Strain-specific antisera to identify Thai Bradyrhizobium japonicum strains in preserved soybean nodules

Strain-specific antisera were produced against six Bradyrhizobium japonicum strains using two immunization procedures. These specific antisera were used for detection of bradyrhizobia in preserved soybean nodules. Antisera specific for two of these strains were either conjugated with a fluorescent dye or used with a fluorescent secondary antibody for identification of bradyrhizobia in soybean nodules that were preserved in four different storage conditions. Results show that soybean nodules dried in the oven, stored under room temperature, or at –20 °C are as suitable as fresh nodules for strain identification using fluorescent antisera.     

Identification of Bradyrhizobium japonicum Nodule Isolates from Wisconsin Soybean Farms

One-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis was a more discriminating method than serotyping for identifying strains of Bradyrhizobium japonicum. Analysis of 543 nodule isolates from southeastern Wisconsin soybean farms revealed that none of the isolates were formed by any of the inoculant strains supplied by either of two inoculant companies. Twenty-nine indigenous strains and six inoculant strains were identified. Strain 61A76, the most competitive indigenous strain, formed 21% of the nodules. Indigenous strains 3030, 3058, 0336, and 3052 formed 15, 11, 9, and 9% of the nodules, respectively. These predominant strains were not associated with a particular soybean cultivar, soil type, or farm location.     

Suppression of nodulation in soybeans by superoptimal inoculation with Bradyrhizobium japonicum

Symbiotic nodulation of the primary roots of soybeans ( Glycine max L. Merrill cv. Pride 216) is regulated by the plant, and is suppressed in response to a high inoculum dose of Bradyrhizobium japonicum USDA strain I–110 (ARS)⁺ applied at one time to the root. If an optimal dose is followed 10 h later by a superoptimal dose, nodules from the first inoculum near the base of the primary root are suppressed in a dose-dependent way similar to that observed after single inoculations. The nodules which appear are probably derived from infections initiated by the bacteria in both inocula.     

Persistence and recovery of introduced Rhizobium ten years after inoculation on Leucaena leucocephala grown on an Alfisol in southwestern Nigeria

Establishment of Leucaena leucocephala was poor at Ibadan (Transition forest-savanna zone) and Fashola (savanna zone, 70 km north of Ibadan) in southwestern Nigeria as a result of low soil fertility and the presence of only a few native rhizobia capable of nodulating it. Inoculation with L. leucocephala at these two locations in 1982 resulted in striking responses with Rhizobium strains IRc 1045 and IRc 1050 isolated from L. leucocephala grown in Nigeria. The persistence of inoculated effective Rhizobium strains after inoculation is desirable since it removes the need for reinoculation. Because of the perennial nature of L. leucocephala and its use in long-term alley farming experiments, we examined the persistence of inoculated rhizobial strains after inoculation, and their ability to sustain N₂-fixation and biomass production at Ibadan. In 1992, ten years after Rhizobium introduction, uninoculated, L. leucocephala fixed about 150 kg N ha^-1 yr^-1 or about 41% of total plant N compared to 180 kg N ha^-1 yr^-1 or 43% measured in 1982. Serological typing of the nodules using the Enzyme-Linked-Immunosorbent Assay (ELISA) and intrinsic resistance to the streptomycin test revealed that most of the nodules (96%) formed on L. leucocephala in 1992 were by Rhizobium strains IRc 1045 and IRc 1050, which were inoculated in 1982. Nodules were absent on uninoculated L. leucocephala grown on the adjacent field with no history of L. leucocephala cultivation. We conclude that the N₂ fixed by Rhizobium strains IRc 1045 and IRc 1050 persisted for many years in the absence of L. leucocephala and sustained effectively fixed N₂ which growth and yield of L. leucocephala after several years, thus encouraging a possible low-input alley farming system by smallholder farmers in Nigeria.     

Effect of drought on Bradyrhizobium japonicum populations in Midwest soils

Background and aims

Bradyrhizobium japonicum and soybean (Glycine max (L.) Merr.) form a symbiotic association which allows for biological nitrogen fixation (BNF) to help meet the nitrogen (N) requirement of soybean plants. Rhizobial inoculants are not always used in soybean production in the Midwestern USA because of high naturalized soil populations, but drought conditions experienced in the region during the 2012 growing season may have led to a decline in numbers resulting in the need for inoculation the following growing season. Therefore, the effect of drought on B. japonicum population size was investigated in this study.

