Rhizobia

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Review of literature

Rhizobia involved in nitrogen fixation are mostly specific to their host. The N fixation depends greatly upon the environmental conditions and the genetic diversity of the Rhizobia as well as the host species. Studies relating to different aspects of the phenotypic characterization and genetic diversity in Rhizobia and their nitrogen fixation potential are given as under.

2.1. Generation time

Working with 536 cowpea nodule bacteria (Lindete et al., 1997) reported fast growth for 27 isolates. They further arranged them in 78 different groups on the base of colony morphology and growth rate. The analysis of the isolates from each region showed an increased proportion of fast growing rhizobia when going from the coast to inland. They concluded that selection pressure on rhizobia leads to the evolution of stress tolerant forms. The high incidence of fast-growing rhizobia in the arid regions suggests that they are better survivors than slow growing strains. Mâatallah et al. (2002a) grouped chickpea rhizobia on the basis of generation times. According to their results 22% of the isolates were fast growers (50 min < GT < 3 h), 32% slow growers (3 h < GT <9 h) and 46% were extra-slow growing bacteria with a generation time of more than 9 h.

Mâatallah et al. (2002b) reported about 8.3% of the chickpea strains as fast growers with a generation time (GT) lower than 3 h, 41.7% as slow-growing with a GT between 3 and 9 h, and 50% as extra-slow grower bacteria with a generation time greater than 9 h. Odee et al. (2004) reported a wide range of growth rates (MGT): very fast growing (1.6-2.5 h), fast growers (2.8-4.8 h), intermediate between fast and slow growing (4.6-5.7 h) and very slow growers (6.4-8.8 h). On the basis of mean generation time (MGT) very fast and fast growers were included in Rhizobium spp., while very slow and intermediates were placed in Bradyrhizobium spp. Kücük et al. (2006) isolated thirty rhizobial strains from bean (Phaseolus vulgaris L.) and classify 10 isolates under the heading of fast growers (60 min), while rest of other isolates were slow growers (12 h).

2.2. pH tolerances

According to Sane (1987) alkaline soils are characterized by high pH values up to 11.5. Kulkarni and Nautiyal (1999) observed considerable growth on pH 9 for majority of strains, except 3 strains that were well adapted to grow on pH 12.0. Rickert et al. (2000) described in their results that adaptive response was dependent on the sub-lethal pH and the strain intrinsic acid tolerance: the lowest pH values tolerated after adaptation were 4.0 for strain LL56 and 5.7 for strain LL22, and the lowest pH values tolerated after adaptation were 3.0 and 4.0 respectively. Raza et al. (2001) tested Bradyrhizobium sp (lupini) strains for pH range (4-10). Their over all results indicated that the isolates were tolerant to extremes of low and high pH since they grew over a range of pH from 4 to 10. All the isolates survived at acidic pH (4-5) and alkaline conditions (pH 9-10). Priefer et al. (2002) working with Tn5 mutants isolated strains that were not only able to grow on extreme pH but also under neutral conditions. Maatallah et al. (2002) isolated rhizobia from chickpea that were effective and able to grow at pH ranging from 5 to 8. Hung et al. (2005) observed majority of strain tolerated extreme pH in their medium from 3.5 to 12. Shamseldin and Werner (2005) investigated pH tolerant strains and found pH 4.7 as the minimum level of pH tolerance for majority of the strains. Rhizobium etli strains from Egypt were resistant at pH 4.7 while Columbian strain Rhizobium tropici CIAT899 survived well at pH 4.

Kücük et al. (2006) tested Rhizobium strains nodulating beans for pH (3.5-9). All isolates were grown in YEM medium with pH values of 5 and 8, but differences were detected at pH 4. All the isolates grow at extremely basic condition as high as 9. Rodrigues et al. (2006) quantify bacterial growth for the pH range (5-9). A positive correlation was observed between the maximum growth pH and the isolate origin soil pH. They further elaborated that some isolates showed changes in the preferential pH of culture medium with temperature. At 28oC two isolates grew more at pH 7, but at 20oC, the growth rate was higher at pH 9. Kücük and Kivanc (2008) noted that all chickpea nodulating strains grew in YEM medium with pH values of 5.0 and 8.0, but differences were detected at pH values of 3.0 and 9.0.

2.3. Temperature

Osa-Aflana and Alexander (1982) compared temperature tolerance of cowpea rhizobia. Their results present no growth at 40oC, but all grew at 29, 31, 33, and 35oC. Most of strains tested have an optimum growth at 33oC. Kulkarni and Nautiyal (1999) described that out of 2500 rhizobial strains 405 strains were selected that had similar growth patterns after 72 h on YEM plates incubated at 30 and 45oC. The second screening resulted in 24 tolerant strains, were able to grow at 47.5oC.

Maatallah et al. (2002a) reported maximum growth between 20 to 30oC. Percentages of isolates that can grow below and above theses limits were 12 % at 5oC and 7% at 45oC. More than 50 % could not tolerate more than 42-45oC. According to the results of Hung et al. (2005) 28 strains grew well between temperatures 37 and 45oC. Kücük et al. (2006) recorded abundant growth at a temperature of 42oC for rhizobia isolated from /root nodules of beans. Rodrigues et al. (2006) observed better growth for most of the isolates at 28oC except one of the strain that shows maximum growth at 20oC. Isolates grew efficiently at 20oC and that at 37oC, except two isolate that were more tolerant to 37oC. Kücük and Kivanc (2008) dealing with temperature tolerance in chickpea rhizobia find that all strain showed growth in YEM medium of 20, 25, 30 and 37oC while 75% of the strains were tolerant to 40oC.

