Page 242. Chapter 11 Enhancement of RhizobiaĀLegumes Symbioses and Nitrogen Fixation for Crops Pr... more Page 242. Chapter 11 Enhancement of RhizobiaĀLegumes Symbioses and Nitrogen Fixation for Crops Productivity Improvement Hamdi Hussein Zahran Abstract Rhizobia form a very interesting symbiotic relationship with leguminous plants. ...
International microbiology : the official journal of the Spanish Society for Microbiology, 2013
Fifty four bacterial strains were isolated from root nodules of the grain legumes Cicer arietinum... more Fifty four bacterial strains were isolated from root nodules of the grain legumes Cicer arietinum, Lens esculentus, Phaseolus vulgaris, Pisum sativum, and Vicia faba grown in cultivated lands of Beni-Suef Governorate (Egypt). Repetitive extragenic palindromic (REP)-polymerase chain reaction (PCR) clustered the strains into 15 REP-PCR groups. The nearly complete sequence of the 16S rRNA gene from a representative strain of each REP-PCR pattern showed that the strains were closely related to members of the family Rhizobiaceae of the Alphaproteobacteria. Pairwise alignments between globally aligned sequences indicated that the strains from V. faba had 99.6% identity with Rhizobium leguminosarum, and those from P. vulgaris 99.76% and 100% with sequences from R. leguminosarum and R. mesosinicum, respectively. Strains from P. sativum had 99.76%, 99.84%, and 99.92% sequence identity with R. leguminosarum, R. etli, and R. pisi, respectively, and those from L. esculentus had 99.61% identity ...
A different combined effects for helium gas discharge such as: magnetic field strength, breakdown... more A different combined effects for helium gas discharge such as: magnetic field strength, breakdown voltage, applied power, applied pressure, cathode fall thickness, edge effect, distribution of the electron temperature and density, and finally exposure time for Staphylococcus aureus substrate over slides at the cathode edge, are discussed under the influence of cold, nonthermal plasmas, ultra low pressure, and presence of the magnetic field for disinfection of bacteria for short exposure times, compatible to International Commission on Non-Ionizing Radiation Protection, Health Phys (ICNIRP) for healing applications. Furthermore, analyses of the experimental data of initial and final densities of cells alive, using survival curves, showed an impressive inhibitory effect of plasma discharge to the remaining survival of bacterial ratio under the influence of the magnetic field.
... For example, 15 isolates of S. meliloti recovered from nodules of wild species of ... associa... more ... For example, 15 isolates of S. meliloti recovered from nodules of wild species of ... association with a wide range of rhizobia (Rhizobium, Mesorhizobium, and Sinorhizobium), welladapted to ... The high sensitivity of the legumeĀRhizobium symbiosis to salinity has been recognized ...
World Journal of Microbiology & Biotechnology, 1994
The effects of osmotic and heat stress on lipopolysaccharides and proteins of rhizobia isolated f... more The effects of osmotic and heat stress on lipopolysaccharides and proteins of rhizobia isolated from the root nodules of leguminous trees grown in semi-arid soils of the Sudan, and of agricultural legumes grown in salt-affected soils of Egypt, were determined by SDS-PAGE. The rhizobia were of three types: (1) sensitive strains, unable to grow in 3% (w/v) NaCl in yeast mannitol medium; (2) tolerant strains which could grow in 3% (w/v) NaC1; and (3) halophytic strains which grew with 3 to 10% (w/v) NaCl. The sensitive strains changed their gel pattern or the amount of lipopolysaccharide they synthesized when grown in 1% (w/v) NaC1. The tolerant and halophytic strains often modified their lipopolysaccharides in 3% NaCl, which was evident by a shift in the banding patterns towards longer chain length. Similar effects were observed in cells incubated with sucrose and, to a lesser extent, in cells incubated at growth temperatures near the recorded maximum temperature for growth. The stress-induced changes in lipopolysaccharides were not associated with specific banding patterns of the lipopolysaccharides. During incubation in medium containing elevated concentrations of NaCI or sucrose, the protein patterns of the rhizobia were also changed. A protein with relative mobility of 65 kDa appeared during temperature stress. The maximum growth temperature of the Sudanese rhizobia were up to 44.2Ā°C.
