Metabolic and physiological characteristics of salt-tolerant strains of Bradyrhizobium spp.

 Biochemical and physiological characteristics of salt-tolerant (88 mM, 264 mM and 440 mM NaCl) strains of Bradyrhizobium were evaluated according to their capacity for using different carbon sources, growth rate, resistance to antibiotics, plasmid profile and exopolysaccharide production. Salt-tolerant strains significantly enhance their capacity to oxidize C sources (about 75–85 compounds) by increasing growth rate and exopolysaccharide production involved in adhesion, resulting in a greater adapting capacity to colonize unfavorable saline environments. However, salt stress could work as a curing agent and thus the gene stability would become critical for the biological nitrogen-fixation information present in plasmids, as is the case in Rhizobium. Received: 5 January 1998     

Soil aggregate as a favorable habitat for Bradyrhizobium japonicum strains

Abstract

Rhizobial cells are present in soils as saprophytes after the decay of host plant nodules, and must survive in the soil until the next encounter with the infection sites of the host plant root. Biotic and abiotic environmental factors affect the population size of these rhizobia in the soil (Vincent 1977). Precise estimation of the population size of the native and the introduced rhizobia in the soil is necessary to study the conditions for the successful nodule formation by introduced strains.     

Determination of competitive abilities of Bradyrhizobium japonicum strains in soils from soybean production regions in South Africa

Bradyrhizobium japonicum strain CB 1809 was recently chosen to replace strain WB 1 in commercial soybean [Glycine max (L.) Merr.] inoculants in South Africa, the selection criterion being N₂-fixing effectiveness. Nodulation competitiveness is an additional characteristic required of inoculants and was determined for CB 1809 and WB 1 as well as two other strains, USDA 110 and a Brazilian strain 965, using the gusA marker gene to identify strains. Initial experiments with plants grown in sterile sand showed that the competitive index of strain WB 1 was less than that of the other strains. Further comparisons used plants grown in five soils containing established populations of B. japonicum. When strains were applied in peat inoculum to seed at a rate of 1,000 cells per seed in a soil containing 300 rhizobia g^–1, significant differences in nodule occupancy were detected and strains ranked in the order 965>CB 1809>USDA 110>WB 1. The remaining four soils each contained about 10⁶ rhizobia g^–1 and 5×10⁶ cells were applied per seed. Nodule occupancy by inoculant strains ranged from 22% to 81% between soils. In this experiment, WB 1 was consistently the poorest performer and its competitiveness was significantly less than CB 1809. The competition results supported the recent decision to replace WB 1 with CB 1809 in commercial inoculants. Although WB 1 had been used in inoculants over a period of 19 years, this strain was detected in only one soil, where it comprised 8% of isolates. In contrast, a substantial proportion (32–78%) of isolates from the soils corresponded serologically to a former inoculant strain WB 66, which had been discontinued in 1966. This illustrates the difficulty of replacing a resident population with an introduced strain. The effect of naturalized populations on the establishment of CB 1809 in South African soils will need monitoring Received: 23 November 1999     

Competitiveness of native Bradyrhizobium japonicum strains in two different soil types

Interstrain competitiveness is a key factor affecting the performance of rhizobium inoculant. In the present study five native strains of Bradyrhizobium japonicum, namely SSF 4, SSF 5, SSF 6, SSF 7 and SSF 8, were assessed for their competitiveness in nodulating soybean using serological methods. The strains were inoculated individually or with the type strain USDA 110 at a 1:1 ratio. Nodule occupancy determined by immunofluorescence and dot immunoblot assay revealed that under in vitro conditions SSF 8 is more competitive than USDA 110 whereas the others were less competitive. The competitive ability of these strains was also estimated in pot culture in the field. In red soil both SSF 8 and USDA 110 were equally competitive whereas in black soil SSF 8 competed better than USDA 110 and produced more nodules. In a black soil field trial using a randomized block design, USDA 110 or SSF 8, when inoculated alone, occupied the majority of the nodules and enhanced nodule dry weight and shoot biomass. SSF 8 was more competitive when the strains were co-inoculated. Received: 1 November 1996     

