Genetic diversity of rhizobia associated with indigenous legumes in different regions of Flanders (Belgium)

We investigated the diversity of rhizobia isolated from different indigenous legumes in Flanders (Belgium). A total of 3810 bacterial strains were analysed originating from 43 plant species. Based on rep-PCR clustering, 16S rRNA gene and recA gene sequence analysis, these isolates belonged to Bradyrhizobium, Ensifer (Sinorhizobium), Mesorhizobium and Rhizobium. Of the genera encountered, Rhizobium was the most abundant (62%) and especially the species Rhizobiumleguminosarum, followed by Ensifer (19%), Bradyrhizobium (14%) and finally Mesorhizobium (5%). For two rep-clusters only low similarity values with other genera were found for both the 16S rRNA and recA genes, suggesting that these may represent a new genus with close relationship to Rhodopseudomonas and Bradyrhizobium. Primers for the symbiotic genes nodC and nifH were optimized and a phylogenetic sequence analysis revealed the presence of different symbiovars including genistearum, glycinearum, loti, meliloti, officinalis, trifolii and viciae. Moreover, three new nodC types were assigned to strains originating from Ononis, Robinia and Wisteria, respectively. Discriminant and MANOVA analysis confirmed the correlation of symbiosis genes with certain bacterial genera and less with the host plant. Multiple symbiovars can be present within the same host plant, suggesting the promiscuity of these plants. Moreover, the ecoregion did not contribute to the separation of the bacterial endosymbionts. Our results reveal a large diversity of rhizobia associated with indigenous legumes in Flanders. Most of the legumes harboured more than one rhizobial endosymbiont in their root nodules indicating the importance of including sufficient isolates per plant in diversity studies.     

Diversity and stress tolerance in rhizobia from Parque Chaqueño region of Argentina nodulating Prosopis alba

The aim of this work was to investigate the genetic diversity, symbiotic effectiveness, drought tolerance, and indole acetic acid production of indigenous rhizobial populations in the Parque Chaqueño of Argentina able to nodulate Prosopis alba, the dominant forest tree of this region. The populations were sampled at five locations from the Arid, Semi-arid, and Humid Chaco in the Parque Chaqueño region. A set of rhizobial strains able to nodulate P. alba was obtained and selected based on their molecular diversity. Data obtained by BOX-PCR indicated that the highest molecular variability was observed in rhizobial isolates from Semi-arid Chaco. High level of indolic compound production and tolerance to osmotic treatment were significantly (p?≤?0.05) correlated with water restrictions of the environments where the strains belonged. A small set of rhizobial strains that stimulate P. alba growth was selected from a large group of strains. The strains were identified by 16S rDNA sequencing as belonging to the genera Mesorhizobium, Bradyrhizobium, and Ensifer. To our knowledge, this is the first report of P. alba nodulation by strains other than Mesorhizobium chacoense, which was already described for the Parque Chaqueño.     

Competition between inoculated and indigenous Rhizobium/Bradyrhizobium spp. strains for nodulation of grain and fodder legumes in Pakistan

Summary The competitive ability of inoculated and indigenous Rhizobium/Bradyrhizobium spp. to nodulate and fix N₂ in grain legumes (Glycine max, Vigna unguiculata, Phaseolus vulgaris) and fodder legumes (Vicia sativa, Medicago sativa, and Trifolium subterraneum) was studied in pots with two local soils collected from two different fields on the basis of cropping history. The native population was estimated by a most-probable-number plant infectivity test in growth pouches and culture tubes. The indigenous rhizobial/bradyrhizobial population ranged from 3 to 2×10⁴ and 0 to 4.4×10³ cells g^-1 in the two soils (the first with, the second without a history of legume cropping). Inoculated G. max, P. vulgaris, and T. subterraneum plants had significantly more nodules with a greater nodule mass than uninoculated plants, but N₂ fixation was increased only in G. max and P. vulgaris. A significant response to inoculation was observed in the grain legume P. vulgaris in the soil not previously used to grow legumes, even in the presence of higher indigenous population (>10³ cells g^-1 soil of Rhizobium leguminosarum bv phaseoli). No difference in yield was observed with the fodder legumes in response to inoculation, even with the indigenous Rhizobium sp. as low as <14 cells g^-1 soil and although the number and weight of nodules were significantly increased by the inoculation in T. subterraneum. Overall recovery of the inoculated strains was 38–100%, as determined by a fluorescent antibody technique. In general, the inoculation increased N₂ fixation only in 3 out of 12 legume species-soil combinations in the presence of an indigenous population of rhizobial/bradyrhizobial strains.     