The effects of sodium chloride and polyethylene glycol (PEG) on the interaction between Rhizobium... more The effects of sodium chloride and polyethylene glycol (PEG) on the interaction between Rhizobium leguminosarum strain 29d and root hairs of field bean (Viciafaba L. cv. Maris Bead) plants were investigated. Two levels each of NaC1 (50 and 100 mol.m -3) and PEG (100 and 200 mol.m -3) were given at the time of root-hair formation. Scanning electron microscopy showed rhizobial attachment and colonization on root-hair tips. Adhesion of rhizobia in both lateral and polar orientation, sometimes associated with microfibrils, occurred mainly in crooks at the root-hair tips; most of the infections also occurred here. Bacterial colonization and root-hair curling were both reduced by stress treatments. Polyethylene glycol but not NaC1 significantly reduced root-hair diameter. The proportion of root hairs containing infection threads was reduced by 30% under NaC1 and by 52% under PEG. The structure of some of the root hairs, epidermal and hypodermal cells, as seen by light microscopy in ultrasections, was distorted as a result of NaC1 and PEG treatments; cells showed plasmolysis and folded membranes. After three weeks of treatment, both NaC1 and PEG inhibited nodule number by about 50% and nodule weight by more than 60%. It is concluded that the root-hair infection process in Vicia faba is impaired by NaC1 and PEG treatments and this in turn results in fewer nodules being produced.
Medicago intertexta and Melilotus indicus, two wild leguminous herbs with different tolerance to ... more Medicago intertexta and Melilotus indicus, two wild leguminous herbs with different tolerance to salinity were investigated for NaCl-induced changes in the expression level of some Na 1 transporters. M. indicus plants grew well at NaCl concentration from 0 to 400 mM, whereas growth of M. intertexta plants was severely inhibited at NaCl concentrations higher than 100 mM. In M. intertexta, increasing NaCl in the growth media caused a strong increase in Na 1 content concomitant with a decrease in K 1 content in leaves and, above all, roots. In comparison, M. indicus plants cultivated in the presence of NaCl accumulated much less Na 1 in leaves and roots and no differences in K 1 content among plants grown in nutrient solution containing 100-400 mM NaCl were detected. The expression levels of four genes coding for NHX-type Na 1 /H 1 antiporters in the above two wild legumes were studied in plants cultivated under the different NaCl concentrations. Expression levels of the genes were higher in M. intertexta as compared with M. indicus plants. In M. intertexta, salt treatments increased MtNHX1, MtNHX3 and MtNHX4 transcript levels in leaves and roots. However, in M. indicus NaCl treatments only induced the expression of MtNHX1 in roots. Our data suggest that two different mechanisms, Na 1 avoidance or accumulation into cellular compartments, are developed by the two wild legumes to cope with salt stress, and that expression of NHX antiporters is linked to the accumulator phenotype.
Inactivating viable cells at very short application times has been studied using Ultra Low Freque... more Inactivating viable cells at very short application times has been studied using Ultra Low Frequency Plasma (ULFP) at one Kilo Hertz, using an RF source. The targeted fashion is to inactivate Escherichia coli (E. coli ) in the absence and in the presence of magnetic field. Adding oxygen (O 2) to argon (Ar) in the discharge leads to a complete bacterial inactivation, where the inactivation rate increased as the concentration of O 2 increases. Analyses of the experimental data of the initial and final densities of viable cells, using survival curves, showed a dramatic inhibitory effect of plasma discharge to the residual survival of microbial ratio due to the influence of the magnetic field.
Wild legumes (herb or tree) are widely distributed in arid regions and actively contribute to soi... more Wild legumes (herb or tree) are widely distributed in arid regions and actively contribute to soil fertility in these environments. The N 2 -fixing activity and tolerance to drastic conditions may be higher in wild legumes than in crop legumes. The wild legumes in arid zones harbor diverse and promiscuous rhizobia in their root-nodules. Specificity existed only in few rhizobia from wild legumes, however, the majority of them are with wide host range. Based on phenotypic characteristics and molecular techniques (protein profiles, polysaccharides, plasmids, DNA-DNA hybridization, 16SrRNA, etc.), the root-nodule bacteria that was isolated from wild legumes had been classified into four genera (Rhizobium, Bradyrhizobium, Mesorhizobium and Sinorhizobium). The rhizobia of wild legumes in arid zones, exhibit higher tolerance to the prevailing adverse conditions, e.g. salt stress, elevated temperatures and desiccation. These rhizobia may be used to inoculate wild, as well as, crop legumes, cultivated in reclaimed desert lands. Recent reports indicated that the wild-legume rhizobia formed successful symbioses with some grain legumes. Moreover, intercropping of some N 2 -fixing tree legumes (e.g. Lablab, Leucaena, Sesbania, etc.) to pasture grasses improved biomass yield and herb quality. In recent years, the rhizobia of wild legumes turn the attention of biotechnologists. These bacteria may have specific traits that can be transferred to other rhizobia through genetic engineering tools or used to produce industrially important compounds. Therefore, these bacteria are very important from both economic and environmental points of view.