2.4. Antibiotic resistance

Multiple drug resistance is common in human and animal pathogenic bacteria but quite uncommon in soil bacteria. Antibiotic resistance is one of the methods used as genetic marker to differentiate bacterial strain. Cole and Elkan (1973a) recorded resistance to penicillin G, neomycin and chloramphenicol is extrachromosomal element mediated. Mahler and Bezdicek (1978) studied diversity for antibiotic resistance. They recorded relative resistant to penicillin, chloramphenicol, polymyxin B and novobiocine and were sensitive to erythromycin, tetracycline and streptomycin. Cole and Elkan (1973b) screened 48 strains of R. japonicum for several commonly used antibiotics. 60% strains were resistant to chloramphenicol, polymyxin B, and erythromycin and 47% or more were resistant to neomycin and penicillin G.

Hagedron (1979) examined 50 Rhizobium trifolii isolates and found resistance to 15 antibiotics. The cultures were resistant to high concentrations of 11 of the antibiotic but were relatively sensitive to streptomycin, tetracycline, vancomycin and chloramphenicol. Kremer and Peterson (1982) determined patterns of intrinsic resistance and susceptibility to different concentrations and combinations of five antibiotics (kanamycin sulfate, streptomycin sulfate, tetracycline hydrochloride, penicillin G, and rifampycin) in legumes. They suggested intrinsic resistance and susceptibility patterns were reliable for identification of nodule strains when strains were first isolated from the nodules to provide a standard inoculum size and type on antibiotic containing media. They further elaborated that high strain recovery was associated directly with high rates of inoculation. Gupta et al. (1983) use intrinsic multiple antibiotic resistance markers in order to study competitive and effectiveness of mung bean rhizobia. They antibiotics tested were erythromycin, 15μg/disc; ampicillin, 10 μg/disc; tetracycline, 30 μg/ disc; gentamycin, 10 μg/disc; streptomycin, 10 μg/disc; kanamycin, 30 μg/disc; nalidixic acid, μg/disc; chloramphenicol 30 μg/disc. The antibiotic spectra showed that a large number of native rhizobia were sensitive to all the test antibiotics (53%). They observed that frequency of resistance to single and double antibiotics was higher than multiple resistances. Selvaraj and Iyer (1984) also tested their rhizobial tn5-insertional derivatives of R. meliloti for minimum inhibitory concentrations for carbencillin, 40 μg/ml; chloramphenicol, 30 μg/ml; kanamycin, 40 μg/ml; rifampycin, 150 μg/ml; streptomycin and kanamycin, 30 μg/ml. Most of the derivatives were resistant to streptomycin (50 μg/ml) where as parental strains were sensitive to streptomycin. While a total of 300 kanamycin transfortmants were then screened for streptomycin resistance but no one was enable to grow in streptomycin. Rokman and Bezicek (1989) determined intrinsic antibiotic resistance (IAR) in for 192 isolates of R. leguminosarum biovar Viceae from nodules of peas (Pisum sativum L.) Cluster analysis of (IAR) data indicated that clusters were dominated by one serogroups. IAR grouped 72% of the isolates similarly. Raza et al. (2001) selected 12 antibiotics to investigate intrinsic antibiotic resistance (IAR) patterns tolerance of Bradyrizobium sp. (Lupini) strains. They found that all the strains were sensitive for clindamycin (2 µg/ml) but divided in to four groups on the basis of to the (IAR) for remaining 11 antibiotics. Group A, include to strains that were resistant to all 11 antibiotics and group C, comprises 3 strains that were sensitive for all the antibiotics tested. Maatallah et al. (2002a) and (Maatallah et al., 2002b) find out intrinsic antibiotic resistance to amphicillin, 50 μg/ml, chloramphenicol, 10 μg/ml; kanamycin, 10 and 100 μg/ml; rifampycin, 10 μg/ml; streptomycin 25 and 100 μg/ml and kanamycin, 30 μg/ml chloramphenicol, 30 μg/ml; kanamycin, 40 μg/ml; rifampicin, 150 μg/ml, nalidixic acid, 50 μg/ml; erythromycin, 100 μg/ml and tetracycline 20 μg/ml. 65% isolates exhibited high resistance to kanamycin, nalidixic acid and erythromycin. While 14 to 25% were resistant for streptomycin, amphicillin, chloramphenicol, rifamycin and tetracycline. Their results indicate that tolerance of strains to the antibiotic did not show correlation with their growth rate, but it could be related to the bacterial species. Young and Chao (2004) observed wide variability in resistance to 10 antibiotics for fast and slow growing rhizobia. The intrinsic antibiotic resistance of fast and slow growing rhizobia was extremely high against nalidixic acid (400 μg/ml) and penicillin (200 μg/ml).

Kücük et al. (2006) reported intrinsic resistance for chloramphenicol (20 and 50 μ/ml) erythromycin (30 μg/ml), kanamycin (10 μg/ml), and streptomycin (40, 80 and 100 μg/ml) in bean rhizobia. Kücük and Kivanc (2008) also tested chickpea rhizobia for intrinsic antibiotics for following concentrations, streptomycin (100 μg/ml), kanamycin (50 μg/ml), erythromycin (30 μg/ml), chloramphenicol (200 μg/ml), and penicillin (25 μg/ml). Majority of strains showed a high level of resistance against streptomycin, erythromycin, kanamycin, penicillin and chloramphenicol.

