Hydrogenase Activity of Soybean Nodules Doubly Infected with Bradyrhizobium japonicum and B. elkanii

Rhizobitoxine (2-amino-4-(2-amino-3-hydropropoxy)-trans-but-3-erioic acid) is a phytotoxin produced by some strains of Bradyrhizobium species. Rhizobitoxine-producing strains often induce chlorosis in new leaves of soybean as a result of the synthesis of the toxin in nodules (Owens and Wright 1964; Owens et al. 1972). Some of the B. japonicum bacteroids possessing the hydrogen uptake (Hup) system are capable of ATP production by recycling H₂ evolved from nitrogenase (Evans et al. 1987). Adequate uptake hydrogenase activity in soybean bacteroids often enhances plant growth, as well as the efficiency of energy utilization during nitrogen fixation (Evans et al. 1987).     

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     

Repeated sequence RSα is diagnostic for Bradyhizobium japonicum and Bradyrhizobium elkanii

The genome of Bradyrhizobium japonicum and B. elkanii contains multiple copies of the repeated DNA sequence RSα. A collection of 18 B. japonicum, 4 B. elkanii and 72 other bacterial strains was screened by polymerase chain reaction (PCR) using a pair of primers specific for RSα. Only strains of B. japonicum and B. elkanii gave the predicted amplification product. Restriction analysis of PCR products obtained from different strains of B. japonicum showed that the RSα sequence was generally conserved. The usefulness of RSα as a specific probe for Bradyrhizobium strains capable of nodulating soybean was also demonstrated. Received: 11 May 1995     

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     

Dual inoculation with strains of Bradyrhizobium japonicum and Azospirillum brasilense to improve growth and biological nitrogen fixation of soybean (Glycine max L.)

The effects of inoculation with Bradyrhizobium japonicum and Azospirillum brasilense strains on the growth of soybean were evaluated with regard to the estimation of N₂ fixation using the ¹⁵N isotope dilution technique. Inoculation, in general, increased the dry mass of soybean as well as nitrogen content. Dual inoculation with a mixture of B. japonicum and A. brasilense strains was superior over single inoculation with B. japonicum. Nitrogen fixed (Ndfa) varied according to inoculant and soil conditions. Percentages of nitrogen derived from air (% Ndfa) using a non-nodulating isoline were 72% and 76% for B. japonicum and B. japonicum plus A. brasilense, respectively, in non-sterile soil. A similar but higher trend was recorded in sterilized soil, in which the percentages of N₂ fixed were 81% and 86% for single and dual inoculation, respectively. The correlation coefficient between N₂ fixed and N uptake (r=0.94) and dry mass (r=0.89) was significant. Application of special bacterial inoculants in agricultural systems of Egypt seems to be a promising technology and could be used for improving soybean growth as well as soil fertility, thus minimizing environmental pollution. Received: 10 January 1996     

高固氮基因工程大豆根瘤菌的研制及其增氮效应

通过构建快生型大豆根瘤菌Ｂ５２的基因文库和三亲本杂交，将增效因子ＤＮＡ片段导入优良的慢生型大豆根瘤菌２２－１０中，获得携带来自快生菌增效因子ＤＮＡ片段的工程菌株ＨＮ３２，经盆栽和小区试验，证明基因工程菌株ＨＮ３２比出发菌株２２－１０平均增产６％，比对照平均增产１３．２％～１６．９％，相当于每公顷施７５～１５０ｋｇ尿素．１９９２～１９９５年，在广西推广应用基因工程大豆根瘤菌ＨＮ３２２．１６万ｈｍ２，每公顷平均增产１９％，投入产出比１：３０。增加经济效益１４０９．８万元。     

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.     

Nodulation and competitiveness of gusA-marked Bradyrhizobium japonicum A1017 in soybean

Attempts to improve the symbiotic nitrogen fixation with effective (Brady) rhizobium strains do not always succeed under field conditions due to the lower nodulation competitiveness of the introduced strains than that of the indigenous rhizobia (Triplett and Sadowsky 1992). An introduced strain needs to be marked for monitoring its nodule occupancy under competitive nodulation conditions.     

Inhibition of growth of Bradyrhizobium japonicum bacteroid by spermidine and spermine in yeast extract

Abstract

Bacteroids are defined as the symbiotic forms of Rhizobium or Bradyrhizobium cells in the root nodules of their legume host. The differentiation to bacteroids involves various physiological changes and may be associated with some genetical changes. Single-colony isolates from a nodule formed by a Bradyrhizobium strain often differed in their effectiveness and intrinsic antibiotic resistance (Weaver and Wright 1987; Ozawa unpublished data). Detailed analysis of the changes in the gene structure requires the isolation of individual bacteroids from a nodule. However it has been reported that the viability of bacteroids in culture is very low and only a small fraction of a bacteroid population could produce colonies on yeast extract-mannitol (YEM) agar (Sutton et al. 1977). Bergersen (1974) concluded that the reversion of bacteroids to the vegetative, growing form is very rare.     