Salt tolerance of five rhizobia strains was examined in broth cultures. Five levels of NaCl conce... more Salt tolerance of five rhizobia strains was examined in broth cultures. Five levels of NaCl concentration were used and the optical density was taken as a measure for the vigour of bacterial growth. Rhizobium leguminosarum and R. meliloti were tolerant to high levels of salinity and growth curves in saline broth showed a similar pattern to the control level. Rhizobium japonicum, cowpea Rhizobium, and R. trifolii were intolerant to salt and showed a strong growth retardation with increasing salt concentration. Growth was inhibited at high levels of salinity. It is suggested that rhizobia sensitivity to salts may be partly responsible to the inhibition of nitrogen fixation by legumes growing under salt stress.
Some characteristics of thirty isolates of root-nodule bacteria. indigenous in the salt-affected ... more Some characteristics of thirty isolates of root-nodule bacteria. indigenous in the salt-affected soil of Egypt, were studied. To recognize different isolates of these bacteria, the electrophoretic pattern of plasmids and of whole cell lipopolysaccharides (LPS) and proteins were analysed and compared using agarose gel electrophoresis (AGE) and sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE). respectively. The bacteria showed patterns of LPS, protein and plasmid which varied among isolates of different plant species as among isolates of a single plant species. Based on LPS and protein profile analysis, isolates from root-nodules of broadbean and lupin plants were more diverse than isolates from root-nodules of berseem plant. Root-nodule bacteria of lupin and berseem were also less diverse than root-nodule bacteria of broadbean when comparcd on the basis of plasmid profiles. This result indicate that root-nodule bacteria of broadbean may have higher surviving ability in the salt-affected soil. Most isolates of the root-nodule bacteria synthesized specific LPS (smooth, ladder-like type) and protein. which were different from LPS and protein of other well-known (recognized) Rhizobium species. These specific forms of LPS and protein could be a prerequisite for the bacterial isolates to tolerate salt stress conditions of the salt-affected soils.
Twenty out of 400 isolates of bacteria mainly from salt marshes and saline soils of Egypt were su... more Twenty out of 400 isolates of bacteria mainly from salt marshes and saline soils of Egypt were successfully grown on mannitol N-free medium. The nitrogen-fixing activity was then demonstrated for the twenty isolates in modified STANIER'S medium using the acetylene reduction assay. All of them possessed appreciable nitrogenase activity (acetylene reduction) under non-saline conditions ; however, at 5% NaCl only 60% of the isolates exhibited a high rate of this activity and 25% were completely negative under these conditions. The bacterial isolates grew well in STANIER'S medium; nevertheless, growth of the majority of isolates was reduced by about 30-80% in the same medium containing 5 % NaCl. Cellulolytic activity was detected in 60% of the twenty strains, amylolytic in 45%, and pectinolytic in 10% of the isolates. The bacterial isolates showed also enzymatic activity under saline conditions (5 % NaCl). The preliminary identification indicated that six isolates were Gram positive spore-forming bacteria of the genus Bacillus, the others were Gram negative rods which remain to be identified.
Members of Bricillcicecir, Rlzi=ohioceae, actinomyeetes and others were isolated from cultivated ... more Members of Bricillcicecir, Rlzi=ohioceae, actinomyeetes and others were isolated from cultivated and non-cultivated saline soils. The high population of bacteria and actinomycetes were almost coincided with the relatively high levels of organic matter whatever the degree of soil salinity.
The effects of application of combined nitrogen fertilizer (ammonium nitrate or urea) on root-hai... more The effects of application of combined nitrogen fertilizer (ammonium nitrate or urea) on root-hair infection and nodulation of four grain legumes were studied. Young roots of each legume were inoculated with their compatible rhizobia. The application of the two forms of combined N either at the early stages of plant growth and/or at the time of nodule formation depressed root-hair curling, infection and nodulation. Infection of hairs on the primary roots was more sensitive to the N fertilizer than hair infection of secondary roots in both l, Tcia faba and Pisum sativum. The nodule number and total fresh mass of the four legumes were drastically affected by fertilizer application. The combined N added both at early and at later stages significantly reduced the nodulation of V. faba, Phaseolus vulgaris and F/gna s/nens/s. The inhibitory effect of urea on nodulation of P. sativum was only observed when the fertilizer was applied at the late stages of plant growth. It is concluded that, although the nodulation of the four legumes was suppressed by combined N, the initial events of Rhizobium-legume symbiosis (infection of roots and nodule initiation) are more sensitive to combined N than the stages after nodule formation.