Water-facilitated dispersal of inoculant Bradyrhizobium japonicum in soils

Summary Studies were performed to assess the influence of percolating water and an advancing wetting front on the transport of Bradyrhizobium japonicum in sand and silt loam soils, and to assess the influence of clay content on water-facilitated dispersal of these bacteria in a sand amended with various amounts of kaolin. The data obtained showed that movement of B. japonicum in soil was dependent upon water movement and that both percolating water and an advancing wetting front readily transported bacteria in coarse-textured soils. Percolation with the equivalent of 10 cm of rainfall dispersed B. japonicum throughout 40-cm columns containing sand and silt loam soils. Percolation with 5 cm of water was sufficient to disperse B. japonicum throughout 20-cm columns of these soils but did not transport these bacteria below the surface 4 cm of a sand amended with 12% kaolin. Our finding that cells of B. japonicum are readily transported by an advancing wetting front indicates that non-saturated flow of soil water contributes to dispersal of inoculum in soils.     

Denitrifying ability of indigenous strains of Bradyrhizobium japonicum isolated from fields under paddy-upland rotation

Summary In Japan some paddy fields are used for upland crops for several years and then returned to paddy fields (paddy-upland rotation). Soybeans (Glycine max L.) are an important summer crop. The ability to denitrify and some characteristics of denitrification by isolated strains of Bradyrhizobium japonicum were investigated to clarify the frequency of denitrifiers in indigenous populations of B. japonicum in fileds under paddyupland rotation. Eight field plots with different cropping systems at two sites were used. The fields consisted of a Gray Lowland Soil, and either soybeans or paddy rice (Oryza sativa L.) was grown as a summer crop, with barley (Hordeum distichum L.) or wheat (Triticum aestivum L.) as a winter crop. All B. japonicum strains present in the plots were able to denitrify. Isolated strains fell into two main groups (groups I and II) according to the rate of denitrification. Strains of group I evolved N₂O with C₂H₂ at a rate comparable to that of Alcaligenes denitrificans IAM 12370, whereas the denitrification activity of group II strains was 100 times lower than that of group I strains. Both group I and II strains occurred in each plot. Amounts of N₂O produced by indigenous strains with and without C₂H₂ suggested that strains of group I and II evolved N₂ or N₂O, respectively, as the end product of denitrification. One strain (S 107) that was isolated had the highest denitrifying ability with an end product of N₂O. These results indicate that indigenous bradyrhizobia may partly contribute to denitrification of field soil under a paddy-upland rotation.     

Transformation of added phosphorus to acid upland soils with different soil properties in Indonesia

Abstract

The transformation of added phosphorus (P) to soil and the effect of soil properties on P transformations were investigated for 15 acid upland soils with different physicochemical properties from Indonesia. Based on oxide-related factor scores (aluminum (Al) plus 1/2 iron (Fe) (by ammonium oxalate), crystalline Al and Fe oxides, cation exchange capacity, and clay content) obtained from previous principal component analyses, soils were divided into two groups, namely Group 1 for soils with positive factor scores and Group 2 for those with negative factor scores. The amounts of soil P in different fractions were determined by: (i) resin strip in bicarbonate form in 30 mL distilled water followed by extraction with 0.5 mol L^?1 HCl (resin-P inorganic (P_i) that is readily available to plant), (ii) 0.5 mol L^?1 NaHCO₃ extracting P_{i and} P organic (P_o) (P which is strongly related to P uptake by plants and microbes and bound to mineral surface or precipitated Ca-P and Mg forms), (iii) 0.1 mol L^?1 NaOH extracting P_i and P_o (P which is more strongly held by chemisorption to Fe and Al components of soil surface) and (iv) 1 mol L^?1 HCl extracting P_i (Ca-P of low solubility). The transformation of added P (300 mg P kg^?1) into other fractions was studied by the recovery of P fractions after 1, 7, 30, and 90 d incubation. After 90 d incubation, most of the added P was transformed into NaOH-P_i fraction for soils of Group 1, while for soils of Group 2, it was transformed into resin-P_i, NaHCO₃-P_i and NaOH-P_i fractions in comparable amounts. The equilibrium of added P transformation was reached in 30 d incubation for soils of Group 1, while for soils of Group 2 it needed a longer time. Oxide-related factor scores were positively correlated with the rate constant (k) of P transformation and the recovery of NaOH-P_i. Additionally, not only the amount of but also the type (kaolinitic) of clay were positively correlated with the k value and P accumulation into NaOH-P_i. Soils developed from andesite and volcanic ash exhibited significantly higher NaOH-P_i than soils developed from granite, volcanic sediments and sedimentary rocks. Soil properties summarized as oxides-related factor, parent material, and clay mineralogy were concluded very important in assessing P transformation and P accumulation in acid upland soils in Indonesia.     