Effect of CO2-free air treatment on nitrogen fixation of soybeans inoculated with Bradyrhizobium japonicum and Rhizobium fredii

Soybean (Glycine max L. Merr.) cultivars Akisirome and Peking were inoculated with Bradyrhizobium japonicum Is-21 and Rhizobium fredii USDA 194, respectively, and were grown in cylindrical pots containing sterilized vermiculite which were aerated with CO₂-free air or ambient air to study the effects of CO₂ deficiency in the rhizosphere on plant growth, nodulation, and nitrogen fixation. The repressive effects of CO₂-free air treatment were more conspicuous in Peking than Akisirome, and nodule number, nodule mass, amount of biologically fixed N and plant growth of Peking were reduced remarkably by the CO₂-free air treatment.

Acetylene reduction activity (ARA) of Peking inoculated with USDA 194 and Akisirome inoculated Is-21 was assayed in the absence and presence of CO₂, ARA of Akisirome was not affected by the absence of CO₂, while that of Peking decreased drastically.

Based on these results, it was concluded that nitrogen fixation by Akisirome inoculated with B. japonicum Is-21 was not appreciably influenced by CO₂ deficiency in the rhizosphere, while that of Peking inoculated with R. fredii USDA 194 was severely repressed, and the decrease was estimated to be due to both the reduction of the nodule mass and specific nitrogen-fixing activity.     

Field performance of three Bradyrhizobium japonicum strains with two soybean (Glycine max. L.) cultivars

Summary A field experiment was condutced in a clay loam soil to study the performance of three Bradyrhizobium japonicum strains; USDA 110, USDA 138 and TAL 379, in relation to their N₂-fixing potential and competitiveness on two soybean cultivars (Clark and Calland). Inoculation of soybean cultivars with these strains, either singly or in combination, induced significant increases in plant dry weight, N₂ fixation and seed yields. However, no significant differences were found between the rhizobial strains and/or their mixtures in N₂ fixation and increased seed yield for both cultivars. The two soybean cultivars gave similar responses to inoculation. No significant differences in seed yield were observed between Clark and Calland cultivars. The interaction between inoculant strain and soybean cultivar was not significant. The competition between strains for nodulation was assessed. Strain USDA 110 was the most competitive, followed by USDA 138. Strain TAL 379 was always less competitive on both cultivars. The incidence of double-strain occupancy of nodules varied from 8% to 40%.     

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     

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     

Evaluation of effective Myanmar Bradyrhizobium strains isolated from Myanmar soybean and effects of coinoculation with Streptomyces griseoflavus P4 for nitrogen fixation

In order to substitute the use of chemical fertilizers in legume production, there is a need for the production of rhizobial inoculants which are capable of being used as biofertilizers. To achieve this, an effective symbiotic nitrogen (N) fixation between legumes and root nodule bacteria will be essential. Evaluation of effective Myanmar Bradyrhizobium (Jordan 1982) strains isolated from Myanmar soybean (Glycine max L. Merr.) and effects of coinoculation with Streptomyces griseoflavus Krainsky 1914 P4 for N fixation were studied in pot experiments using sterilized vermiculite and Hoagland solution in the Phytotron (25°C and 70% relative humidity) with completely randomized design and three replicates. N fixation ability of soybean was evaluated by acetylene reduction activity (ARA) by gas chromatography. It was found that MAS23 showed a relatively high degree of stability and a high level of ARA per plant on both Yezin-3 and Yezin-6 soybean varieties. In the symbiotic relationship between Bradyrhizobium strains and P4 experiments, the treatments consisted of six Bradyrhizobium strains (MAS23, MAS33, MAS34, MAS43, MAS48 and USDA110) and Streptomyces griseoflavus P4 were evaluated with four Myanmar soybean varieties (Yezin-3, Yezin-6, Hinthada and Shan Sein). In the Yezin-3 soybean variety, the best treatment for ARA per plant was found in the dual inoculation of P4 and MAS34. In the Yezin-6 soybean variety, the highest nodule dry weight was found in dual inoculation of P4 with MAS34 but the highest ARA per plant was observed in the dual inoculation of P4 and MAS23. On the other hand, single inoculation of MAS43 and coinoculation of P4 with MAS48 were significantly higher in N fixation of Hinthada, and coinoculation of P4 with MAS33 was significant improvement of ARA per plant (P < 0.05) in Shan Sein soybean.     