Plant needles of Casuarina equisetifolia were collected and analyzed in parallel with soil analys... more Plant needles of Casuarina equisetifolia were collected and analyzed in parallel with soil analysis. In three strains of Frankia -symbionts of Casuarina -their infectivity and plant performance was determined in vitro after soil amendment with different leaf litter concentrations. Only one strain was able to nodulate the plant at all litter concentrations (0.5, 3 and 5 %) although the nodules were very small. However, all treated plants grew poorly; their growth was reduced by approximately 90 % (for 5 % litter concentration) compared to plants grown on untreated soil, on the basis of total dry mass. Inhibition of nodulation can be attributed to high concentrations of some elements and compounds that were either found in C. equisetifolia litter or originally found in soil (i.e. chloride, cyanide, copper, manganese and phenols). In general, plant growth decreased as more litter was added. Plant total nitrogen content was also reduced after increasing the litter concentration. The inhibitory effect of high litter concentrations was mainly on plant growth and to a lesser extent on plant nodulation by Frankia strains. 430 W.F. SAYED et al.
A total of about 50 strains of rhizobia from two leguminous trees (Acacia and Prosopis) were desc... more A total of about 50 strains of rhizobia from two leguminous trees (Acacia and Prosopis) were described and compared with 20 reference strains of rhizobia from other tree and herb legumes on the basis of protein, fatty acid and plasmid profiles, and DNA-DNA hybridization. The rhizobia formed thirteen clusters based on protein profile analysis. These clusters were not in complete agreement with a previously published cluster analysis based on numerical taxonomy of phenotypic characteristics and lipopolysaccharide (LPS) profile analysis (Zhang et al., Int~.Syst.Bacteriol. 41, 104, 1991; Lindstr6m and Zahran, FEMS Microbial.Lett. 107, 327, 1993). The fatty acid methyl esters (FAME) of representative strains of rhizobia were analyzed.
The DNA-DNA hybridization was used to characterize thirty isolates of root-nodule bacteria indige... more The DNA-DNA hybridization was used to characterize thirty isolates of root-nodule bacteria indigenous to the salt-affected soils of Egypt. Total DNA from different bacterial isolates lacked homology with total DNA probes of the effective strains of Rluzobium leguminosarum and R. meliloti. It is suggested that the genomic structure of the root-nodule bacteria may be modified by salt stress and/or that the effective strains of these bacteria are to be eliminated from the salt-affected soil.
About 20 strains of rhizobia from wild legumes were characterized based on numerical analysis of ... more About 20 strains of rhizobia from wild legumes were characterized based on numerical analysis of phenotypic characteristics, nodulating ability, fatty acid methyl esters (FAME) and SDS-PAGE profiles of whole cell proteins. FAME analysis revealed that palmitic (16:0), stearic (18:0) and arachidonic (20:0) were detected in most of wild-legume rhizobia, the latter being uncommon in fatty acid profiles of Rhizobium and Sinorhizobium. Numerical analysis of FAME classified strains of wild-legume rhizobia into 9 clusters and one heterogeneous group. There was both agreement and disagreement with the clustering data based on phenotypic analysis and FAME analysis. Four strains were grouped together in the same cluster based on both methods. However, 4 another strains, which were placed in one cluster of phenotypic analysis, were distributed in several clusters after FAME analysis. SDS-PAGE of whole-cell proteins revealed that the rhizobial strains exhibited protein profiles with peptide bands ranging from 5-19 band per profile and showed molar mass of 110-183 kDa. As in the case of FAME analysis, numerical analysis of protein bands was compared with clustering of phenotypic analysis. Agreement of the two methods was obvious when clustering some strains but conflicted in the classification of some other strains. However, integration of the three methods could be the basis of a polyphasic taxonomy. The twenty strains of wild-legume rhizobia were finally classified as follows: 12 strains related to Rhizobium leguminosarum, 5 strains related to Sinorhizobium meliloti and 3 strains to Rhizobium spp. Rhizobia nodulating wild herb legumes are among indigenous strains nodulating crop legumes in cultivated as well as noncultivated lands.
