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.     

Competitiveness of Rhizobium leguminosarum bv. phaseoli strains in relation to environmental stress and plant defense mechanisms

Summary Six Rhizobium leguminosarum bv. phaseoli strains (Ciat 151, Ciat 895, Ciat 899, CE3, H2C, Kim5s) were tested for nodule occupancy in different bean cultivars at two field sites (one fertile, one acid tropical soil) and in the greenhouse. The effects of several environmental factors such as low pH, high temperature, Al and Mn toxicity, iron deficiency, bean tannins, and bean phytoalexins were tested in vitro. Strain Kim5s was competitive under all tested conditions while strains CE3 and H2C had consistently low nodule occupancy levels. Strain Ciat 151 was superior to the other inoculant strains in the acid soil but competed poorly in the fertile soil. Strain Ciat 895 was more competitive in the fertile soil. There was a decline in nodule occupancy for all strains tested from the first trifoliate leaf stage to the pod-filling stage. No plant genotype effect on nodule occupancy was observed. There were significant (P<0.05) plant genotype and location effects, but no significant strain effect on acetylene reduction activity, plant dry weight, and nodule number. The greenhouse experiments confirmed, at least partially, the results from the field trials. In Leonard jars with an acid soil, strains Ciat 151 and Kim5s were amongst the most competitive strains. In coinoculation experiments, Kim5s was the most competitive strain, followed by Ciat 899 and Ciat 895. The competitiveness of a given strain was affected by the coinoculant strain. Tolerance in vitro to low pH, high growth temperature, Al or Mn toxicity, or Fe limitation was not related to competitiveness of the inoculum strains. The sensitivity of the strains towards bean tannins or bean phytoalexins also was not correlated with their competitiveness.     

Rhizobiophage effects on nodulation,nitrogen fixation,and yield of field-grown soybeans (Glycine max L. Merr.)

Summary Previous laboratory and greenhouse studies have shown that phages significantly reduce soil populations of homologous rhizobia. Reductions in nodulation and N₂ fixation have also been observed. The purpose of the current study was to examine the effect of a phage specific ofBradyrhizobium japonicum USDA 117 on nodulation, nodule occupancy, N₂ fixation and soybean growth and yield under field conditions. The phage was inoculated in combination withB. japonicum USDA 117 and/orB. japonicum USDA 110 (resistant strain) into a rhizobia-free sandy loam soil and planted toGlycine max (L.) Merr. Williams. When the phage was applied to soil inoculated withB. japonicum USDA 117 alone, significant reductions in nodule weight and number, shoot weight, foliar N, nitrogenase activity, and seed index were observed. When, however, the soil also contained the non-homologous strain,B. japonicum USDA 110, no significant effects on any of these parameters were found. Nodule occupancy by competing strains ofB. japonicum USDA 110 and USDA 117 was also affected by the phage. In soil which did not contain the phage, 46% and 44% of the identified nodules were occupied by USDA 110 and 117, respectively. When the phage was present in the soil, nodule occupancy byB. japonicum USDA 117 was reduced to 23%, while occupancy byB. japonicum USDA 110 was increased to 71%. These results suggest that nodulation by selected strains of rhizobia can be restricted and nodulation by more effective, inoculated strains can be increased through the introduction of a homologous phage to soils.     

Competitiveness and persistence of strains ofRhizobium phaseoli introduced into a Moroccan sandy soil

Summary Sixteen strains ofRhizobium phaseoli were isolated from the Loukkos sandy soil and were compared with four selected strains ofR. phaseoli (CIAT 676, CIAT 57, Olivia, Viking 1) for their N₂-fixing ability and for their serological affinities by means of the fluorescent antibody technique. Two indigenous strains were rated as highly effective, six as moderately effective, and eight were ineffective. Viking 1 was ranked as highly effective while the other inoculum strains were moderately effective. None of the 4 inoculum strains cross-reacted with the 16 indigenous strains. The indigenous strains were grouped into only two serogroups but showed a high degree of heterogeneity in regard to the strength of the immunofluorescence response. The inoculum strains outcompeted but did not eliminate the resident population for nodule occupancy on two French bean cultivars (Royalnel and Fetiche). The competitiveness, however, differed between inoculum strains. It was influenced by the host cultivar and by the host cultivar growth stage. Viking 1 was consistent in its high competitive ability whether it was inoculated singly or in a mixture with the other strains. It showed high persistence and formed more than 50% of the nodules 1 year after it was introduced. CIAT 57, Olivia, and CIAT 676 were mediocre in their persistence.     