Immediate Effects of Time and Method of Alfalfa Termination on Soil Mineral Nitrogen,Moisture, Weed Control,and Seed Yield,Quality, and Nitrogen Uptake

Three field experiments were conducted on Gray Luvisol (Typic Cryoboralf) soils in northeastern Saskatchewan to compare the effects of alfalfa (Medicago sativa Leyss) stand termination with tillage and herbicides at different times on mineral nitrogen (N) (ammonium-N and nitrate-N) and moisture content of soil in spring (experiments 1 and 2), soil moisture, volunteer alfalfa and dandelion control, plant density, seed yield, protein concentration and N uptake for wheat (Triticum aestivum L.), barley (Hordeum vulgare L), canola (Brassica rapa L.), and pea (Pisum sativum L.) crops (experiment 3). Termination treatments included combinations of times (in mid-June after cut 1, in mid-August after cut 2 and in mid-May during spring) and methods [tillage alone, herbicides alone (glyphosate + 2,4-D amine and also clopyralid + 2,4-D ester in experiment 3) and these herbicides + tillage]. Tillage alone significantly increased spring soil nitrate-N levels over herbicides alone or herbicides + tillage. Termination after cut 1 had the highest levels of soil nitrate-N. There was little effect of time and method of termination on soil ammonium-N and moisture content in spring. Herbicides + tillage generally provided better control of both volunteer alfalfa and dandelion in the four crops than tillage or herbicides alone. In general, alfalfa termination with herbicides alone significantly reduced plant density, seed yield, and N uptake of all crops and protein concentration of cereals only due to effects on levels of soil nitrate-N, dandelion control, and crop injury by clopyralid or 2,4-D residues in soil. Plant density, seed yield, N uptake and protein concentration of crops tended to decline with delay in termination time. The results of this study support the use of some tillage in alfalfa stand termination in helping to control volunteer alfalfa and dandelion and optimize annual crop yields and quality.     

Nodulation of Lotus pedunculatus in acid rooting solution by fast- and slow-growing rhizobia

The nodulation of Lotus pedunculatus and the multiplication of three Rhizobium loti (fast-growing, acid-producing) and two Bradyrhizobium (slow-growing, alkali-producing) strains was investigated in acidified rooting solution. R. loti strains multiplied at pH 4.5 but Bradyrhizobium strains failed to multiply. No difference in growth rate between R. loti and Bradyrhizobium strains was apparent in rooting solution at pH 6.7. Similar responses to pH were observed in yeast extract-mannitol broth except that Bradyrhizobium strains multiplied more slowly than R. loti at pH 6.7. All strains nodulated L. pedunculatus growing in acid (pH 4.5) rooting solution when presented as single cultures. Following inoculation with 1:1 mixtures of R. loti and Bradyrhizobium strains, R. loti formed 93% of nodules at pH 4.5 and significantly fewer nodules (66%) at pH 6.7. These results demonstrate a competitive advantage for acid-tolerant strains over acid-sensitive strains in nodulation of their lost legume at pH 4.5.     

Effects of resident rhizobial communities and soil type on the effective nodulation of pulse legumes

Communities of resident rhizobia capable of effective nodulation of pulse crops were found to vary considerably over a range of soil environments. These populations from soils at 50 sites in Southern Australia were evaluated for nitrogen fixing effectiveness in association with Pisum sativum, Vicia faba, Lens culinaris, Vicia sativa, Cicer arietinum and Lupinus angustifolius. The values for nitrogen fixing effectiveness could be related to soil pH as determined by soil type and location. It was found that 33% of paddocks had sufficient resident populations of Rhizobium leguminosarum bv viciae for effective nodulation of faba bean, 54% for lentils, 55% for field pea and 66% for the effective nodulation of the vetch host plant. Mesorhizobium cicer populations were very low with only 7% of paddocks surveyed having sufficient resident populations for effective nodulation. Low resident rhizobial populations (<10 rhizobia g⁻¹ soil) of R. leguminosarum bv viciae and M. cicer were found in acid soil conditions. In contrast, Bradyrhizobium populations increased as soil pH decreased. Inoculation increased faba bean yields from 0.34 to 4.4 t ha⁻¹ and from 0.47 to 2.37 t ha⁻¹ for chickpeas on acid soils. On alkaline soils, where resident populations were large there was no consistent response to inoculation. Observations at experimental field sites confirmed the findings from the survey data, stressing the importance of rhizobial inoculation, especially on the acid soils in south-eastern Australia.     