2.5. Heavy metal resistance

Heavy metals adversely influence microorganisms, not only affect their growth, but also their morphology and activities. Metals also can exert a selective pressure on the organisms that increases heavy metal tolerance in microbial populations. Due to importance of legumes in animal and human consumption and their use in maintaining soil fertility, some attention has been given to the effects that heavy metals exert on Rhizobium isolates.

Diaz-Ravina et al. (1993) determined the tolerance of a soil bacterial community to Cu, Cd, Zn, Ni, Pb. They artificially contaminated an agricultural soil in laboratory. An increase to tolerance to the metal added to soil was observed for the bacterial community obtained from each polluted soil was compared with non polluted soil. An increase in the tolerance to metal added to soil was observed, indicating that there was multiple heavy metal tolerance at the community level. They found significant positive relationships between changes in Cd, Zn and Pb tolerance and to a lesser degree, between changes in Pb and Ni. Saxena et al. (1996) stated that imposition of any stress to bacteria results in adaptive responses that lead to changes in the regular metabolic process in the cells, which are then reflected in the alteration of the protein profiles. Metals influenced their protein profiles, most of the alterations corresponding to decreases in polypeptide expression However, in tolerant isolates these alterations corresponding basically to increase (Pereira et al., 2006).

2.6. Carbon utilization patterns

Carbon utilization patterns have also been used to distinguish isolates and strains among the Rhizobiaceae family. If we look at the past work a few researchers focused this parameter for chickpea and garden pea.

Kulkarni and Nautiyal (1999) selected 7 strains of root nodule bacteria isolated from Prosopis juliflora for 17- carbon sources utilization. They observed positive results for all the carbon sources (glucose, mannitol, sorbitol, galactose, sucrose, fructose, lactose, xylose, raffinose, acetate, formate, citrate, propionate, tartarate, ethanol, glycerol) except Na tartarate. According to the findings of Maatallah et al. (2002a) all tested strains of chikpea rhizobia grew on glycerol, D-fructose, N acetylglucosamine, sorbitol, mannitol, surose, trehalose, L- fructose, gluconate, L- arabitol, maltose and cellibiolose, while L- xylose, glycogen, inulin or α-methyl-D-mannoside was not utilized by any strain. Kücük et al. (2006) being presenting biochemical properties if bean rhizobia found that Eskiseshir isolates were able to use several compounds as sloe source of carbon. All isolates show growth for D (-) fructose, D (+) mannitol, sucrose, D (+) galactose, starch α- L-rhamnose and malate. The isolates were unable to use tartrate and dulcitol. Kuuk and Kivanc (2008) tested for rhizobial growth of chickpea nodule isolates for against 12 carbon sources and observed positive result for all compound used as carbon source.

2.8. Plasmid profiles

Toro and Olivares et al. (1986) reported two cryptic plasmids of 140 and 114 MDa in reference strain R. meliloti GR4. Velazauez et al. (1995) revealed that wild-type strains of R. meliloti had pSym plasmids with a molecular weight above 1,400 X 10 and exhibited three different plasmid profiles distinguished by the presence or absence of various smaller sized cryptic plasmids. Loccoz and Weaver (1996) observed that plasmids play important role in saprophytic characteristics and sodium chloride tolerance of W14-2 in vitro. No plasmid-cured derivative grew better than the wild-type overall, suggesting that he extra chromosomal genome contributes to the saprophytic competence W14-2 in soil. Zou et al. (1998) working with Tephrosia candida observed that plasmid profile of strain S25  harbors only one plasmid with an estimated size o 150 kb. Lakzian et al. (2002) investigated populations of Rhizobium legueminosarum bv viciae from plots of a long-term sewage sludge experiment. They observed three and nine plasmids for rhizobial isolates which vary in size from approximately 100 to 850 kb. A total of 49 plasmid profile groups were identified among isolates they studied. Castro et al. (2003) studied the role of plasmids in the ecology of these rhizobia strains in the presence of heavy metal (mainly Zn and Hg). They concluded that plasmids could be important for the adaptation of rhizobia to stressful conditions. Romero and Broom (2004) reported plasmid number from 0 to 11 and size from 150 to 1,683 kb in family Rhizobeaceae. They also reported that proportion of plasmids to total genome size represent even from 25 to 50% of genome size. Vessey and  Cheminging'wa (2006) studied that Rhizobium legueminosarum bv. viciae strains varied in number of plasmid from 1 to 8. Inoculant strains had three to five plasmid bands, while most field isolates exhibited one to five plasmid bands. Single (27.8%)  and four (27.4%) plasmid band isolates were the most common The percentage of isolates with two, three and five bands was 6.1, 14.3 and 19.1, respectively; isolates with six to eight plasmid bands constituted just about 5% of the total isolates. Strains varied in plasmid size from less than 50 kb to more than 1000 kb.

Lakzian et al. (2007) investigated plasmid profiles among isolates of Rhizobium legueminosarum bv. viciae in heavy metal contaminated soils and found  three, four or five bands and 10 to 14 banding patterns among the 50 isolates . They described that one isolates originally, from uncontaminated soil that had five large plasmids was the most abundant type re-isolated from all of the soils. They further demonstrate that the transfer of naturally-occurring plasmids is important in conferring enhanced tolerance to elevated zinc concentrations in rhizobia.