Nodulation of African yam bean (Sphenostylis stenocarpa) by Bradyrhizobium sp. isolated from Erythrina brucei

African yam bean (Sphenostylis stenocarpa), which is widely cultivated in Africa because of its growth capability on marginal soils, was nodulated by an endosymbiont (characterized and designed Bradyrhizobium sp. AUEB20) isolated from the Ethiopian tree Erythrina brucei with the formation of a small number of large, indeterminate N₂-fixing nodules. In contrast, 24 other isolates from Ethiopian woody legumes were ineffective. Strain AUEB20 promiscuously nodulated a number of tropical legumes, but none out of five European crop plants tested. Received: 17 September 1996     

Diversity of indigenous Bradyrhizobium japonicum in North Carolina soils

Summary The diversity of Bradyrhizobium japonicum in agricultural fields has not been well characterized. Therefore a study was conducted to determine the serotypic diversity of B. japonicum both within and among six fields in the Coastal Plain and Piedmont of North Carolina where soybeans [Glycine max (L.) Merr.] are grown. Nodule samples were collected from non-inoculated standing soybean crops. Both nodules and isolates were typed by the enzyme-linked immunosorbent assay (ELISA) technique. Serotypes and their proportions varied both within and among locations. Common serotypes in order of abundance across all sites were 76, M1 (multiple reaction beyween 31 and 94), 94, 24, and 122, and together accounted for over 66% of the typable reactions. No cultivar effect on serotype distribution was observed. Unknown types ranged from 4 to 24%. Based on the total number of serotypes identified and the Shannon diversity index (H), the mean population diversity was 0.76 for the Piedmont sites and 0.91 for the Coastal Plain sites.Paper no. 12315 of the Journal Series of the North Carolina Agricultural Research Service, Raleigh, NC 27695-7643. The use of trade names in this publication does not imply endorsement by the North Carolina Agricultural Research Service of the products named or criticism of similar ones not mentioned     

Survival and symbiotic properties of Bradyrhizobium japonicum in the presence of thiram: isolation of fungicide resistant strains

The fungicide thiram, widely used as a chemical seed protectant, induces a strong inhibition of primary nodulation in the crown zone of soybean roots. The present work reports on the isolation of Bradyrhizobium japonicum strains resistant to thiram, some of which (T3B, A86 and A2) maintained their capacity for nodulation and were still efficient symbionts, but some (A1, C1 and C6) lost the ability to stimulate nodulation. Characterization tests such as growth at different pH, denitrifying ability, salt tolerance, production of siderophores and phosphate solubilization were performed on the resistant strains. Inoculants produced from these strains could be appropriate for use with thiram-treated seeds, without causing a loss of bacteria viability. Received: 16 September 1996     

Comparison of aluminum tolerance and phosphate absorption between rape (Brassica napus L.) and Tomato (Lycopersicum esculentum Mill.) in relation to organic acid exudation

Aluminum (Al) tolerance and phosphate absorption in rape and tomato were compared under water culture and field conditions. The relative growth rate in the Al treatment compared with -A1 treatment was similar in the two crops under water culture conditions, while under field conditions, the growth rate was 2- to 3-fold higher in rape than in tomato in spite of the higher Al concentration in the soil solution than in the culture solution. The relative amount of phosphate absorbed in the Al treatment compared with - Al was not appreciably different between rape and tomato under water culture conditions, while under field conditions, it was 3- to 6-fold larger in rape than in tomato. The exudation rate of citric acid by roots was much higher in rape than in tomato. The plant growth, root elongation, and amount of phosphate absorbed in rape were inhibited in the 150 µM Al in the culture solution. However, the inhibition was alleviated by the addition of 200 µM citric acid or 500 µM malic acid. The P concentration in the culture solution decreased by the presence of Al as aluminum phosphate. However, addition of citric and malic acids increased the amount of phosphate released from the precipitated aluminum phosphate. In conclusion, one of the mechanisms for the higher Al tolerance and larger phosphate absorption in rape than in tomato under field conditions was ascribed to the higher concentration of exuded citric acid by Al in the rhizosphere. It was suggested that the exudation of citric acid might contribute to the detoxification of Al and to the increase phosphate availability in the rhizosphere in rape.     