Page 242. Chapter 11 Enhancement of RhizobiaĀLegumes Symbioses and Nitrogen Fixation for Crops Pr... more Page 242. Chapter 11 Enhancement of RhizobiaĀLegumes Symbioses and Nitrogen Fixation for Crops Productivity Improvement Hamdi Hussein Zahran Abstract Rhizobia form a very interesting symbiotic relationship with leguminous plants. ...
International microbiology : the official journal of the Spanish Society for Microbiology, 2013
Fifty four bacterial strains were isolated from root nodules of the grain legumes Cicer arietinum... more Fifty four bacterial strains were isolated from root nodules of the grain legumes Cicer arietinum, Lens esculentus, Phaseolus vulgaris, Pisum sativum, and Vicia faba grown in cultivated lands of Beni-Suef Governorate (Egypt). Repetitive extragenic palindromic (REP)-polymerase chain reaction (PCR) clustered the strains into 15 REP-PCR groups. The nearly complete sequence of the 16S rRNA gene from a representative strain of each REP-PCR pattern showed that the strains were closely related to members of the family Rhizobiaceae of the Alphaproteobacteria. Pairwise alignments between globally aligned sequences indicated that the strains from V. faba had 99.6% identity with Rhizobium leguminosarum, and those from P. vulgaris 99.76% and 100% with sequences from R. leguminosarum and R. mesosinicum, respectively. Strains from P. sativum had 99.76%, 99.84%, and 99.92% sequence identity with R. leguminosarum, R. etli, and R. pisi, respectively, and those from L. esculentus had 99.61% identity ...
A different combined effects for helium gas discharge such as: magnetic field strength, breakdown... more A different combined effects for helium gas discharge such as: magnetic field strength, breakdown voltage, applied power, applied pressure, cathode fall thickness, edge effect, distribution of the electron temperature and density, and finally exposure time for Staphylococcus aureus substrate over slides at the cathode edge, are discussed under the influence of cold, nonthermal plasmas, ultra low pressure, and presence of the magnetic field for disinfection of bacteria for short exposure times, compatible to International Commission on Non-Ionizing Radiation Protection, Health Phys (ICNIRP) for healing applications. Furthermore, analyses of the experimental data of initial and final densities of cells alive, using survival curves, showed an impressive inhibitory effect of plasma discharge to the remaining survival of bacterial ratio under the influence of the magnetic field.
... For example, 15 isolates of S. meliloti recovered from nodules of wild species of ... associa... more ... For example, 15 isolates of S. meliloti recovered from nodules of wild species of ... association with a wide range of rhizobia (Rhizobium, Mesorhizobium, and Sinorhizobium), welladapted to ... The high sensitivity of the legumeĀRhizobium symbiosis to salinity has been recognized ...
World Journal of Microbiology & Biotechnology, 1994
The effects of osmotic and heat stress on lipopolysaccharides and proteins of rhizobia isolated f... more The effects of osmotic and heat stress on lipopolysaccharides and proteins of rhizobia isolated from the root nodules of leguminous trees grown in semi-arid soils of the Sudan, and of agricultural legumes grown in salt-affected soils of Egypt, were determined by SDS-PAGE. The rhizobia were of three types: (1) sensitive strains, unable to grow in 3% (w/v) NaCl in yeast mannitol medium; (2) tolerant strains which could grow in 3% (w/v) NaC1; and (3) halophytic strains which grew with 3 to 10% (w/v) NaCl. The sensitive strains changed their gel pattern or the amount of lipopolysaccharide they synthesized when grown in 1% (w/v) NaC1. The tolerant and halophytic strains often modified their lipopolysaccharides in 3% NaCl, which was evident by a shift in the banding patterns towards longer chain length. Similar effects were observed in cells incubated with sucrose and, to a lesser extent, in cells incubated at growth temperatures near the recorded maximum temperature for growth. The stress-induced changes in lipopolysaccharides were not associated with specific banding patterns of the lipopolysaccharides. During incubation in medium containing elevated concentrations of NaCI or sucrose, the protein patterns of the rhizobia were also changed. A protein with relative mobility of 65 kDa appeared during temperature stress. The maximum growth temperature of the Sudanese rhizobia were up to 44.2Ā°C.