2.10. RAPD analysis

De Oleveria et al. (2000) isolated efficient bean nodulating Rhizobium strains, and characterized by 27 RAPD primers of opern kit. Their study showed great genetic heterogeneity between R. tropici and R. leguminosarum biovar phaseoli.They concluded that genetic grouping of strains could be used to select appropriate Rhizobium strains of respective crops. Priefer et al. (2001) analyzed rhizobial strains nodulating Phaseolus vulgaris for their phylogenetic relationship. Strain RP163, exhibiting high nodulation efficiency and a broad pH tolerance was mutagenised by Tn5 and mutants unable to grow on extreme pH media were isolated. Sequenced mutants generated in this region were found to be impaired in growth at low pH, but also under neutral conditions.

Kumar et al. (2006) investigated genetic diversity of five Sinorhizobia nodulating Mucuna pruries using randomly amplified polymorphic DNA (RAPD) analysis. They recommended (RAPD) as new tools for investigating genetic polymorphism for genomic DNA of the bacterial isolates. They used 15 arbitrary chosen primers of OP series (A, B, C and E) were used. They found little polymorphism among isolates utilized. Sajjad et al. (2008) studied genetic diversity in rhizobial strains by using random amplified polymorphic DNA (RAPD) markers. They observed two distinct clusters by using two series of OP primers (A & C). A total of 1480 bands were amplified in the PCRs of 12 strains, out of which 663 were polymorphic, showing 44.80% of overall polymorphism. Number of bands produced per genotype ranged for 93 to 147 with an average of 123 bands per genotype. Among the strains, maximum number of bands were 147 (70 polymorphic), while minimum number of bands were 93 (45 polymorphic). The number of amplification products produced per primer varied from 4 to 9 with an average of 6 bands per primer. Rajasundari et al. (2009) subjected nine soil isolates from different field locations to RAPD analysis. They observed four clusters with more than 50 per cent similarly. Their results indicated that RAPD proved a very discriminative and efficient method for differentiating and studying genetic diversity of Rhizobium strains as they observed 90% dissimilarity for a strain that it formed separate cluster.

2.11. Symbiotic effectiveness

Since last two decades considerable work on symbiotic effectiveness of rhizobia that nodulate wild legumes and Rhizobium japonicum have been reported. Friedricks et al. (1990) isolated strains of Rhizobium leguinosarum (biovar trifolii) from two Ethiopian soils and were tested for symbiotic effectiveness. Numerous Rhizobium trifolii strains that exhibited varying levels of symbiotic effectiveness were isolated. The soil isolates were compared with commercial strain and found superior in symbiotic effectiveness. Several Rhizobium trifolii strains were found to be effective on more than one clover species, and there appeared to be at least two and possibly three distinct cross-inoculation effectiveness groups. Valzquez et al. (1995) indicate in their results that Rhizobium. moliloti  wild-type strain SAF22 has the genetic capability to develop fully effective root nodules on alfafa, but this phenotype is attenuated by its cryptic plasmid pRmSAF22c, which interferes with the nodule development required for fully effective nitrogen fixing symbiosis. Lindete et al. (1996) investigated fixation efficiency of rhizobia in symbiosis with soybean. They found that high effectiveness was generally associated with the presence of an active hydrogenase uptake system (Hup). Hup+ strains were capable of recycling the hydrogen evolved by nitrogenase. Twenty-five group isolates were not able to nodulate soybeans. Some isolates also form pseudo-nodules. Baraibar et al. (1999) working with symbiotic effectiveness and ecological characterization of Rhizobium loti come to the conclusion that shoot dry weight was lower for all the soils populations compared to the N or U-226 treatment.

Burdon et al. (1999) found significant variation in 22 Acacia species and nearly all of 67 populations. They observed 70% effective Acacia host-rhizobium strain combination. Many combinations were poor resulted in plants less than one-tenth of the best combination. Significant host based variability in the ability to form effective symbiotic interactions were detected. Rhizobium population of soil 'S' neither differs statistically from U-226 nor from the nitrogen treatment. Although the soil 'Y' presented the highest rhizobia density related to their nutritional levels and textural properties it was not the most efficient. The REI of the indigenous populations of R. loti, except soil 'S', was lower than 50%. In relation to REI of the 50 isolates of R. loti, 6% was between 100-119% and another 6% were located in the 0-40% range. The mean of the REI of the isolates from each soil compared with the efficiency determined from the indigenous populations of the soils. Isolates from five soils showed REI superior to 70% and the mean of the REI of the other five soils was 60%. REI values of the isolates in relation to REI of the indigenous population were visibly higher in all cases, except in the soils 'G' and 'S'. In two cases the REI of isolates was up to four times higher than that of the respective soil Rhizobium population.

The nodules in Lotus pedunculatus and Lotus subbiflorus were small, red on the surface and ineffective in nitrogen fixation. The symbiotic effectiveness of the 17 strains as P. juliflora as host was evaluated in green house by Kulkarni and Nautiyal (1999). The effectiveness of the host Rhizobium strain combinations was determined from measurements of plant dry weight, five strains were tolerant to 60 °C in YEB for 6 h and established effective symbiosis, as determined by plant top dry weight i-e, 29.2, 32.2, 60.2, 72.2 and 88.6 % higher, respectively compared with un-inoculated control plants.

Thrall et al. (2000) working with variations in the effectiveness of symbiotic associations between rhizobia and Australian legumes. They found a range of different nodule type i.e., large red, large white and many small white; only nodules showing to red and pink to red centers due to presence of leg-haemoglobin were capable of forming effective symbiotic relationship. Their results generally revealed an inverse relationship between above-ground biomass and root/ shoot ratios. Host-rhizobial combinations resulting in poor growth showed the highest root/shoot ratios; there were also some combinations that had the highest percentage of infective nodules or lacked nodules.