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.     

Genetic variability of soybean bradyrhizobia populations under different soil managements

Due to their ecologic and economic importance, bradyrhizobia have been extensively studied in recent years. Since 1992, Bradyrhizobium elkanii SEMIA 587 and SEMIA 5019 and Bradyrhizobium japonicum SEMIA 5079 and SEMIA 5080 have been widely used in most Brazilian soybean fields. The objective of this work was to estimate the genetic variability of bradyrhizobial isolates recovered from soils under rhizobial inoculation and different soil managements. Only 25% of the isolates demonstrated high similarities to the original strains, and a strong correlation was obtained between the bradyrhizobial genetic variability and soil management. A high level of genetic diversity was observed both within isolates (H = 5.46) as well as among the different soil practices. Soil under no-tillage presented a higher bradyrhizobia diversity compared with bradyrhizobia isolated from soil under conventional tillage. Serological characterization also indicated that B. elkanii strains SEMIA 587 and SEMIA 5019 were more competitive and presented a higher nodular occupancy capacity than strains belonging to B. japonicum species in Southern Brazilian soils.     

Competitive interaction among strains of Rhizobium leguminosarum bv. phaseoli in the nodulation of kidney beans (Phaseolus vulgaris L.)

We examined the competitiveness of five effective Rhizobium leguminosarum biovar phaseoli strains in the nodulation of kidney beans (Phaseolus vulgaris L.), either alone or in pairwise combination, against the indigenous strains. The results showed that the introduced Rhizobium sp. strains (B2, B17, B36, T2, or CIAT 652) occupying 64–79% of the total nodules (as single inocula) were more competitive in nodulation than the native rhizobia. However, the competitiveness of the individual Rhizobium sp. strain either increased or decreased when used in a pairwise combination of double-strain inocula. For example, strain B17, although quite competitive against the indigenous population (68% nodule occupancy), became poorly competitive in the presence of strain B2 (reduced from 68 to 2.5%). A similar reduction in nodule occupancy by strain B17 was observed in the presence of B36 or CIAT 652, indicating that two competitive strains may not always be compatible. These results suggest that it is important to co-select competitive as well as compatible rhizobia for multistrain inoculant formulation.     

Competitive ability of selected Cyclopia Vent. rhizobia under glasshouse and field conditions

This study tested the competitive ability of three locally isolated Cyclopia rhizobia and strain PPRICI3, the strain currently recommended for the cultivation of Cyclopia, a tea-producing legume. Under sterile glasshouse conditions, the three locally isolated strains were equally competitive with strain PPRICI3. In field soils, the inoculant strains were largely outcompeted by native rhizobia present in the soil, although nodule occupancy was higher in nodules growing close to the root crown (the original inoculation area). In glasshouse experiments using field soil, the test strains again performed poorly, gaining less than 6% nodule occupancy in the one soil type. The presence of Cyclopia-compatible rhizobia in field soils, together with the poor competitive ability of inoculant strains, resulted in inoculation having no effect on Cyclopia yield, nodule number or nodule mass. The native rhizobial population did not only effectively nodulate uninoculated control plants, they also out-competed introduced strains for nodule occupancy in inoculated plants. Nonetheless, the Cyclopia produced high crop yields, possibly due to an adequate supply of soil N.     

Direct monitoring of Rhizobium sp. (Cicer) in nodules and soil using lac Z fusions

We constructed lacZ fusions in Rhizobium sp. (Cicer) by random Tn5-lacZ mutagenesis. The lacZ⁺ fusants formed blue colonies on a Rhizobial minimal medium containing 5-bromo-4-chloro-3-indolyl--D-galactopyranoside (X-gal). Rhizobium sp. (Cicer) fusant HSL-2 was identified in nodules and soil in a mixed population on the basis of the lacZ⁺ phenotype. Nodule occupancy of inoculated Rhizobium sp. (Cicer) HSL-2 (lacZ⁺) was assessed by directly streaking the nodule sap on X-gal plates. This method revealed differences between rhizobia carrying identical antibiotic markers. The rhizobial population in soil was estimated by direct plate counts using a medium containing X-gal. Introduction of lacZ into the Rhizobium sp. thus provided a simple and direct method for identifying strains from nodules and soil.     