The effects of sodium chloride and polyethylene glycol (PEG) on the interaction between Rhizobium... more The effects of sodium chloride and polyethylene glycol (PEG) on the interaction between Rhizobium leguminosarum strain 29d and root hairs of field bean (Viciafaba L. cv. Maris Bead) plants were investigated. Two levels each of NaC1 (50 and 100 mol.m -3) and PEG (100 and 200 mol.m -3) were given at the time of root-hair formation. Scanning electron microscopy showed rhizobial attachment and colonization on root-hair tips. Adhesion of rhizobia in both lateral and polar orientation, sometimes associated with microfibrils, occurred mainly in crooks at the root-hair tips; most of the infections also occurred here. Bacterial colonization and root-hair curling were both reduced by stress treatments. Polyethylene glycol but not NaC1 significantly reduced root-hair diameter. The proportion of root hairs containing infection threads was reduced by 30% under NaC1 and by 52% under PEG. The structure of some of the root hairs, epidermal and hypodermal cells, as seen by light microscopy in ultrasections, was distorted as a result of NaC1 and PEG treatments; cells showed plasmolysis and folded membranes. After three weeks of treatment, both NaC1 and PEG inhibited nodule number by about 50% and nodule weight by more than 60%. It is concluded that the root-hair infection process in Vicia faba is impaired by NaC1 and PEG treatments and this in turn results in fewer nodules being produced.
Medicago intertexta and Melilotus indicus, two wild leguminous herbs with different tolerance to ... more Medicago intertexta and Melilotus indicus, two wild leguminous herbs with different tolerance to salinity were investigated for NaCl-induced changes in the expression level of some Na 1 transporters. M. indicus plants grew well at NaCl concentration from 0 to 400 mM, whereas growth of M. intertexta plants was severely inhibited at NaCl concentrations higher than 100 mM. In M. intertexta, increasing NaCl in the growth media caused a strong increase in Na 1 content concomitant with a decrease in K 1 content in leaves and, above all, roots. In comparison, M. indicus plants cultivated in the presence of NaCl accumulated much less Na 1 in leaves and roots and no differences in K 1 content among plants grown in nutrient solution containing 100-400 mM NaCl were detected. The expression levels of four genes coding for NHX-type Na 1 /H 1 antiporters in the above two wild legumes were studied in plants cultivated under the different NaCl concentrations. Expression levels of the genes were higher in M. intertexta as compared with M. indicus plants. In M. intertexta, salt treatments increased MtNHX1, MtNHX3 and MtNHX4 transcript levels in leaves and roots. However, in M. indicus NaCl treatments only induced the expression of MtNHX1 in roots. Our data suggest that two different mechanisms, Na 1 avoidance or accumulation into cellular compartments, are developed by the two wild legumes to cope with salt stress, and that expression of NHX antiporters is linked to the accumulator phenotype.
Inactivating viable cells at very short application times has been studied using Ultra Low Freque... more Inactivating viable cells at very short application times has been studied using Ultra Low Frequency Plasma (ULFP) at one Kilo Hertz, using an RF source. The targeted fashion is to inactivate Escherichia coli (E. coli ) in the absence and in the presence of magnetic field. Adding oxygen (O 2) to argon (Ar) in the discharge leads to a complete bacterial inactivation, where the inactivation rate increased as the concentration of O 2 increases. Analyses of the experimental data of the initial and final densities of viable cells, using survival curves, showed a dramatic inhibitory effect of plasma discharge to the residual survival of microbial ratio due to the influence of the magnetic field.
Wild legumes (herb or tree) are widely distributed in arid regions and actively contribute to soi... more Wild legumes (herb or tree) are widely distributed in arid regions and actively contribute to soil fertility in these environments. The N 2 -fixing activity and tolerance to drastic conditions may be higher in wild legumes than in crop legumes. The wild legumes in arid zones harbor diverse and promiscuous rhizobia in their root-nodules. Specificity existed only in few rhizobia from wild legumes, however, the majority of them are with wide host range. Based on phenotypic characteristics and molecular techniques (protein profiles, polysaccharides, plasmids, DNA-DNA hybridization, 16SrRNA, etc.), the root-nodule bacteria that was isolated from wild legumes had been classified into four genera (Rhizobium, Bradyrhizobium, Mesorhizobium and Sinorhizobium). The rhizobia of wild legumes in arid zones, exhibit higher tolerance to the prevailing adverse conditions, e.g. salt stress, elevated temperatures and desiccation. These rhizobia may be used to inoculate wild, as well as, crop legumes, cultivated in reclaimed desert lands. Recent reports indicated that the wild-legume rhizobia formed successful symbioses with some grain legumes. Moreover, intercropping of some N 2 -fixing tree legumes (e.g. Lablab, Leucaena, Sesbania, etc.) to pasture grasses improved biomass yield and herb quality. In recent years, the rhizobia of wild legumes turn the attention of biotechnologists. These bacteria may have specific traits that can be transferred to other rhizobia through genetic engineering tools or used to produce industrially important compounds. Therefore, these bacteria are very important from both economic and environmental points of view.