Khokhar et al. (2001) dealing with chickpea nodulating Rhizobia native to Thal soils observed variability in symbiotic effectiveness for all isolates differed significantly in effectiveness in terms of nodule dry mass, shoot dry mass and N- content of shoots. Sindhu and Dadarwal (2001) also reported variability in symbiotic effectiveness while working on antibiotic resistant mutants relative to parent strains of chickpea nodulating bacteria. Some mutant strains showed Nod (+) and Fix (-) phenotype. Other mutants also showed decreased nodule number and reduction in nitrogenase activity as well as in shoot dry weight as compared to inoculation with parental strains and they showed the acquisition of streptomycin resistance in Rhizobium sp. Cicer strains is associated with decreased symbiotic effectiveness in chickpea, suggesting that antibiotic resistant mutants first should be analyzed for symbiotic effectiveness before using these mutants for ecological studies or nodulation competitiveness.

Siddiqui and Mahmood (2001) conducted a single three-factor experiment for effect of root symbionts, rhizobacteria and nematodes on the growth of chickpea. They find significant (P<0.05) effect of root symbionts on nodulation. Lanarjo et al. (2001) detected a low symbiotic efficiency (30-50%) for the native isolates and for the reference strain CP31 when compared with the nitrogen supplemented plants. While among Beja rhizobia analyzed some of the isolates were with higher symbiotic efficiency values.

Icgen et al. (2002) also evaluate five local and seven standard strains of Rhizobium for symbiotic effectiveness. Strains were compared in terms of their efficiency in increasing the nitrogen content of the chickpea. Shoot dry weight, nodule number, nodule dry weight, was taken as the parameters of plant productivity. Out of twelve isolates, only one local and three standard strains were selected as effective in root nodulation.

Mâatallah et al. (2002a) dealing with chickpea rhizobia come to know a large diversity in their capacity to infect the host plant and to fix atmospheric nitrogen. According to their findings nodule mean number varied from 5 for isolate Rch 30b to 62 for isolate Rch 8, being the most infective isolate. In comparison to TN control which represents s the 100% level of shoot dry matter and T0 control, which represents 36%. Most of the tested isolates showed a dry matter yield higher than T0. Tamimi (2002) working on symbiotic effectiveness of rhizobia isolated from root nodules of common bean (Phaseoulus valgaris L.) reported diversity for symbiotic effectiveness. Out of ten, three gave highest readings for nodule number per plant, nodule mass, shoot dry matter and N2 fixation.

Mhadhbi et al. (2004) found different symbiotic performance of M. ciceri, M mediterraneum, and S. medicae, when inoculated to chickpea at unstressed conditions and under salt conditions. Saini (2004) observed significant higher value of nodule dry weight in the treatments receiving Rhizobium inoculation. Sattery et al. (2004) studied effect of resident rhizobial communities for nitrogen fixing effectiveness. Fifty different soils were evaluated for Vicia faba, Lens calinaris, Vicia sativa, Cicer arietinum and Lupins angustifolus. They stated that soil pH is positively correlated with the values for nitrogen fixation effectiveness. They found that 33% of paddock had sufficient resident populations of Rhizobium leguminosarum bv. Viciae for effective nodulation of faba bean, 54% for lentil, 55% for field pea and 66% for the effective nodulation of the vetch host plant.

Thiao et al. (2005) investigated effectiveness of rhizobial strains from Gliricidia sepium. Their result showed that G. sepium establishes an effective symbiosis only with fast-growing rhizobia, although infective nodules were observed on root systems of plants inoculated with slow growing rhizobia. Brigido et al. (2007) estimated symbiotic effectiveness of rhizobia isolates 29 (Neutrophils) and 64 (moderately acidophile) was evaluated under pH 5 and 7. The symbiotic effectiveness values obtained ranged from 21.4 to 59.8%. The number and dry weight of nodules were also analyzed. They found no correlation between the number of nodules and symbiotic effectiveness. The moderately acidophilic isolate showed a higher symbiotic effectiveness values under pH5, and the neutrophile isolates demonstrated a higher symbiotic effectiveness at pH7.

Lagurre et al. (2007) studied variability of the developmental responses of pea (Pisum sativum). They found contrasting effects of nodule isolates on the development of nodules, roots and shoots dry weights. They had identified nod gene that induced very large, branched nodules, smaller nodule numbers, high nodule biomass, but reduced aerial parts and root development. The plants associated with this genotype accumulated less N in shoots, but N concentration in leaves was not affected.

2.12. Shoot total protein

Garge et al. (1990) compared Hup positive transformants for total N concentration. The higher accumulation of nitrogen was observed in plants inoculated with the Hup transconjugant Vc4 (2.17±0.06) than its parent Vml (1.82±0.07). Velázquez et al. (1995) calculated total shoot nitrogen of alfalfa inoculated by rhizobial strain. They found significant variation for all the strains from the corresponding means of R. meliloti SAF22 at a level of P<0.001. They concluded that SAF22 is less effective than other wild type strains.