Methods

Drought conditions, 8 weeks long or 4 weeks long preceded (STOP) or followed (START) by 4 weeks of normal watering, were simulated in two contrasting soil types in a greenhouse setting with soybeans as host plants. Drought conditions were monitored by measuring water content. Population size of B. japonicum was quantified using quantitative real-time polymerase chain reaction (qPCR) and most probable number (MPN) methods and compared to population from non-drought treatment.

Results

Using both quantification methods, the response of B. japonicum to drought treatments was minimal.

Conclusions

Drought conditions 4 to 8 weeks long did not reduce B. japonicum population size to levels which would affect soybean growth and development.     

Variability in molybdenum uptake activity in Bradyrhizobium japonicum strains.

Twenty naturally occurring strains of Bradyrhizobium japonicum in 11 serogroups were screened for the ability to take up Mo as bacteroids from soybean root nodules. The strains varied greatly in their ability to take up Mo in a 1-min period. The best strain was USDA 136, which had an Mo uptake activity of almost 3.0 pmol/min per mg of bacteroid (dry weight). In contrast, the poorest strain, USDA 62, had an Mo uptake activity of 0.35 pmol of Mo per min per mg of bacteroid. There were similarities in Mo uptake ability among most of the same serogroup members. The variability in Mo uptake rates between the best (USDA 136 and USDA 122) and poorest (USDA 62 and USDA 140) strains was attributed to their differing affinities for Mo. Double-reciprocal plots of velocity versus substrate indicated a Km for USDA 136 and USDA 122 of 0.045 and 0.054 microM, respectively, whereas strains USDA 62 and USDA 140 both exhibited an apparent Km for MoO42- of about 0.36 microM. The two strains with the higher-affinity Mo binding also accumulated four to five times as much Mo over a 30-min period as the other strains. Soybeans were grown in Mo-deficient and Mo-supplemented conditions after inoculation with the three top-ranking Mo uptake strains and the three poorest Mo uptake strains. Two separate greenhouse studies indicated that Mo supplementation significantly increased the N2 fixation activity of USDA 140 nodules; up to a 35% increase in specific nitrogen fixation activity of nodules due to Mo supplementation was observed. Strain USDA 62 nodule N2 fixation responded positively to Mo supplementation in one of the two experiments. The results indicate that MoO42- transport and, specifically, affinity for Mo by the bacteroid may ultimately affect symbiotic N2 fixation activity. Attempts to reactivate nitrogenase by adding molybdate to bacteroids from plants grown in Mo-deficient conditions were unsuccessful.     

Soybean cultivars affect nodulation competition of Bradyrhizobium japonicum strains

The influence of five Thai soybean cultivars on nodulation competitiveness of four Bradyrhizobium japonicum strains was investigated. Cultures of B. japonicum strains THA5, THA6, USDA110 and SEMIA5019 were mixed with each other prior to inoculating germinated soybean seeds growing in Leonard jars with nitrogen-free nutrient solution. At harvest, nodule occupancy by each strain was determined by a fluorescent antibody technique. The term ‘general competitive ability’ was introduced to describe the average competitive nodule occupancy of a strain in paired co-inoculation with a number of strains on soybean. The nodule occupancies by an individual strain were directly correlated with the proportions of that strain in the inoculum mixtures. USDA110 showed higher nodulation competitiveness than the other strains on three of the five cultivars. The Thai strain THA6 appeared to be more competitive than USDA110 on cultivar SJ5. Thus, nodulation competitiveness of the B. japonicum strains was affected by the cultivars of soybean used. This revised version was published online in August 2006 with corrections to the Cover Date.     

Response of introduced Bradyrhizobium strains infectinga promiscuous soybean cultivar

Two field experiments were established to assess the competitiveness of foreign bradyrhizobia in infecting the promiscuous soybean cultivar TGX 536-02D. Seeds were inoculated with antibiotic mutants of the bradyrhizobia strains before planting after land preparation. Soybean plants were harvested at pre-determined days after planting for estimating nodule number, nodule dry weight, nodule occupancy, shoot dry weight and seed yield. Results show that nodule number and dry weight significantly increased and showed great variability at 84 days after planting (DAP), probably due to differences in the ability of inoculant bradyrhizobia to form nodules with the soybean cultivar TGX 536-02D. Increased shoot dry weight, %N, total N and seed yield were a result of increased nodulation by the effective and competitive inoculant Bradyrhizobium strains. Strain USDA 110 occupied the highest percentage of nodule sites because it was more competitive than the other Bradyrhizobium strains. These results show that there was high potential for increasing growth and seed yield of the promiscuous soybean cultivar TGX 536-02D by inoculation with foreign Bradyrhizobium strains.     