Salt tolerance of five rhizobia strains was examined in broth cultures. Five levels of NaCl conce... more Salt tolerance of five rhizobia strains was examined in broth cultures. Five levels of NaCl concentration were used and the optical density was taken as a measure for the vigour of bacterial growth. Rhizobium leguminosarum and R. meliloti were tolerant to high levels of salinity and growth curves in saline broth showed a similar pattern to the control level. Rhizobium japonicum, cowpea Rhizobium, and R. trifolii were intolerant to salt and showed a strong growth retardation with increasing salt concentration. Growth was inhibited at high levels of salinity. It is suggested that rhizobia sensitivity to salts may be partly responsible to the inhibition of nitrogen fixation by legumes growing under salt stress.
Some characteristics of thirty isolates of root-nodule bacteria. indigenous in the salt-affected ... more Some characteristics of thirty isolates of root-nodule bacteria. indigenous in the salt-affected soil of Egypt, were studied. To recognize different isolates of these bacteria, the electrophoretic pattern of plasmids and of whole cell lipopolysaccharides (LPS) and proteins were analysed and compared using agarose gel electrophoresis (AGE) and sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE). respectively. The bacteria showed patterns of LPS, protein and plasmid which varied among isolates of different plant species as among isolates of a single plant species. Based on LPS and protein profile analysis, isolates from root-nodules of broadbean and lupin plants were more diverse than isolates from root-nodules of berseem plant. Root-nodule bacteria of lupin and berseem were also less diverse than root-nodule bacteria of broadbean when comparcd on the basis of plasmid profiles. This result indicate that root-nodule bacteria of broadbean may have higher surviving ability in the salt-affected soil. Most isolates of the root-nodule bacteria synthesized specific LPS (smooth, ladder-like type) and protein. which were different from LPS and protein of other well-known (recognized) Rhizobium species. These specific forms of LPS and protein could be a prerequisite for the bacterial isolates to tolerate salt stress conditions of the salt-affected soils.
Twenty out of 400 isolates of bacteria mainly from salt marshes and saline soils of Egypt were su... more Twenty out of 400 isolates of bacteria mainly from salt marshes and saline soils of Egypt were successfully grown on mannitol N-free medium. The nitrogen-fixing activity was then demonstrated for the twenty isolates in modified STANIER'S medium using the acetylene reduction assay. All of them possessed appreciable nitrogenase activity (acetylene reduction) under non-saline conditions ; however, at 5% NaCl only 60% of the isolates exhibited a high rate of this activity and 25% were completely negative under these conditions. The bacterial isolates grew well in STANIER'S medium; nevertheless, growth of the majority of isolates was reduced by about 30-80% in the same medium containing 5 % NaCl. Cellulolytic activity was detected in 60% of the twenty strains, amylolytic in 45%, and pectinolytic in 10% of the isolates. The bacterial isolates showed also enzymatic activity under saline conditions (5 % NaCl). The preliminary identification indicated that six isolates were Gram positive spore-forming bacteria of the genus Bacillus, the others were Gram negative rods which remain to be identified.
Members of Bricillcicecir, Rlzi=ohioceae, actinomyeetes and others were isolated from cultivated ... more Members of Bricillcicecir, Rlzi=ohioceae, actinomyeetes and others were isolated from cultivated and non-cultivated saline soils. The high population of bacteria and actinomycetes were almost coincided with the relatively high levels of organic matter whatever the degree of soil salinity.
The effects of application of combined nitrogen fertilizer (ammonium nitrate or urea) on root-hai... more The effects of application of combined nitrogen fertilizer (ammonium nitrate or urea) on root-hair infection and nodulation of four grain legumes were studied. Young roots of each legume were inoculated with their compatible rhizobia. The application of the two forms of combined N either at the early stages of plant growth and/or at the time of nodule formation depressed root-hair curling, infection and nodulation. Infection of hairs on the primary roots was more sensitive to the N fertilizer than hair infection of secondary roots in both l, Tcia faba and Pisum sativum. The nodule number and total fresh mass of the four legumes were drastically affected by fertilizer application. The combined N added both at early and at later stages significantly reduced the nodulation of V. faba, Phaseolus vulgaris and F/gna s/nens/s. The inhibitory effect of urea on nodulation of P. sativum was only observed when the fertilizer was applied at the late stages of plant growth. It is concluded that, although the nodulation of the four legumes was suppressed by combined N, the initial events of Rhizobium-legume symbiosis (infection of roots and nodule initiation) are more sensitive to combined N than the stages after nodule formation.