Khokhar et al. (2001) also found significant differences for N-content of shoot. The total shoot nitrogen varies from (PAC-28) 1.6 to (PAC-19/3) 23.5/mg. Only one strain possessed high mean values for total nitrogen accumulated in shoot in comparison to reference strain. Rest of strains accumulates less total N. Icgen et al. (2002) compared five local and seven standard strains of Rhizobium ciceri in terms of their efficiency in increasing the nitrogen content of the chickpea. Nitrogen concentration was taken as one of the parameter to calculate plant productivity. The maximum increase in total nitrogen content was only 3.5-fold in single infection whereas an increase as great as 35-fold was recorded for multiple infections. The double infection with Y-29 and 385 as well as the triple infection with Y-29, 620 and 3379 gives rise to the maximum values.

Tamimi (2002) investigated best symbiotic performance for 10 bean strains in terms of plant nitrogen content. Best results were recorded for three isolates; JOV1 (S.E., 62.3±2.5), JOV3 (S.E., 54.6±1.2) and JOV10 (S.E., 51.2±1.8). Bhasker and Kashyap (2004) working with azide resistant Rhizobium ciceri strains reported that (complemented mutant) M126 containing C4 clone infected plants had similar amount of nitrogen (17.49 ± 0.96 mg of nitrogen/g dry wt; n=5) as that of plants infected with (Azide resistant) strain 18-7 (18.61±0.96 mg on nitrogen/g dry wt; n=5). M126 nodulated plants had significantly lesser nitrogen (13.38±0.96 mg nitrogen dry wt; n=5). They concluded that 18-7 and M126 (C4) infected plants had 34.56 and 26.46% higher nitrogen per g dry wt respectively than plants infected with M126 alone.

Satterly et al. (2004) estimated effect of inoculums on the effective nodulation of pluses. Like former worker they also calculate total N (%) as an important parameter in Pisum sativm.  All nodulated tested grow vigorously fixing N, indicating effectiveness of the populations. Laguerre et al. (2007) observed no significant strain (Rhizobium leguminosarum) effect on shoot N concentration of (Pisum sativum). The values of accumulated shoot N varied according to shoot DW. The ratio between shoot N accumulated per unit of nodule DW varied by a factor of 20 with very low nodule efficiency for the BNO plants inoculated with strains LRBA7 and P1Np2K.

2.13. Symbiotic effectiveness under salt stress

2.13.1. Growth and symbiotic characteristics

Hafeez et al. (1988) reported the effect of salinity and inoculation on growth, ion uptake and nitrogen fixation by Vigna radiata. They found 60% decline in dry matter and grain yield of mung bean for EC level of 7.5 dS m ~ . Most of their studied strains of Rhizobium were salt tolerant while nodulation, nitrogen fixation and total nitrogen concentration of the plant and acetylene reduction activity was drastically affected at 7.5 dS m -l. Loccoz and Weaver (1996) studied sodium chloride tolerance of clover rhizobia. They reported wild- type W 14-2 and all derivatives LD50 values comprised between 12.4 and 12.9 g NaCl l-1 and were more tolerant to sodium chloride than any derivatives lacking plasmid b and d. While latter derivatives displayed LD50 values comprised between 6.6 and 7.9 g NaCl l-1, with the exception of derivatives with only plasmid d, which had a LD50 of 2.0 g NaCl l-1.

Cordovilla et al. (1999) reported that the adverse effect of salinity was more on nodules than on vegetative parts and N2-fixation was more sensitive to salinity than plant growth. Raza et al. (2001) examined Bradyrhizobium sp (Lupini) strains for their ability to survive under different levels of NaCl (1-8% w/v). All the strains sustain 5% NaCl where as 8% NaCl inhibit growth. They found two tolerant groups, the first group of six isolates failed to tolerate more than 5% NaCl, where as, the second group of four isolates were able to tolerate up to 7% NaCl.

Wahab et al. (2002) concluded that high salts levels depressed the nodule number to weight to about 30% and 35%. Ashraf and Bashir (2003) reported that salt stress caused a marked reduction in nodule fresh mass and nodule number in both leguminous species, but nodules dry weight did not decrease significantly in both species under salt stress. Present reduction in nodule number due to salt stress was more in P. vulgaris than that in S. aculeata, but the reverse was true for nodule size. Comparison between vegetative parts and nodules shows that nodules were more sensitive to salt stress than shoot and roots while, studying the affect of salinity on growth, nodulation and nitrogen assimilation in nodules of faba bean.

Jenkins (2003) working with rhizobial and bradyrhizobial isolates observed that eight isolates showed decreased specific growth rates at NaCl concentration of 100, 300 and 500 mM, but nevertheless remained viable at 500 mM NaCl concentration. Tejera et al. (2004) working with CIAT889-derived mutants observed that all of them established symbiosis with reduced nitrogen-fixing capacity. These mutants were able to form nodules that looked normal in size and shape compared with wild-type nodules. However, the dry weights of nodules formed by DST strains were significantly reduced with respect to the wild-type strains, particularly in the case of mutants HB8 and HB13 in the presence of 25 or 50 mM/L NaCl.

Shamseldin and Werner (2005) observed no significant difference between the nodule numbers formed by the salt-sensitive strain EBRI 2 without salt and under stress of salt (0.2% NaCl). Aurag et al. (2005) observed differential response for NaCl treatment with in genotype, as shown by shoot and root dry weights. The growth of plants of the five cultivars tested was very much affected by salinity at 25 mM, whereas significant decrease in shoot and root dry weight was noticed. Bouhmouch et al. (2005) described nodule differentiation was also affected by salt, as evidenced by the appearance of white nodules which lost their pink colour (leghaemoglobin content). Application of salt completely inhibited nodule formation of salt-tolerant variety, and reduced significantly (P<0.05) the number of nodules and nodule dry weight over the control by 50-90% respectively. Dry weight reduction of nodules resulted from the low number low nodules and/or from the reduction of the size of nodules. Nodule weight per plant was more sensitive than nodule number.