Nodulation status of native woody legumes and phenotypic characteristics of associated rhizobia in soils of southern Ethiopia

The nodulation of provenances of Acacia seyal, Acacia tortilis and Faidherbia albida, and other indigenous multipurpose tree species were tested in 14 different soil samples collected from diverse agro-ecological zones in southern Ethiopia. Associated rhizobia were isolated from these and from excavated nodules of field standing mature trees, and phenotypically characterized. Indigenous rhizobia capable of eliciting nodules on at least one or more of the woody legume species tested were present in most of the soils. Tree species were markedly different in nodulation in the different site soils. Sesbania sesban and Acacia abyssinica showed higher nodulation ability across the different sites indicating widespread occurrence of compatible rhizobia in the soils. The nodulation patterns of the different provenances of Acacia spp. suggested the existence of intraspecific provenance variations in rhizobial affinity which can be exploited to improve N fixation through tree selection. Altogether, 241 isolates were recovered from the root nodules of trap host species and from excavated nodules. Isolates were differentiated by growth rate and colony morphology and there were very fast-, fast-, slow-, and very slow-growing rhizobia. The bulk of them (68.5%) were fast-growing acid-producing rhizobia while 25.3% were slow-growing alkali-producing types. Fast-growing alkali-producing (2.9%) and slow-growing acid-producing strains (3.3%) were isolated from trap host species and excavated nodules, respectively. All isolates fell into four colony types: watery translucent, white translucent, dull glistering and milky (curdled) type. The diversity of indigenous rhizobia in growth rate and colony morphology suggested that the collection probably includes several rhizobial genera.     

<Emphasis Type="Italic">Rhizobium</Emphasis>,<Emphasis Type="Italic"> Bradyrhizobium</Emphasis> and<Emphasis Type="Italic"> Agrobacterium</Emphasis> strains isolated from cultivated legumes

The present study was conducted to isolate and characterize rhizobial strains from root nodules of cultivated legumes, i.e. chickpea, mungbean, pea and siratro. Preliminary characterization of these isolates was done on the basis of plant infectivity test, acetylene reduction assay, C-source utilization, phosphate solubilization, phytohormones and polysaccharide production. The plant infectivity test and acetylene reduction assay showed effective root nodule formation by all the isolates on their respective hosts, except for chickpea isolate Ca-18 that failed to infect its original host. All strains showed homology to a typical Rhizobium strain on the basis of growth pattern, C-source utilization and polysaccharide production. The strain Ca-18 was characterized by its phosphate solubilization and indole acetic acid (IAA) production. The genetic relationship of the six rhizobial strains was carried out by random amplified polymorphic DNA (RAPD) including a reference strain of Bradyrhizobium japonicum TAL-102. Analysis conducted with 60 primers discriminated between the strains of Rhizobium and Bradyrhizobium in two different clusters. One of the primers, OPB-5, yielded a unique RAPD pattern for the six strains and well discriminated the non-nodulating chickpea isolate Ca-18 from all the other nodulating rhizobial strains. Isolate Ca-18 showed the least homology of 15% and 18% with Rhizobium and Bradyrhizobium, respectively, and was probably not a (Brady)rhizobium strain. Partial 16S rRNA gene sequence analysis for MN-S, TAL-102 and Ca-18 strains showed 97% homology between MN-S and TAL-102 strains, supporting the view that they were strains of B. japonicum species. The non-infective isolate Ca-18 was 67% different from the other two strains and probably was an Agrobacterium strain.     

Substrate specificity of chlorophenoxyalkanoic acid-degrading bacteria is not dependent upon phylogenetically related tfdA gene types