Competition between effective and less effective strains of Bradyrhizobium spp. for nodulation on Vigna radiata

Local and exotic cowpea bradyrhizobial strains were examined for their effectiveness and competitive ability on the cultivated Vigna radiata (mungbean) variety NM-51. The seven most efficient N₂-fixing strains were then evaluated for their competitiveness against two less effective strains in a pair-wise inoculation experiment. Nodule occupancy was determined using fluorescent antibodies. The most efficient strain, Vr-2, could not compete with less effective strains for nodule occupancy, so the biomass and N yield were low. Bradyrhizobial strain M-55 had good competitive ability, as it occupied almost 50% of nodules in comparison with less effective strains and improved the biomass and N yield of mungbean significantly. Competitiveness/compatibility is one of the vital parameters for the selection of inocula strains, therefore, M-55, K-92 and Vr-6 are recommended as good inocula of commercial quality because of their competitiveness along with their highly efficient N₂-fixation.     

Competitive abilities of common field isolates and a commercial strain of Rhizobium leguminosarum bv. trifolii for clover nodule occupancy

We previously reported that commercial Rhizobium leguminosarum bv. trifolii inoculants failed to outcompete naturalized strains for nodule occupation of clover sown into an alkaline soil [Aust. J. Agric. Res. 53 (2002) 1019]. Two field isolates that dominated nodule occupancy at the field site were labeled with a PnifH-gusA marker. Marked strains were chosen on the basis that they were equally competitive and fixed similar amounts of nitrogen in comparison to their parental strain. The minitransposon insertions were cloned and sequence analysis revealed that neither lesion disrupted the integrity of any known gene. The marked strains were then used to follow nodule occupancy of Trifolium alexandrinum in competition against the commercial inoculant TA1 under a range of experimental conditions. In co-inoculation experiments in sand-vermiculite, TA1 outcompeted each marked field isolate for nodule occupancy. However, using TA1-inoculated seed sown into alkaline soil containing a marked field strain, it was demonstrated that by increasing the cell number of marked rhizobia in the soil and reducing the cell number of the commercial inoculant, the proportion of nodules occupied by TA1 was reduced. These studies indicate that the ability of the field isolates to dominate nodule occupancy in the alkaline field soils was most likely caused by poor commercial inoculant survival providing the advantage for naturalized soil rhizobia to initiate nodulation.     

Enumeration of Bradyrhizobium japonicum in soil subjected to high temperature: Comparison of plate count, most probable number and fluorescent antibody techniques

The survival of BradyrMzobium japonicum strains in autoclaved and non-autoclaved soil at various temperatures for up to 18 days was determined by plate count, plant infectivity-most probable number (MPN) and modified fluorescent antibody (FA) techniques. The high-temperature tolerant strains studied were BR587, NC1005 and NC1033, and the high-temperature intolerant strains studied were NCI028, 3Ilb24 and 311b123. Each strain was exposed individually in a sandy loam soil at 28, 33 or 37°C under humidity controlled conditions. Those strains reported to be tolerant to elevated temperature in pure culture decreased by <10^1.5 cells g⁻¹ when enumerated by plate count after high-temperature exposure in autoclaved soil. Based on plate counts, the population of two of the three high-temperature intolerant strains added to autoclaved soil decreased by more than 10³ cells g⁻¹ soil after exposure to elevated temperatures. In non-autoclaved soil, the population of all inoculated strains decreased after exposure to elevated temperature when enumeration was by the plant infectivity-MPN technique. In autoclaved soil at 28°C, the correlation between plate count and FA enumerations was significant at the 0.05 probability level for four of the six strains (r² = 0.65). After high-temperature incubation, counts based on the FA technique were not correlated with plate counts of intolerant strains or MPN enumeration of tolerant and intolerant strains (r² = 0.004). Differential survival of B. japonicum strains in autoclaved soil at high temperature as enumerated by plate counts confirms the designation of temperature tolerance made by Munevar and Wollum (1981). However. MPN enumeration of bradyrhizobia in the non-autoclaved system did not follow the original temperature tolerance classifications. Enumeration by FA procedures did not show a decrease in population after exposure to high temperature as observed with the plate and MPN counts. Thus, the FA technique may not adequately indicate population dynamics in short-term ecological studies.     

