix- nodules in which bacteroids could not develop into the last two ultrastructural types. The use of mutant strains deleted of DNA fragments bearing functional reiterated pSym fix genes and complemented with recombinant plasmids, each carrying one of these fragments, strengthened the correlation between the occurrence of type 4 bacteroids and acetylene reduction. A new nomenclature is proposed to distinguish the histological areas in alfalfa nodules which account for and are correlated with the multiple stages of bacteroid development.
In nitrogen-poor soils, rhizobia elicit nodule formation on legume roots, within which they differentiate into bacteroids that fix atmospheric nitrogen. Protection against reactive oxygen species (ROS) was anticipated to play an important role in Rhizobium-legume symbiosis because nitrogenase is extremely oxygen sensitive. We deleted the sodA gene encoding the sole cytoplasmic superoxide dismutase (SOD) of Sinorhizobium meliloti. The resulting mutant, deficient in superoxide dismutase, grew almost normally and was only moderately sensitive to oxidative stress when free living. In contrast, its symbiotic properties in alfalfa were drastically affected. Nitrogen-fixing ability was severely impaired. More strikingly, most SOD-deficient bacteria did not reach the differentiation stage of nitrogen-fixing bacteroids. The SOD-deficient mutant nodulated poorly and displayed abnormal infection. After release into plant cells, a large number of bacteria failed to differentiate into bacteroids and rapidly underwent senescence. Thus, bacterial SOD plays a key protective role in the symbiotic process.
A simple glass slide technique has been devised for the continuous microscopical observation of growth and infection of root hairs of clover seedlings. The method involves an aseptic cultivation of seedlings on microscope slides which are partly immersed in a mineral salts medium. The roots are protected by a cover- slip. By this procedure, the root hairs of white clover inoculated with nodule bacteria were studied. The earliest infection was observed to take place within 48 hr. of inoculation, on 4-day-old seedlings. In branched hairs the growth of the thread from a lateral branch towards the hair tip is tentatively explained as an effect of the position of the hair nucleus relative to the site of infection. The infection of leguminous plants by nodule bacteria has, as a rule, been studied with fixed and sectioned material. The first stages of this process may also be studied in vivo, but no suitable technique has yet been described for a continuous microscopical examination of these stages. A method for the study of growing root hairs (of wheat) was devised by Lundegkrdh (1946) and modified by Ekdahl(1953). In their work, a chamber containing flowing salt solution and resting on a microscope stage was em- ployed. However, difficulties arise with this method if aseptic conditions are also necessary. The present author has therefore used a technique for the aseptic cultivation of young seedlings on microscope slides in a manner which permits periodic observations under the microscope of the growth and infec- tion of individual root hairs. The arrangement also offers good conditions for photomicrography . The details of the method are as follows.
Effects of 24-epibrassinolide on plant growth, antioxidants defense system, and endogenous hormones in two wheat varieties under drought stress
- I Khan
- S A Awan
- R Ikram
- M Rizwan
- N Akhtar
- H Yasmin
- R Z Sayyed
- S Ali
- N Ilyas
- I. Khan