Plant needles of Casuarina equisetifolia were collected and analyzed in parallel with soil analys... more Plant needles of Casuarina equisetifolia were collected and analyzed in parallel with soil analysis. In three strains of Frankia -symbionts of Casuarina -their infectivity and plant performance was determined in vitro after soil amendment with different leaf litter concentrations. Only one strain was able to nodulate the plant at all litter concentrations (0.5, 3 and 5 %) although the nodules were very small. However, all treated plants grew poorly; their growth was reduced by approximately 90 % (for 5 % litter concentration) compared to plants grown on untreated soil, on the basis of total dry mass. Inhibition of nodulation can be attributed to high concentrations of some elements and compounds that were either found in C. equisetifolia litter or originally found in soil (i.e. chloride, cyanide, copper, manganese and phenols). In general, plant growth decreased as more litter was added. Plant total nitrogen content was also reduced after increasing the litter concentration. The inhibitory effect of high litter concentrations was mainly on plant growth and to a lesser extent on plant nodulation by Frankia strains. 430 W.F. SAYED et al.
A total of about 50 strains of rhizobia from two leguminous trees (Acacia and Prosopis) were desc... more A total of about 50 strains of rhizobia from two leguminous trees (Acacia and Prosopis) were described and compared with 20 reference strains of rhizobia from other tree and herb legumes on the basis of protein, fatty acid and plasmid profiles, and DNA-DNA hybridization. The rhizobia formed thirteen clusters based on protein profile analysis. These clusters were not in complete agreement with a previously published cluster analysis based on numerical taxonomy of phenotypic characteristics and lipopolysaccharide (LPS) profile analysis (Zhang et al., Int~.Syst.Bacteriol. 41, 104, 1991; Lindstr6m and Zahran, FEMS Microbial.Lett. 107, 327, 1993). The fatty acid methyl esters (FAME) of representative strains of rhizobia were analyzed.
The DNA-DNA hybridization was used to characterize thirty isolates of root-nodule bacteria indige... more The DNA-DNA hybridization was used to characterize thirty isolates of root-nodule bacteria indigenous to the salt-affected soils of Egypt. Total DNA from different bacterial isolates lacked homology with total DNA probes of the effective strains of Rluzobium leguminosarum and R. meliloti. It is suggested that the genomic structure of the root-nodule bacteria may be modified by salt stress and/or that the effective strains of these bacteria are to be eliminated from the salt-affected soil.
About 20 strains of rhizobia from wild legumes were characterized based on numerical analysis of ... more About 20 strains of rhizobia from wild legumes were characterized based on numerical analysis of phenotypic characteristics, nodulating ability, fatty acid methyl esters (FAME) and SDS-PAGE profiles of whole cell proteins. FAME analysis revealed that palmitic (16:0), stearic (18:0) and arachidonic (20:0) were detected in most of wild-legume rhizobia, the latter being uncommon in fatty acid profiles of Rhizobium and Sinorhizobium. Numerical analysis of FAME classified strains of wild-legume rhizobia into 9 clusters and one heterogeneous group. There was both agreement and disagreement with the clustering data based on phenotypic analysis and FAME analysis. Four strains were grouped together in the same cluster based on both methods. However, 4 another strains, which were placed in one cluster of phenotypic analysis, were distributed in several clusters after FAME analysis. SDS-PAGE of whole-cell proteins revealed that the rhizobial strains exhibited protein profiles with peptide bands ranging from 5-19 band per profile and showed molar mass of 110-183 kDa. As in the case of FAME analysis, numerical analysis of protein bands was compared with clustering of phenotypic analysis. Agreement of the two methods was obvious when clustering some strains but conflicted in the classification of some other strains. However, integration of the three methods could be the basis of a polyphasic taxonomy. The twenty strains of wild-legume rhizobia were finally classified as follows: 12 strains related to Rhizobium leguminosarum, 5 strains related to Sinorhizobium meliloti and 3 strains to Rhizobium spp. Rhizobia nodulating wild herb legumes are among indigenous strains nodulating crop legumes in cultivated as well as noncultivated lands.
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