Mesquite rhizobia isolated from the Sonoran Desert: Competitiveness and survival in soil

The competitiveness of a mesquite Rhizobium (AZ-M1) and its ability to survive in desert soils was compared to a selected commercial strain (31A5). In a greenhouse study, the native isolate out-competed strain 31A5 in nodule occupancy, when applied as a mixed inoculant to seed germinated and grown in sand culture, and irrigated with N-free nutrients. A high incidence of nodule double occupancy was found when double strain inoculants were used. The survival rate of the two strains was tested in three desert soils in a controlled laboratory study. The desert strain AZ-M1 grew and survived in all the soils for 1 month. The commercial strain 31A5, did not grow, and the population decreased in 14 days from 10⁸ cells g^?1 dry soil to below 10⁴ cells g^?1. Both strains survived to a lesser extent in a saline-sodic soil. A significant morphological change from a rod to a coccus was observed 2 days after strain 31A5 had been introduced into the desert soils.     

Benefits of inoculation of the common bean (<Emphasis Type="Italic">Phaseolus vulgaris</Emphasis>) crop with efficient and competitive<Emphasis Type="Italic"> Rhizobium tropici</Emphasis> strains

Cropping in low fertility soils, especially those poor in N, contributes greatly to the low common bean (Phaseolus vulgaris L.) yield, and therefore the benefits of biological nitrogen fixation must be intensively explored to increase yields at a low cost. Six field experiments were performed in oxisols of Paraná State, southern Brazil, with a high population of indigenous common bean rhizobia, estimated at a minimum of 10³ cells g^–1 soil. Despite the high population, inoculation allowed an increase in rhizobial population and in nodule occupancy, and further increases were obtained with reinoculation in the following seasons. Thus, considering the treatments inoculated with the most effective strains (H 12, H 20, PRF 81 and CIAT 899), nodule occupancy increased from an average of 28% in the first experiment to 56% after four inoculation procedures. The establishment of the selected strains increased nodulation, N₂ fixation rates (evaluated by total N and N-ureide) and on average for the six experiments the strains H 12 and H 20 showed increases of 437 and 465 kg ha^–1, respectively,in relation to the indigenous rhizobial population. A synergistic effect between low levels of N fertilizer and inoculation with superior strains was also observed, resulting in yield increases in two other experiments. The soil rhizobial population decreased 1 year after the last cropping, but remained high in the plots that had been inoculated. DGGE analysis of soil extracts showed that the massive inoculation apparently did not affect the composition of the bacterial community.     

Establishment of <Emphasis Type="Italic">Bradyrhizobium japonicum</Emphasis> and <Emphasis Type="Italic">B. elkanii</Emphasis> strains in a Brazilian Cerrado oxisol

The competition with established soil populations of Bradyrhizobium able to nodulate soybean has been one of the major constraints to the introduction of more efficient strains in Cerrados soils. The effects of nodulation establishment and persistence of four serologically distinct strains of Bradyrhizobium japonicum (CPAC 15 and CPAC 7, belonging to serogroups USDA 123 and CB 1,809) and B. elkanii (29 W and SEMIA 587, belonging to serogroups 29 W and 587) were examined. These strains were introduced in a dark-red oxisol, without indigenous populations of soybean bradyrhizobia, and were evaluated for 6 years. The experimental design was a completely randomized block with four replicates. In the first year, besides the inoculation treatments, there was also an uninoculated control. In the second year, the main plots were split into three sub-plots and treatments consisted of an uninoculated control, CPAC 7 and CPAC 15. In the third year, the entire area was inoculated with CPAC 7. In the fourth and sixth years, the plots were planted with soybean without inoculation, and in the fifth year the plots were left fallow. The strains introduced in the first year influenced nodule occupancy by strain CPAC 7 until the third successive growing season. By the fourth and sixth years, as a consequence of the dispersal of strains serologically related to serocluster 123 in the entire experimental area, this serogroup dominated the nodulation, occurring, on average, in more than 50% of the nodules of the treatments where it had never been inoculated.     