Shamseldin and Werner (2005) found a high degree of diversity. They recoded two strains that were highly tolerant to salt concentration up to 4% NaCl. A positive correlation between the salt tolerance and the adaptation to alkaline pH was determined. Nitrogen fixation was much more affected by high salt concentration (0.4% NaCl). Hung et al. (2005) reported 28 salt tolerant rhizobial strains for 4.5% NaCl out of 83 strains of native shrubby legumes in Taiwan. Bolanos et al. (2006) reported that plants treated with salt developed about half number of nodules than plants growing in the absence of NaCl. Nodule weight also diminished in salt-stressed nodules and most of them appeared pale in the contrast with the control (without salt) pink nodules.

Kücük et al. (2006) studied 30 nodule isolates from Phaseolus vlgaris (L.) and find most of the isolates tolerant to high salt concentrations (5% NaCl). Bacem et al. (2007) isolated salt tolerant rhizobia from Tunisian oasis that are highly effective for symbiotic N2-fixation with Phaseolus vlgaris. Lopez et al. (2007) observed marked reduction in plant biomass and nitrogen fixation parameters of Lotus japonicus and Medicago truncatula under NaCl stress. They reported at harvest time (lowering stage), a decrease of approximately 40% in plant by dry weight (PDW) and root dry weight (RDW) with 25 mM NaCl. However, no significant differences were observed for PDW and RDW in L. japonicus between 25 and 50 mM between 25 and 50 mM treatments. M. truncatula nodule dry weight (NDW) was unaffected by salt stress, while L. japonicus NDW showed 40% decrease under salinity (Bouhmouch et al., 2005).

2.13.2. Total shoot nitrogen

Wahab and Zahran (1981) working with Vicia faba (L.), Medicago sativa (L.) Merrill,Glycine max and Vigna sinensis (L.) observed that salt stress retarded growth of both inoculated and N-fertilized plants. The nitrogen content of both treatments was also affected by salinity and the effect was more severe for inoculated than N-fertilized plant. Pessarakli and Zhou (1990) reported marked decrease in total shoot nitrogen content with NaCl stress in green beans.

2.13.3. Nitrogenase activity

It has also been shown that NaCl stress inhibited nitrogenase activity and nodule respiration (Serraj et al., 1994). Reduction in absolute nitrogenase activity, leghaemglobin content of nodules was found by Wahab et al. (2002). Jabara et al. (2005) also reported increasing inhibition of nitrogenase activity with increasing age of plant under salt stress. Shamseldin et al. (2005) reported strongly depressed nitrogenase activity for salt sensitive as well as salt resistant bacterial strain. Shamseldin and Werner (2005) determined that nitrogenase activity was strongly depressed at 0.4% NaCl with salt-sensitive EBRI 2 as well as with the salt-resistant EBRI 26(3.9 and 3.8 nM C2H4 h-1 mg-1, nodules). Tejera et al. (2005) investigated total nitrogenase activity. They found diversity for symbioses tested. Nodules formed by ILC1919 registered the highest specific nitrogenase activity, and nodules of Lechoso the lowest value, 44 and 18 mol C2H4 (g NDW) −1 h−1.

Bolanos et al. (2006) concluded that acetylene reduction activity, as a measurement of nitrogenase activity, was never detected in pea nodules developing in plants treated with 75 mM NaCl. Tejera et al. (2006) measured nitrogenase activity (ARA), on the flowering period, and was found more affected by NaCl than plant growth. The negative effect of salt was also observed in the total nitrogenase activity (ARAP) that decreased more than 90% with 100 mM NaCl treatment in all cultivars tested except in ILC1919 (60%). Lopez et al. (2007) working with growth and nitrogen fixation in Lotus, Medicago truncatula under NaCl stress reported marked decline in nitrogenase activity. Zilli et al. (2008) measured nitrogenase activity as indicators of nodules effectiveness for three salts concentrations. 60% decrease was recorded in nitrogenase activity at 200 mM salt, while no changes was observed neither in 50 mM nor in 100 mM NaCl, with respect to control values.

2.13.4. Leaf chlorophyll content

El-Hafid et al. (1998) observed a positive association between growth and photosynthetic capacity and a high salt tolerance has also been found in a number of crop species. In durum wheat Soussi et al. (1998) investigated affect of salt stress on growth, photosynthesis and nitrogen fixation in chickpea reported marked inhibition of chlorophyll accumulation. Yamane et al. (2004) working with salinity-induced chloroplast damages in rice leaves (Oryza sativa L.) reported drastic decrease in chlorophyll content and swelling as well as destruction of thylakoid membranes in leaves when subjected to 200 mM NaCl. Abdelkader et al. (2007) reported inhibition of chlorophyll accumulation in wheat under salt stress. Shahid et al. (2008) also reported significant decrease in chlorophyll concentration with increasing NaCl in pea (Pisum sativum cv. Meteor). Beinsan et al (2009) dealing with the physiology of tolerance to osmotic stress of some local landraces of Phaseolus vulgaris L. reported that in plants that grown under saline conditions, photosynthetic activity decreases leading to reduced plant growth, leaf area and chlorophyll content. The increase of NaCl concentrations produced a decrease of chlorophyll a and b concentrations. Taffouo et al. (2009) reported significant decrease in total chlorophyll (P<0.05) by addition of NaCl in the soil in cowpea.