The phenoxyalkanoic acid herbicides constitute a group of chemically related molecules that have been widely used for over 50 years. A range of bacteria have been selected from various locations for their ability to degrade these compounds. Previously reported strains able to utilise 2,4-dichlorophenoxyacetic acid (2,4-D) include, Ralstonia eutropha JMP134, Burkholderia sp. RASC and Variovorax paradoxus TV1 and Sphingomonas sp. AW5 able to utilise 2,4,5-trichlorophenoxyacetic acid (2,4,5-T). In addition a novel set of mecoprop-degrading strains including Alcaligenes denitrificans, Alcaligenes sp. CS1 and Ralstonia sp. CS2 are here described. It has been reported recently that TfdA enzymes, initially reported to have a role in 2,4-D catabolism are also involved in the first-step cleavage of related phenoxyalkanoate herbicides. However, a diversity of tfdA gene sequences have been reported. We relate the tfdA gene type to the metabolic ability of these strains. The tfdA-like genes were investigated by polymerase chain reaction amplification using a set of specific tfdA primers. Degradation ability was observed via phenol production from a range of unsubstituted and substituted phenoxyalkanoics including, 2,4-D, 2-methyl 4-chlorophenoxyacetic acid (MCPA), racemic mecoprop, (R)-mecoprop, 2-(2,4-dichlorophenoxy) propionic acid (racemic 2,4-DP), 2,4,5-T, 2,4-dichlorophenoxybutyric acid (2,4-DB), 4-chloro-2-methylphenoxybutyric acid (MCPB) and phenoxyacetate. Mecoprop-degrading strains showed partial tfdA sequences identical to the one described for V. paradoxus TV1 (a strain isolated on 2,4-D). However, substrate specificity was not identical as V. paradoxus exhibited greatest activity towards 2,4-D and MCPA only, whereas the mecoprop-degrading strains showed intense activity towards 2,4-D, MCPA, racemic mecoprop and (R)-mecoprop as substrates. However, Sphingomonas sp. AW5 which has been shown to carry a very different tfdA-like gene was the only strain to utilise the phenoxybutyric acid MCPB as a sole carbon source. In this study, we thus demonstrate that sequence diversity is not related to substrate specificity within the tfdA-like gene family. However, phylogenetically unrelated sequences may govern substrate specific activity.     

Simultaneous Biodegradation and Detoxification of the Herbicides 2,4-Dichlorophenoxyacetic Acid and 4-Chloro-2-Methylphenoxyacetic Acid in a Continuous Biofilm Reactor

The herbicides 2,4-diclorophenoxiacetic and 4-chloro-2-methylphenoxyacetic acids (2,4-D and MCPA) are widely used in agricultural practices worldwide. Not only are these practices responsible of surface waters contamination, but also agrochemical industries through the discharge of their liquid effluents. In this investigation, the ability of a 2,4-D degrading Delftia sp. strain to degrade the related compound MCPA and a mixture of both herbicides was assessed in batch reactors. The strain was also employed to remove and detoxify both herbicides from a synthetic effluent in a continuous reactor. Batch experiments were conducted in a 2-L aerobic microfermentor, at 28 °C. Continuous experiments were carried out in an aerobic downflow fixed-bed reactor. Bacterial growth was evaluated by the plate count method. Degradation of the compounds was evaluated by UV spectrophotometry, gas chromatography (GC), and chemical oxygen demand (COD). Toxicity was assessed before and after the continuous process by using Lactuca sativa seeds as test organisms. Delftia sp. was able to degrade 100 mg L^?1 of MCPA in 52 h. When the biodegradation assay was carried out with a mixture of 100 mg L^?1 of each herbicide, the process was accomplished in 56 h. In the continuous reactor, the strain showed high efficiency in the simultaneous removal of 100 mg L^?1 of each herbicide. Removals of 99.7, 99.5, and 95.0% were achieved for 2,4-D, MCPA, and COD, respectively. Samples from the influent of the continuous reactor showed high toxicity levels for Lactuca sativa seeds, while toxicity was not detected after the continuous process.     

Isolation and characterization of plant growth promoting endophytic bacterial isolates from root nodule of Lespedeza sp.

Thirty-nine endophytic bacterial strains were isolated from the nodule of Lespedeza sp. grown in two different locations of South Korea. All strains were checked for their plant growth promoting (PGP) abilities under in vitro conditions. Most of the isolates showed multiple PGP activity, i.e., indole acetic acid production, ACC deaminase activity, siderophore production, and phosphate solubilization. The strains were identified by using 16S rRNA gene sequence analysis as belonging to Alphaproteobacteria, Betaproteobacteria, Actinobacteria, and Firmicutes phylum with nine different genera Arthrobacter, Bacillus, Bradyrhizobium, Burkholderia, Dyella, Methylobacterium, Microbacterium, Rhizobium, and Staphylococcus. Gene nodA amplification showed positive results only for strains from Bradyrhizobium and Rhizobium genera. The strains from Bradyrhizobium and Rhizobium genera enhanced plant growth, nodulation, and acetylene reduction activity when inoculated on Vigna unguiculata L. (cowpea), whereas other strains did not induce nodule formation but enhanced plant growth. Herbaceous legume Lespedeza sp. formed root nodules with diverse bacterial group, and probably, these bacteria can be used for stimulating plant growth.     