Efficiency of different formulations of Bradyrhizobium japonicum and effect of co-inoculation of Bacillus subtilis with two different strains of Bradyrhizobium japonicum

A key constraint in successfully obtaining an effective inoculant is overcoming difficulties in formulating a viable and user-friendly final product and maintaining the microbial cells in a competent state. Co-cultures of rhizobia and PGPR (Plant Growth Promoting Rhizobacteria) are a logical next subject for formulation researchers as they can influence the efficacy of rhizobia. A greenhouse experiment was set to assess the formulation effect of one strain i.e. Bradyrhizobium japonicum, 532c (granules, liquid and broth) and also to determine the efficiency of co-inoculation of Bacillus with two commercial strains of B. japonicum (532c and RCR 3407) on 2 soybean (Glycine max L.) varieties. PCR-RFLP analysis was used to determine the nodule occupancy in each treatment. Most of the inoculants showed increased nodulation and biomass yields (by approximately 2-5 and 4-10 g plant(-1) respectively) as compared to the uninoculated controls. TGx1740-2F showed no significant differences in nodule fresh weights for the formulation effect while the co-inoculants increased the nodule fresh weights by up to 4 g plant(-1). The liquid and granule-based inoculants induced higher biomass yields (4-8 g plant(-1)) suggesting a possible impact of formulation on the effectiveness of the inoculants. The co-inoculants also gave higher yields but showing no significant differences to the rhizobial inoculants. Nodule occupancy was 100 % for the rhizobial inoculants as well as the co-inoculants emphasizing the infectivity and high competitiveness of 532c and RCR 3407 strains despite the high population of indigenous rhizobia.     

Establishment of Bradyrhizobium japonicum for soybean by inoculation of a preceding wheat crop

On fields with no history of soybean (Glycine max (L.) Merr.) production, inoculation alone is often inadequate to provide for adequate nodulation the first time this crop is grown. The objective of this study was to determine if inoculation of spring wheat (Triticum aestivum L.) seed with Bradyrhizobium japonicum would lead to an increase of B. japonicum numbers in the soil, and improve nodulation of a subsequent soybean crop. In the greenhouse, wheat seed inoculation increased B. japonicum numbers from undetectable numbers to >9000 g^–1 of soil, whereas the numbers of introduced B. japonicum declined in unseeded pots. In the field, inoculation of wheat seed increased B. japonicum numbers in the soil from undetectable levels to >4000 g^–1 the following year. When soybean seed was inoculated, but grown in soil devoid of B. japonicum, nodules formed only near the point of seed placement. The heaviest nodulation, and widest distribution of nodules in the topsoil were found whenB. japonicum was established the year before by wheat seed inoculation, plus soybean seed inoculation. Wheat seed inoculation the year before growing soybean, combined with proper soybean seed inoculation, should provide for abundant nodulation the first time soybean is grown on a field.     

A succinate transport mutant of Bradyrhizobium japonicum forms ineffective nodules on soybeans.

Biochemical evidence has shown that dicarboxylic acids actively support symbiotic nitrogen fixation by both fast- and slow-growing Rhizobium. Mutants defective in the active uptake of succinate have been previously described only in species of the fast-growing rhizobium. This article is a report on the isolation of mutants defective in dicarboxylate transport in a slow-growing species of rhizobium, Bradyrhizobium japonicum. One of these presumptive dicarboxylate transport mutants, GTS, was characterized further. Cultured GTS was unable to accumulate [14C]succinate above background levels but possessed normal rates of malate dehydrogenase, fumarase, and hydroxybutyrate dehydrogenase activities. When inoculated onto soybeans, GTS produced a Nod+, Fix- phenotype. The bacteroids isolated from these nodules failed to accumulate labelled succinate. Electron micrographs of nodules formed by inoculation with GTS appeared normal with the exceptions of more prominent peribacteroid spaces in the infected cells and the appearance of starch granules in the noninfected cells. The phenotypical and morphological changes observed for B. japonicum are similar to those previously reported for the fast-growing species.     