Nodulation competition among Bradyrhizobium japonicum strains as influenced by rhizosphere bacteria and iron availability

Summary Bacteria isolated from the root zones of field-grown soybean plants [Glycine max (L.) Merr.] were examined in a series of glasshouse experiments for an ability to affect nodulation competition among three strains of Bradyrhizobium japonicum (USDA 31, USDA 110, and USDA 123). Inocula applied at planting contained competing strains of B. japonicum with or without one of eleven isolates of rhizosphere bacteria. Tap-root nodules were harvested 28 days after planting, and nodule occupancies were determined for the bradyrhizobia strains originally applied. Under conditions of low iron availability, five isolates (four Pseudomonas spp. plus one Serratia sp.) caused significant changes in nodule occupancy relative to the corresponding control which was not inoculated with rhizosphere bacteria. During subsequent glasshouse experiments designed to verify and further characterize these effects, three fluorescent Pseudomonas spp. consistently altered nodulation competition among certain combinations of bradyrhizobia strains when the rooting medium did not contain added iron. This alteration typically reflected enhanced nodulation by USDA 110. Two of these isolates produced similar, although less pronounced, effects when ferric hydroxide was added to the rooting medium. The results suggest that certain rhizosphere bacteria, particularly fluorescent Pseudomonas spp., can affect nodulation competition among strains of R. japonicum. An additional implication is that iron availability may be an important factor modifying interactions involving the soybean plant, B. japonicum, and associated microorganisms in the host rhizosphere.Paper No. 10648 of the Journal Series of the North Carolina Agricultural Research Service, Raleigh, NC 27695-7601, USA     

菌根与根瘤菌联合应用对复垦矿区根际土壤环境的改良后效

为了进一步确定微生物复垦对土壤的持续修复能力,在神东矿区活鸡兔开采沉陷试验基地,接种丛枝菌根真菌和大豆根瘤菌改良农用地土壤,已取得了较好的微生物复垦效果,经过2 a的退耕撂荒以后,对微生物复垦后效进行系统地研究。研究结果表明,撂荒后,接菌小区根外菌丝密度、根系侵染率和pH值均显著高于对照小区,分别比对照提高了90%、52%和1.3%;接菌小区细菌数量、微生物总量、有效磷含量、电导率和酸性磷酸酶活性也明显高于对照小区,但差异不显著。撂荒后与撂荒前相比,土壤呈退化趋势,但接菌小区菌丝密度几乎增加了1倍,差异显著,同时侵染率、细菌数量和酸性磷酸酶活性均明显升高,差异不显著;而在对照小区中,菌丝密度稍有增加,侵染率、细菌数量和酸性磷酸酶活性均明显降低,且差异不显著。利用丛枝菌根真菌与大豆根瘤菌联合作为绿色环保肥料,能够有效改善根际土壤环境,减轻土壤的退化程度,在弃耕状态下也能发挥良好的效用,对土地贫瘠的开采沉陷地进行生态修复具有重大意义。     

Cloning and characterization of a NADP+-malic enzyme gene from Bradyrhizobium japonicum USDA110

Malic enzymes have been considered to play a key role in energy metabolism for nitrogenase reaction in bacteroids. To elucidate the physiological role of the malic enzymes in Bradyrhizobium japonicum bacteroids, a putative malic enzyme gene Bjtme1 was cloned by polymerase chain reaction (PCR) using degenerated primers from conserved regions of the protein sequences of bacterial malic enzymes and draft sequence data of the Bradyrhizobium japonicum USDA110 genome sequence project. To confirm the characteristics of the Bjtme1 gene, the protein encoded by this gene was over-expressed using a pET32a(+) system and it exhibited a NADP⁺-malic enzyme (EC 1.1.1.40) activity, indicating that Bjtme1 was the gene of the NADP⁺-malic enzyme. This is the first report on the cloning and characterization of the NADP⁺-malic enzyme gene from B. japonicum, and the gene structure was compared with that of NADP⁺-malic enzyme genes of other rhizobia.     

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.     

丛枝菌根真菌与根瘤菌接种对大豆根瘤分布及磷素吸收的影响

在温室条件下,采用石英砂盆栽试验研究了大豆(冀豆6号)接种丛枝菌根真菌（Glomous mosseae）与根瘤菌 （Bradyrhizobium japonicum）对根瘤的形成、分布以及磷素吸收效率的影响。结果表明,大豆生长至开花期（接种后56 d）,与单接种根瘤菌处理相比,双接种AM真菌和根瘤菌显著增加大豆生物量、氮、磷含量、根系上的总根瘤数。单接种根瘤菌条件下,总根瘤数的48.4%分布在主根上,51.6%分布在侧根上;根瘤菌与AM真菌双接种时,总根瘤数的32.5%分布在主根上,67.5%分布在侧根上。双接种处理的侧根根瘤的固氮酶活性显著高于单接种处理的。双接种条件下大豆侧根中AMF侵染增强,尤其是结根瘤侧根上的AM真菌的侵染率高于未结瘤的侧根的菌根侵染率。接种后28 d单接种菌根真菌处理显著高于双接种处理的植株磷的吸收效率;而56 d 时趋势相反。以上结果表明,AM真菌侵染改变根瘤在大豆根系上的分布,根瘤数量、分布与结根瘤侧根上AM真菌的侵染强度存在正相关关系。