Survey of genes identified in Sinorhizobium meliloti spp., necessaryfor the development of an efficient symbiosis

The symbiosis between the soil bacteria Rhizobium, Sinorhizobium, Azorhizobium, Mesorhizobium or Bradyrhizobium and leguminous plants is characterised by a specific multistep signal exchange. Only when a compatible rhizobial strain encounters its leguminous host, nodules will be formed on the roots of the host. During infection of this nodule, the microsymbiont evolves into a bacteroid form which, when provided with plant-derived carbon sources, is able to convert atmospheric nitrogen to ammonia that subsequently is supplied to the plant. The developmental programme underlying nodule organogenesis and functioning has been studied intensively for several decades. In this review, several observed plant phenotypes resulting from an ineffective symbiosis between plants and mutant rhizobial strains are represented. Besides the influence of the bacterial nodulation, nitrogen fixation and surface polysaccharide genes on symbiosis, the role of other genes important for the formation of effective nitrogen fixing nodules will be explained.     

Low pH and aluminum tolerance of Bradyrhizobium strains isolated from acid soils in Indonesia

Sixty-seven strains of Bradyrhizobium isolated from soybean plants growing on acid soils in West Java and Sumatra, Indonesia, were examined for the effect of the pH and aluminum concentration on their growth in nutrient media, compared with 61 strains of Bradyrhizobium from soils in Japan. The results in this study indicated that the indigenous population of Bradyrhizobium in the soils of Indonesia showed a large difference in acid- and Al-tolerance from that of Japan. Eighty-five and 48% of the isolates from Japanese soils and Indonesian soils, respectively, were unable to grow in YEM broth at pH below 4.5. The acid-tolerance was correlated with AI-tolerance of the isolates on YEM agar plates at pH 4.4. Seventy-five percent of the isolates that grew in YEM broth at pH 4.5 were also resistant to 400 µM Al on the YEM plates. Acetylene reduction assay of the root nodules revealed that 3 of the acid- and Al-tolerant isolates from Indonesian soils showed a significantly high nitrogen fixation activity.     

Utilization of trehalose, benzoate, valerate, and seed and root exudates by genotypes of 2,4-diacetylphloroglucinol producing Pseudomonas fluorescens

Isolates of Pseudomonas fluorescens producing the antibiotic 2,4-diacetylphloroglucinol (2,4-DAPG) are effective biocontrol agents against soilborne pathogens. A previous study showed that the superior (“premier”) root colonizer P. fluorescens Q8r1-96 (genotype D) utilized trehalose, benzoate and valerate as sole carbon sources but average colonizers Q2-87 (genotype B) and 1M1-96 (genotype L) did not. We tested the utilization of these three carbon sources by a collection of 55 2,4-DAPG-producing P. fluorescens strains from 17 genotypes and found no correlation between a strain's ability to utilize these carbon sources and superior rhizosphere competence on wheat and pea. Of the strains tested, 73%, 48% and 69% were able to utilize trehalose, benzoate and valerate as sole carbon sources, respectively. With some exceptions, we found a correlation between the utilization of these compounds and previous groupings of these strains by BOX-PCR; genotype D strains utilized all three compounds. Twenty-three strains grew efficiently on root and seed exudates from wheat and pea, with doubling times between 0.9 and 1.6 h generation⁻¹ and lag phases between 5 and 8 h, comparable to growth on glucose as a sole carbon source. Only 10 strains, including those with “premier” (Q8r1-96) and “average” (Q2-87) rhizosphere competence, showed slower growth in wheat root exudates, with lag phases between 16 and 22 h. Results were the same when soil was added to the culture medium. Growth of four strains in media containing glucose or wheat or pea seed exudates as a sole carbon source was not influenced by whether the bacterial cells used as inoculum were harvested from wheat seeds or broth culture. We conclude that the superior ability of some strains to colonize the roots of certain crops cannot be explained by the utilization of the carbon sources tested in our study.     