Stability of Bradyrhizobium japonicum Inoculants after Introduction into Soil

Bradyrhizobium japonicum USDA 125-Sp, USDA 138, and USDA 138-Sm had been used as inoculants for soybean (Glycine max (L.) Merr.) in soils previously free of B. japonicum. At 8 to 13 years after their release, these strains were reisolated from soil samples. A total of 115 isolates were obtained through nodules, and seven colonies were obtained directly by a serological method. The stability of the inoculants was confirmed by comparing the reisolated cultures with their respective parental strains which had been preserved by being lyophilized or stored on a yeast extract-mannitol agar slant at 4°C. Comparisons were made on morphological and serological characters, carbon compound utilization (8 tested), intrinsic antibiotic resistance (9 tested), and enzymatic activity (19 tested). Mucous and nonmucous isolates of serogroup 125 were analyzed for symbiotic effectiveness and restriction fragment hybridization with a DNA probe. Our data suggest that the B. japonicum inoculants have survived for up to 13 years in the soils without significant mutation except for two reisolates with a slightly increased kanamycin resistance level.     

Differential symbiotic response of phage-typed strains of Bradyrhizobium japonicum with soybean cultivars

In this study, native Bradyrhizobium strains were isolated from the host plant, Glycine max, harvested from fields in Madhya Pradesh, India, and were typed by lytic rhizobiophages. Eight indigenous (Soy2, ASR011, ASR031, ASR032, MSR091, ISR050, ISR076 and ISR078) and two exotic strains (USDA123 and CB1809), all of which evidenced a distinct reaction with six phages, were employed in this study. The symbiotic interaction of these strains was studied initially using soybean cultivar JS335 in a sand culture in a controlled environment, and the efficiency was assessed based on the nodule number, nodule dry weight, plant dry weight, nitrogenase activity, and total accumulation of N per plant. Symbiotic effectiveness was found to be highest with the native phage-sensitive isolate ASR011, whereas it was at a minimum with the phage-resistant isolates, ISR050 and ISR078. Additionally, the effectiveness of these strains was evaluated using six soybean cultivars belonging to different maturity groups; namely, Bragg, Lee, Pusa20, PK416, JS335 and NRC37. Analysis of variance data evidenced significant differences due to both symbionts, for the majority of the tested parameters. The CB1809, USDA123, and ASR011 strains evidenced relatively superior symbiotic effectiveness with soybean cultivars Bragg, Lee and JS335. Strain ISR078 evidenced no significant responses with any of the cultivars. The ASR031 strain performed moderately well with all tested cultivars. The symbiotic response of all the strains was quite poor with cultivar PK416. Our studies showed that a significant relationship existed between the phage sensitivity and symbiotic efficiency of the bacterial strains with the host-cultivars.     

The Bradyrhizobium japonicum hsfA gene exhibits a unique developmental expression pattern in cowpea nodules.

The Bradyrhizobium japonicum host-specific fixation gene hsfA was identified as essential for nitrogen fixation on cowpea, but not required for nitrogen fixation on soybean or siratro. The DNA sequence of the hsfA promoter contains a consensus RpoN, -24/-12 binding site, suggesting the involvement of a regulatory protein that binds to an upstream activating sequence (UAS). To further explore the regulation of this interesting gene, serial deletions of the hsfA promoter were made and fused with the beta-glucuronidase (GUS) gene. The HsfA3 deletion, containing 60 bp 5' of the -24/-12 sequence, showed a similar level of GUS expression to that shown by the longest fusion construct (HsfA1), containing 464 bp of upstream sequence. In contrast, the HsfA4-GUS fusion, containing only 20 bp 5' of the -24/-12 region, showed no GUS activity, delimiting the location of a putative UAS to a 40-bp region. During nodule development, GUS expression first appeared in nodules 12 days postinoculation (dpi) and reached a maximum level of expression in approximately 17-day-old nodules. By 28 dpi, HsfA-GUS expression had returned to a low, basal level. These data were consistent with the detection of hsfA mRNA by in situ hybridization in 17-day-old nodules, but not in 28-day-old nodules. In contrast to the stage-specific expression in cowpea, HsfA-GUS expression increased with nodule development in HsfA3-inoculated soybean. These data indicate that HsfA expression is regulated in cowpea in a unique developmental manner and that the DNA regulatory regions that control this expression are confined to a short, promoter-proximal region.     