Interaction of Bradyrhizobium sp. with an antagonistic actinomycete in culture medium and in soil

Summary Experiments were conducted to determine the extent to which populations of Bradyrhizobium sp. strain Tal 209SR were suppressed by the antagonistic activity of a Streptomyces sp. isolated from a highly weathered tropical soil. The activity of the actinomycete was evaluated in culture medium in the presence or absence of clay minerals and in the soil from which the actinomycete was isolated after sterilization by gamma-irradiation. The presence of the actinomycete in culture medium was associated with a significant reduction in the density of Bradyrhizobium sp. (Tal 209SR). Nevertheless, the density of the Bradyrhizobium sp. surviving in the medium in the presence of the actinomycete was much higher than that normally observed when comparable densities of rhizobia are introduced into non-sterile soils. The presence of the antagonistic actinomycete in culture medium was associated with a drastic decrease in the optical density of the medium. This decrease was accompanied by a significant decrease in the insoluble exopolysaccharide content of the medium in addition to the significant decrease in bradyrhizobial cells. The actinomycete did not appear to significantly influence the growth and survival of the test Bradyrhizobium sp. in sterile soil. The inability of the actinomycete to significantly antagonize the test bacterium in sterile soil was not adequately explained by the presence of clay minerals.Hawaii Institute of Tropical Agriculture and Human Resources Journal Series No. 3129     

Influence of a vesicular-arbuscular mycorrhizal fungus on the competitive ability of Bradyrhizobium spp. for nodulation of cowpea Vigna unguiculata (L.) Walp in non-sterilized soil

Summary We examined the influence of a vesicular-arbuscular (VAM) fungus (Glomus pallidum Hall) on the competitive ability of introduced and native Bradyrhizobium strains to nodulate cowpeas [Vigna unguiculata (L) Walp]. Our experiments in non-sterilized soil revealed that in the presence of VAM fungus, introduced Bradyrhizobium spp. strains become more competitive than native rhizobia. For example, strain JRC29 occupied 59.2% of the total nodules when inoculated alone, but this figure increased to 71.2% when JRC29 was used in dual inoculations with VAM fungus. A similar pattern of enhanced competitiveness for nodule formation was observed with the two other strains in the presence of the VAM fungus. Our results suggest that the competitiveness of rhizobia can be enhanced by co-inoculating with a selected strain of a VAM fungus.     

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.     

A proposal for re-evaluating the most probable number procedure for estimating numbers of Bradyrhizobium spp.

The reliability of the most probable number (MPN) method for estimating bradyrhizobial numbers was evaluated by comparison with the plate count procedure. MPN estimates increased with time of nodulation scoring after seedling inoculation through 6 weeks of incubation. Ratios of MPN to plate counts increased as the bradyrhizobial cell suspension concentration increased. The MPN method could not detect Bradyrhizobium japonicum numbers at concentrations of 10³ colony forming units (CFU) ml^-1 and below. A proposal for re-evaluating MPN estimates is discussed.     

Intrinsic antibiotic resistance and competition in fast- and slow-growing soybean rhizobia on a hybrid of Asian and US cultivars

Summary Six fast-growing soybean rhizobia (Rhizobium fredii) and thirteen slow-growing soybean rhizobia (Bradyrhizobium japonicum) were examined for resistance to 10 antibiotics. Axenic studies were carried out to determine the competitiveness of dual-strain inocula consisting of fast- and slow-growing rhizobia isolated from subtropical-tropical soils for nodule occupancy on a hybrid of Asian and US soybean cultivars. Nodule occupancy was determined by intrinsic resistance to erythromycin and neomycin. The results showed 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 g ml^–1) and penicillin (200 g ml^–1). The competitive ability of inoculant strains for nodule occupancy varied for different combination sets and with the plant growing media. Our results show that fast-growing rhizobia nodulate a hybrid of Asian and US soybean cultivars. Fast-growing soybean rhizobia did not completely exclude nodulation by the slow-growing strains, which formed 0–79% nodules, depending on the strain used in the inoculum.