Genetic diversity and relationship between Bradyrhizobium strains isolated from blackgram and cowpea

The genetic diversity of bradyrhizobial strains associated with blackgram and cowpea grown in two different agricultural soils (non-saline and saline) along the coastline of Tamil Nadu has been analysed. Phenotypically indistinguishable isolates were analysed for DNA polymorphism using random amplification of polymorphic DNA (RAPD) and polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) of 16S rDNA and nifD. Although these bacteria belong to a group with a broad host range, RAPD analysis showed a considerable level of genetic diversity among the strains isolated from different host plants. Soil pH and salinity seem to have an effect on the selection of natural populations as revealed by PCR-RFLP of 16S rDNA. A combination of PCR-RFLP genotyping with nodulation studies indicates that monocropping of blackgram and the salinity of the soil have made ineffective rhizobia the dominant genotype, thereby creating an ecological burden on their other compatible hosts. A group of strains and a type strain sharing three different 16S PCR-RFLP types were shown to have the same set of symbiotic genes as inferred from the PCR-RFLP pattern of nifD. Another group of cowpea rhizobia that were found to be effective nitrogen fixers and sharing distinct 16S profiles were found to have a different set of symbiotic genes.     

Genetic diversity within Aspergillus flavus strains isolated from peanut-cropped soils in Argentina

The genetic diversity of Aspergillus flavus populations isolated from the peanut-cropped soils in the peanut-growing region at Cordoba Province was evaluated by analysis of vegetative compatibility group (VCG). VCG_s were determined through complementation assays between nitrate-nonutilizing (NNO) mutants. Fifty-six VCG_s were identified from 100 isolates. Twenty-five VCG_s contained two or more isolates and 31 VCG_s contained only a single isolate. In general, there were significant differences among VCG_s in aflatoxin and CPA production. One VCG group included a single atoxigenic strain since it was neither aflatoxin nor cyclopiazonic acid producer. This isolate could be useful as a biological control agent, since it was unable to form a stable heterokaryon in the complementation test with the other isolates. Seven A. flavus isolated from soil were atypical because they simultaneously produced aflatoxins B, G and CPA.     

Beneficial effect of aluminum on growth of plants adapted to low pH soils

Plants in which growth was reduced by low and high Al applications were designated as Al-sensitive plant (Hordeum vulgare) and Al-medium tolerant plants (Leucaena leucocephala, Ischaemum barbatum, Stylosanthes guianensis, and Fagopyrum esculentum), respectively, while plants in which growth was not affected or was stimulated by Al application were designated as Al-tolerant plant (Brachiaria ruziziensis) and Al-stimulated plants (Melastoma malabathricum, Melaleuca cajuputi, Acacia mangium, Hydrangea macrophyila, Vaccinium macrocarpon, Polygonum sachalinense, and Oryza sativa), respectively. Plants tolerant to or stimulated by Al were further classified based on the criteria of Al accumulation: 1) Al-excluders such as M. cajuputi, A. mangium, L. leucocephala, I. barbatum, S. guianensis, and O. sativa, 2) Al root-accumulators such as V. màcrocarpon, B. ruziziensis, and P. sachalinense, and 3) Al-accumulators such as M. malabathricum, H. macrophylla, and F. esculentum. The growth and N, P, and K uptake in M. malabathricum, M. cajuputi, A. mangium, L. leucocephala, H. macrophylla, V. macrocarpon, I. barbatum, P. sachalinense, F. esculentum, and O. sativa were stimulated by Al application, especially P uptake, while in H. vulgare (Al-sensitive plant) they were reduced by Al application. Ca and Mg uptake of many plants was inhibited by Al application, while that of some plants adapted to low pH soils was not affected at all (Ca and Mg: M. cajuputi, H. macrophylla, V. macrocarpon, I. barbatum, and S. guianensis; Mg: B. ruziziensis and P. sachalinense). In M. malabathricum, the relationship between Al and Ca (or Mg) was antagonistic because the Ca and Mg contents decreased by Al application even though dry matter, N, P, and K accumulation was stimulated by Al application. Plants adapted to low pH soils grew poorly in the no-Al treatment. Since the effect of the pH on plant growth was less conspicuous than that of Al, growth stimulation by Al application was ascribed not only to the alleviation of H⁺ toxicity but also to the increase of root activity such as P uptake.     