2.14. Total soluble protein

Mothes (1956) suggested that decrease in soluble protein is due to protein break down under stress conditions. Udovenko et al. (1970) working with changes of root cell ultra structure under salinization in plants of different salt resistance states that salt stress reduces amino acid incorporation into proteins in V. faba and P. sativum. The effect of salt on soluble protein in the nodule is less when plants are grown with high KNO3 concentrations. Stewart and Lee (1979) investigated rate of proline accumulation in halophytes suggested that decrease in soluble protein may be due to an alteration in the incorporation of amino acids into proteins. Bourgeais-Chaillou et al. (1992) reported that decrease in soluble protein content of the nodules is a common response to salt stress.

2.15. Antioxidant enzyme

Puppo and Rigaud (1986) concluded that SOD is involved in the protection of cellular components that are crucial to the overall process of nitrogen fixation. As SOD appears generally correlated with superoxide anion production, the high levels of this enzyme can result either from higher superoxide anion production by the metabolism of nitrogen-fixing microorganisms. Dalton et al. (1996) observed that during early period of nodule development, ascorbate peroxidase, dehydroascorbate reductase activities and total glutathione contents of nodule extracts increased strikingly and were positively correlated with acetylene reduction rates and nodule leghemogloubin contents. According to these results ascorbate peroxidase, dehydroascorbate reductase activities, glutathione, dehydroascorbate reductase and glutathione reductase play an important role in defense mechanism in soybean root nodules.

According to Comba et al. (1998), antioxidant defense systems of soybean (Glycine max (L.) Merr) nodules responded differently to 50 and 200 mM NaCl. At 50 mM NaCl, leghemogloubin content and nitrogenase activity remained unchanged but there was an overall increase in the antioxidant enzymes (ascorbate peroxidase, catalase, glutathione reductase and superoxide dismutase) and in reduced glutathione. They further elaborated that salt treatment reduced the leghemogloubin content and nitrogenase activity by 31% and 50%, respectively. Ascorbate peroxidase (APX), catalase and glutathione reductase activities decreased between 30 and 100% while superoxide dismutase and reduced glutathione increased over the controls by 19% and 30% respectively. These results suggest that under mild saline stress, the elevated levels of the antioxidant enzymes and reduced glutathione protect nodules against the activated oxygen species thus avoiding lipid and protein peroxidation, and leghemogloubin breakdown. However, severe saline treatment produced an irreversible decay in the leghemogloubin content and nitrogenase activity despite the high reduced glutathione level and glutathione reductase activity.

Mittova et al. (2000) reported that higher SOD and APX ratio in all Lpa organelles contributes to the inherently better protection of Lpa from salt stress. Several researchers (Lee et al., 2000; Rubio et al., 2002) reported increase in SOD activity in plant tissues under salt stress. This enzyme converts superoxide radical to hydrogen peroxide (H2O2) and molecular oxygen (O2). Madhbi et al. (2004) worked out antioxidant enzyme activity in chickpea rhizobacteria. Their studies showed no affect of salt stress on the nodular (SOD) activity of the symbiosis implicating the latter strain. They reported least decrease for the (CAT) and the highest increase of (POD) activity. They related this with the tolerance to salt. Tejera et al. (2004) observed that over all, mutant nodules lower antioxidant enzyme activities that the wild type nodules. They found that the levels of nodule catalase correlate with symbiotic nitrogen fixation efficiency. Superoxide dismutase and dehydroacarbate reductase seem to function in the molecular mechanisms underlying the tolerance of nodules salinity.

Jebara et al. (2005) recorded changes in antioxidant enzyme activities in common bean and analyzed superoxide dismutase catalase, ascorbate peroxidase and peroxidase in nodule, roots and a free rhizobial strain. The result indicated that SOD and CAT nodular isozymes had bacterial and root origins. The SOD was expressed the same CuZn, Fe and MnSOD isoforms in nodules and root profiles. They concluded that plant growth; nitrogen fixation and antioxidant (defense) enzymes in nodules were affected by salt stress. According to them NaCl stress led to a differential regulation of SOD and POD isozymes.

Loscos et al. (2008) studied regulation of ascorbate and homoglutathione biosynthesis in common bean (Phaseolus vulgaris) nodules under stress conditions and during aging. They not only found ascorbate and glutathione as major antioxidants and redox buffers in plant cells but also involved in the growth, development and stress responses. Dehydroascorbate reductase activity was post translationally suppressed, ascorbate oxidase showed strong transcriptional up-regulation, and dehydroascorbate content increased moderately in the first stage, ascorbate decreased by 60% and homoglutathione and antioxidant activities during stress conditions remained fairly constant, whereas in the second stage ascorbate and homoglutathione, their redox state, and their associated enzyme activities significantly decreased.

2.16. SDS under salt

Protein content of bacteroids and cytosol were moderately affected by mild levels of NaCl and drought but significantly reduced to about 25-35% of the control treatments (Wahab et al., 2002). Shamseldin et al. (2006) studied that six proteins are highly expressed after induction by 4% NaCl compared to the non-salt-stressed cells with masses of approximately 22, 25, 40, 65, 70, and 95 kDa. Soluble proteins from salt-induced on non-salt-induced cultures from R. etli strain EBRI 26, separately were compared by 2D gel. Result revealed that 49 proteins are differently expressed after the addition of sodium. Fourteen proteins are over-expressed and 35 were downgraded.

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