Root-temperature effects on competition for nodule occupancy between two Rhizobium meliloti strains

Summary Axenically grown alfalfa (Medicago sativa L. var. Peace) was simultaneously inoculated with Canadian commercial Rhizobium meliloti strains NRG-185 and BALSAC. The plants were grown for 7 weeks in sealed units at five different root temperatures (8°, 13°, 17°, 21°, and 25°C) and at a relatively constant air temperature (24°–30°C). Nodule occupancy by each strain was determined by enzyme-linked immunosorbent assay (ELISA). Nitrogenase activity, nodule fresh weight, and plant dry weight were also measured. The lowest root-temperature regime (8°C) resulted in substantially lower nodule numbers and weights, and plant dry weights, than the higher temperature regimes. Development of nitrogenase activity was completely inhibited at 8°C. The immunoassay of nodule-strain occupancy showed markedly different strain-nodulation responses to the various root-temperature regimes. At 8°C, 63% of nodules were occupied by both strains. Dual strain occupancy decreased from 63% to 2% with increasing root-growth temperature, while the proportion of nodules containing only strain NRG-185 increased from 9% to 75%. Nodules containing only strain BALSAC remained relatively constant at 25% from 8° to 21°C, decreasing slightly at 25°C. The results suggest that root-environment temperatures during the period of nodule formation may have major differential effects on the success of competing rhizobial strains. If this is so, then selection of Rhizobium strains with enhanced low-temperature nodulation capabilities should be possible.     

Responses of Lentil to Co-Inoculation with Phosphate-Solubilizing Rhizobial Strains and Arbuscular Mycorrhizal Fungi

The purpose of this study was to evaluate the responses of lentil (Lens culinariscv. ‘Ziba’) to co-inoculation with arbuscular mycorrhizal (AM) fungi and some indigenous rhizobial strains varying in phosphorus (P)-solubilizing ability in a calcareous soil with high pH and low amounts of available P and nitrogen (N). A factorial experiment with completely randomized block design was conducted under controlled greenhouse conditions. The treatments consisted of (1) three inoculants of Rhizobium leguminosarum bv. viciae strains and a mixed rhizobial inoculant with an effective P-solubilizer strain of Mesorhizobium ciceri, (2) two AM fungal species, Glomus mosseae and Glomus intraradices, (3) two P sources, superphosphate and phosphate rock. Four replications were prepared for each treatment and a related control. After the growth period of three months, the dry matter of shoots plus seeds, their P and N contents, and percent of root colonized by AM fungus were measured. The results showed that the effects of AM fungi, rhizobial strains, and P fertilizers were highly significant (p < 0.01) for all the characteristics studied. The rhizobial strain with P-solubilizing ability showed a more beneficial effect on plant growth and nutrient uptake than the strain without this ability, although both strains had similar effectiveness for N₂-fixation in symbiosis with lentil. Synergistic relationships were observed between AM fungi and some rhizobial strains that related to the compatible pairing of these two microsymbionts. The P-uptake efficiency was increased when P fertilizers were applied along with AM fungi and/or P-solubilizer rhizobial strains.     

Host specificity and phenotypic diversity of Rhizobium strains nodulating Leucaena, Acacia, and Sesbania in Egypt

Phenotypic diversity was studied among 13 Rhizobium strains selected from a total of 160 Rhizobium isolates from root nodules of Leucaena leucocephala. Two strains from Acacia saligna and two strains from Sesbania sesban plants were included in the examination for host range in the greenhouse. The Rhizobium sp. (Leucaena) strains were different from the reference strains and fell into three distinct groups for the utilization of 95 different carbon sources. Four of the best symbionts constituted a group, the majority of the strains fell into a second group, and strain DS 91 was the only member of the third group. Strains were effective symbionts for their original hosts. Nine strains were tolerant to elevated temperature (>42°C), and three strains were resistant to high salinity (>3% NaCl). All Rhizobium sp. (Leucaena) strains effectively nodulated L. leucocephala and L. culensii, but nitrogen fixation was greater with L. leucocephala than with L. culensii. These strains failed to form effective symbioses with two other species of Leucaena (L. retusa and L. divursiflora) or with alfalfa, Medicago sativa. Rhizobium sp. (Leucaena) strains DS 65, DS 78, and DS 158 nodulated and efficiently fixed nitrogen with Phaseolus vulgaris, with DS 65 showing the highest symbiotic capability. Strain DS 65 also nodulated and fixed nitrogen with Glycine max and Vigna sinensis. Nodulation of Leucaena by two Bradyrhizobium sp. (Acacia) strains was sparse. Strain DS 101 from Sesbania formed nodules on Leucaena, whereas the other strain from Sesbania, DS 110, failed to nodulate this genus. Received: 30 September 1996     