Bradyrhizobium canariense and Bradyrhizobium japonicum are the two dominant rhizobium species in root nodules of lupin and serradella plants growing in Europe

Forty three Bradyrhizobium strains isolated in Poland from root nodules of lupin species (Lupinus albus, L. angustifolius and L. luteus), and pink serradella (Ornithopus sativus) were examined based on phylogenetic analyses of three housekeeping (atpD, glnII and recA) and nodulation (nodA) gene sequences. Additionally, seven strains originating from root-nodules of yellow serradella (O. compressus) from Asinara Island (Italy) were included in this study. Phylogenetic trees revealed that 15 serradella strains, including all yellow serradella isolates, and six lupin strains grouped in Bradyrhizobium canariense (BC) clade, whereas eight strains from pink serradella and 15 lupin strains were assigned to Bradyrhizobium japonicum (BJ1). Apparently, these species are the two dominant groups in soils of central Europe, in the nodules of lupin and serradella plants. Only three strains belonged to other chromosomal lineages: one formed a cluster that was sister to B. canariense, one strain grouped outside the branch formed by B. japonicum super-group, and one strain occupied a distant position in the genus Bradyrhizobium, clustering with strains of the Rhodopseudomonas genus. All strains in nodulation nodA gene tree grouped in a cluster referred to as Clade II, which is in line with earlier data on this clade dominance among Bradyrhizobium strains in Europe. The nodA tree revealed four well-supported subgroups within Clade II (II.1-II.4). Interestingly, all B. canariense strains clustered in subgroup II.1 whereas B. japonicum strains dominated subgroups II.2-II.4.     

TnphoA-induced symbiotic mutants of Bradyrhizobium japonicum that impair cell and tissue differentiation in Glycine max nodules

Bradyrhizobium japonicum WM1, an ethylmethane-sulfonate-induced derivative of B. japonicum 110spc4 with reduced phosphatase activity but normal symbiotic properties, was randomly mutagenized using TnphoA. From about 1000 purified single colonies, approximately 300, preferentially those with enhanced phosphatase activity, were inoculated onto soybean seedlings to test their symbiotic traits. Sixteen strains were either completely Fix? or possessed markedly reduced acetylene reduction activity (Fix^red). Contrary to expectations, hybridization of total DNA of these strains to a transposonspecific DNA probe showed that many contained no transposon. Apparently these strains had gained resistance towards kanamycin spontaneously rather than through the introduction of TnphoA. However, in five mutant strains, two hybridizing BamHI fragments of different sizes were detected, as expected. All strains performed acetylene reduction under ex planta conditions, indicating that mutations had not occurred in nif or fix genes. A more than 50-fold increased specific activity of alkaline phosphatase was observed in strain 132, indicating the synthesis and secretion of a polypeptide fused to 'PhoA. Light and electron-microscopic analyses showed that in nodules induced by strain 132 (Fix^red) the infected cells of the central tissue were vacuolated. In some of these cells callose deposition was observed, indicating plant defense reactions. Nodules induced by mutant 184 were infected by bacteroids only in a few cells of the central tissue as isolated clusters, whereas the majority of cells remained uninfected. The concentration of phosphoenolpyruvatecarboxylase protein within the infected tissue was significantly reduced and starch granules accumulated. In both strains TnphoA insertions were identified to be the reasons for the observed phenotypes. These mutant strains should be helpful for studying the influence of the microsymbiont on the differentiation and colonization of infected cells in soybean nodules.     

Expression of nir, nor and nos denitrification genes from Bradyrhizobium japonicum in soybean root nodules

Expression of Bradyrhizobium japonicum wild-type strain USDA110 nirK , norC and nosZ denitrification genes in soybean root nodules was studied by in situ histochemical detection of β -galactosidase activity. Similarly, P_nirK- lacZ , P_norC- lacZ , and P_nosZ- lacZ fusions were also expressed in bacteroids isolated from root nodules. Levels of β -galactosidase activity were similar in both bacteroids and nodule sections from plants that were solely N₂-dependent or grown in the presence of 4 m M KNO₃. These findings suggest that oxygen, and not nitrate, is the main factor controlling expression of denitrification genes in soybean nodules. In plants not amended with nitrate, B. japonicum mutant strains GRK308, GRC131, and GRZ25, that were altered in the structural nirK , norC and nosZ genes, respectively, showed a wild-type phenotype with regard to nodule number and nodule dry weight as well as plant dry weight and nitrogen content. In the presence of 4 m M KNO₃, plants inoculated with either GRK308 or GRC131 showed less nodules, and lower plant dry weight and nitrogen content, relative to those of strains USDA110 and GRZ25. Taken together, the present results revealed that although not essential for nitrogen fixation, mutation of either the structural nirK or norC genes encoding respiratory nitrite reductase and nitric oxide reductase, respectively, confers B. japonicum reduced ability for nodulation in soybean plants grown with nitrate. Furthermore, because nodules formed by each the parental and mutant strains exhibited nitrogenase activity, it is possible that denitrification enzymes play a role in nodule formation rather than in nodule function.