Effects of nitrogen source and aluminum on growth of tropical tree seedlings adapted to low pH soils

The effects of N-source and Al on the growth of seedlings of Melastoma malabathricum, Acacia mangium, and Melaleuca cajuputi, which are tropical woody plants and are very tolerant to Al, and barley (Hordeum vulgare), which is a typical Al-sensitive plant, were investigated. The Al and N treatments consisted of the application of either 0 or 0.5 mM Al, and 2 mM NH₄ ⁺ or N0₃ ^-, respectively. Growth of the tropical plants was enhanced by Al and NH₄ application. In all the plant species, the pH of the culture solution decreased and the concentrations of soluble Al and P increased with the + NH₄ treatment, which positively affected the growth of the tropical plant species. Excised roots of M. malabathricum dissolved insoluble Al with NH₄ application and absorbed Al mainly from root tips. Al did not affect the leaf N concentration except in the case of barley. Roots of M. cajuputi exuded a large amount of citrate, which slightly increased by the + Al treatment. In A. mangium, the reactivity of soluble Al to PCV (pyrocatecholviolet) decreased in the culture solution of the + Al + NH₄. treatment and Al concentration of roots in this treatment was very low. Roots of M. malabathricum released H+ along with Al uptake as well as NH₄ ⁺ uptake. It is concluded that Al and NH₄ ⁺ exert beneficial effects on the growth of tropical tree seedlings.     

Effect of soybean inoculation with Bradyrhizobium and wheat inoculation with Azotobacter on their productivity and N turnover in a Vertisol

A long-term field experiment was conducted for 8 years on a Vertisol in central India to assess quantitatively the direct and residual N effects of soybean inoculation with Bradyrhizobium and wheat inoculation with Azotobacter in a soybean–wheat rotation. After cultivation of soybean each year, its aerial residues were removed before growing wheat in the same plots using four N levels (120, 90, 60 and 30 kg ha^?1) and Azotobacter inoculation. Inoculation of soybean increased grain yield by 10.1% (180 kg ha^?1), but the increase in wheat yields with inoculation was only marginal (5.6%; 278 kg ha^?1). There was always a positive balance of soil N after soybean harvest; an average of +28 kg N ha^?1 yr^?1 in control (nodulated by native rhizobia) plots compared with +41 kg N ha^?1 yr^?1 in Rhizobium-inoculated plots. Residual and direct effects of Rhizobium and Azotobacter inoculants caused a fertilizer N credit of 30 kg ha^?1 in wheat. Application of fertilizers or microbial inoculation favoured the proliferation of rhizobia in crop rhizosphere due to better plant growth. Additional N uptake by inoculation was 14.9 kg N ha^?1 by soybean and 20.9 kg N ha^?1 by wheat crop, and a gain of +38.0 kg N ha^?1 yr^?1 to the 0–15 cm soil layer was measured after harvest of wheat. So, total N contribution to crops and soil due to the inoculants was 73.8 kg N ha^?1 yr^?1 after one soybean–wheat rotation. There was a total N benefit of 13.8 kg N ha^?1 yr^?1 to the soil due to regular long-term use of microbial inoculants in soybean–wheat rotation.     

Use of the fluorescent antibody technique to characterize equilibrium survival concentrations of Bradyrhizobium japonicum strains in soil

Summary The ability of Bradyrhizobium strains to survive saprophytically in soil was studied by means of fluorescent antibodies (FA). It was found that the recovery rate may be considered a constant value although the limit of detection by the FA technique is approximately 10³ bacteria g^–1 soil. By studying the survival kinetics of B. japonicum strains introduced into soils, we observed that whichever soil-strain combination was tested in a given soil during the incubation all the different populations of a strain reached the same survival balance level, generally about 10³–10⁴ Bradyrhizobium g^–1 soil. When we reintroduced strains into a soil containing rhizobia of the same specificity, the new inhabitants reached the same equilibrium level as that of the initial population. The balance threshold level does not appear to be a very sensitive way to classify, strains on their saprophytic ability. We suggest that survival kinetics should be characterized by the rate at which the population reaches equilibrium.