Temperature Effects on Bradyrhizobium spp. Growth and Symbiotic Effectiveness with Pigeonpea and Cowpea

Temperature is a limiting factor on legume-Bradyrhizobium symbiosis of subtropical plants in the temperate region. Twelve strains of Bradyrhizobium spp. that nodulate pigeonpea [Cajanus cajan (L.) Millsp], and cowpea [Vigna unguiculata (L.) Walp], were evaluated for tolerance to three temperature regimes (20°C/10°C, 30°C/20°C, and 38°C/25°C day/night temperature) by determining their growth following exposure to the regimes. The five most temperature-tolerant strains were further evaluated for symbiotic effectiveness with pigeonpea and cowpea under controlled temperatures. These strains were USDA 3278, USDA 3362, USDA 3364, USDA 3458, and USDA 3472. Plant heights of both crops were generally independent of Bradyrhizobium strains and were dependent mainly on temperature regimes. Plant heights were the shortest at the lowest temperature. At the lowest temperature regime, biological nitrogen (N) fixation by pigeonpea was almost completely inhibited. Cowpea genotype IT82E-16 inoculated with USDA 3458 formed the most effective symbiosis. The 30°C/20°C temperature regime was optimum for effective symbiotic association in both crops, and also for Bradyrhizobium survival.     

Investigation on Phosphate Solubilization Potential of Agricultural Soil Bacteria as Affected by Different Phosphorus Sources,Temperature, Salt,and pH

Ten phosphate-solubilizing bacterial strains belonging to genera Pseudomonas, Burkholderia, Enterobacter, Serratia, Klebsiella, and Aeromonas were tested for mineral phosphate solubilization activity in Pikovskaya's broth using different phosphate sources at four temperatures (15, 25, 35, and 45 ^οC). Dicalcium and tricalcium phosphate were solubilized more effectively (≥1000 mg L^?1) than ferric and rock phosphate (≥100 mg L^?1) and 35 °C was found to be the optimum temperature. Although Klebsiella and Aeromonas spp. are well known for their dinitrogen (N₂)–fixing ability, to the best of our knowledge, this is the first report of inorganic phosphate solubilization by Klebsiella terrigena and Aeromonas vaga. Interestingly, A. vaga BAM-77 is the most efficient strain at solubilizing inorganic phosphorus (P) even in the presence of 8% sodium chloride (NaCl) at pH 10. These findings indicate that all four strains are efficient P solubilizers under variable conditions of temperature, pH, and P source and thus can be recommended for P fertilization in different soils.     

Genetic diversity of rhizobia associated with indigenous legumes in different regions of Flanders (Belgium)

We investigated the diversity of rhizobia isolated from different indigenous legumes in Flanders (Belgium). A total of 3810 bacterial strains were analysed originating from 43 plant species. Based on rep-PCR clustering, 16S rRNA gene and recA gene sequence analysis, these isolates belonged to Bradyrhizobium, Ensifer (Sinorhizobium), Mesorhizobium and Rhizobium. Of the genera encountered, Rhizobium was the most abundant (62%) and especially the species Rhizobium leguminosarum, followed by Ensifer (19%), Bradyrhizobium (14%) and finally Mesorhizobium (5%). For two rep-clusters only low similarity values with other genera were found for both the 16S rRNA and recA genes, suggesting that these may represent a new genus with close relationship to Rhodopseudomonas and Bradyrhizobium. Primers for the symbiotic genes nodC and nifH were optimized and a phylogenetic sequence analysis revealed the presence of different symbiovars including genistearum, glycinearum, loti, meliloti, officinalis, trifolii and viciae. Moreover, three new nodC types were assigned to strains originating from Ononis, Robinia and Wisteria, respectively. Discriminant and MANOVA analysis confirmed the correlation of symbiosis genes with certain bacterial genera and less with the host plant. Multiple symbiovars can be present within the same host plant, suggesting the promiscuity of these plants. Moreover, the ecoregion did not contribute to the separation of the bacterial endosymbionts. Our results reveal a large diversity of rhizobia associated with indigenous legumes in Flanders. Most of the legumes harboured more than one rhizobial endosymbiont in their root nodules indicating the importance of including sufficient isolates per plant in diversity studies.