Soybean preference for Bradyrhizobium japonicum for nodulation

For examining the probability of increase in the occupation ratio of inoculated rhizobium in nodules, various Rj-soybean cultivars including the Rj ₂ Rj ₃ Rj ₄-lines of soybean were grown in a field of the Kyushu University Farm. Bradyrhizobium japonicum USDA110 that carries uptake hydrogenase (Hup⁺) was used as an inoculum. The relative efficiency of nitrogen fixation generally increased by the inoculation. However, there were no significant differences in the effects among the genotypes of the host plants. The occupation ratio of serogroup USDA110 in the nodules on the taproot of the inoculated plants was in the range of 77–100%, suggesting that the B. japonicum strain USDA110 infected taproots immediately after inoculation. The occupation ratios in the nodules on the lateral roots were 53–67, 40–86, 63–83, and 62–77% in inoculated plants of the non-Rj-, Rj ₂ Rj ₃-, Rj ₄-, and Rj ₂ Rj ₃ Rj ₄-genotypes, respectively, and they decreased in all the genotypes with the progression of growth. At the time of the first sampling, the occupation ratios on the lateral roots of these Rj ₂ Rj ₃ Rj ₄-genotypes showed values intermediate between those of IAC-2 (Rj ₂ Rj ₃) and Hill (Rj ₄) , which were the parent cultivars of the Rj ₂ Rj ₃ Rj ₄-lines, B340, B349, and C242. The reduction in the occupation ratio of the serogroup USDA110 for about 1 month after the first sampling was the lowest (0.13–0.16) in the Rj ₂ Rj ₃ Rj ₄-genotypes, excluding B349, followed by the non-Rj- and Rj ₂ Rj ₃-genotypes and highest (0.52–0.69) in the Rj ₄-genotypes, excluding Hill. Therefore, it was considered that the population of compatible rhizobia with host soybean plants increased in the rhizosphere with the progression of the development and growth. The results showed that with the expansion of the root area of host plants, the occupation ratio of type A rhizobia including the serogroup USDA110 was high. Therefore, the Rj ₂ Rj ₃ Rj ₄-genotypes were superior to other Rj-genotypes in terms of the inoculation effects of nodulation type A rhizobium, B. japonicum USDA110. However, the preference of the Rj ₂ Rj ₃ Rj ₄-genotype for serogroup USDA110 is not sufficient to rule out the competition with the other serogroups in this study. Therefore, the study should be centered on the isolation of more efficient (Hup⁺) and highly compatible rhizobial strains with the Rj ₂ Rj ₃ Rj ₄- genotypes.     

Soybean preference for Bradyrhizobium japonicum for nodulation

For the increase of the occupation ratio of inoculum strain in the competition with indigenous rhizobia, the relationship between Rj-genotypes of soybean and the preference of Rj-cultivars for various types of rhizobia for nodulation was investigated by using the Rj ₂ Rj ₄-genotype of soybean isolated from the cross between the Rj ₂ Rj ₃-cultivar IAC-2 and Rj ₄-one Hill (Ishizuka et al. 1993: Soil Sci. Plant Nutr., 39, 79-86). Firstly, these Rj ₂ Rj ₄-genotypes were found to harbor the Rj ₃-gene. The Rj ₂Rj₃Rj₄-genotypes of soybean were considered to exhibit a more narrow microsymbiont range for nodulation than the Rj ₂ Rj ₃-and Rj₄-cultivars. Therefore, rhizobia were isolated from the nodules of various Rj-genotypes of soybeans grown in soils, and the preference of the Rj ₂ Rj ₃ Rj ₄-genotype for indigenous rhizobia was examined. The nodule occupancy of serotype 110 was significantly higher in the bacteroids of the nodules from the Rj ₂ Rj ₃ Rj ₄-rgenotypes than in those from the other genotypes, non Rj-, Rj ₂ Rj _3-, and Rj ₄-cultivars. These results demonstrated that the Rj ₂ Rj ³ Rj ⁴-genotype prefers more actively serogroup USDA110 to the others of rhizobia. Thus, Rj ₂ Rj ₃ Rj ₄-genotype is superior to non- Rj-, Rj ₂ Rj ₃-, and Rj ₄-genotypes for the formation of efficient nodules for nitrogen fixation.     

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).     

Improved Soybean Production after Inoculation with Bradyrhizobium japonicum

Fruit firmness testing of strawberries was carried out with five methods. Four instruments, Instron, Texture Analyser, penetrometer PNR10 (SUR Berlin) and Bareiss HHP-2001-Fff, were tested. In addition "Eisenhuth's method" was tried, which involves putting the fruits into a cylinder, placing a load on the top and measuring the compression. Eisenhuth's method was found to be cheap, time-saving and easy to use, and is a promising method for firmness testing of strawberries. Bareiss is a small instrument, which was found to be easy and quick to use, and gave reliable results. The results were easily transmitted to a personal computer. Instron and Texture Analyser are good and advanced instruments with reliable results, but they are expensive, complicated and time-consuming. The PNR10 is quite easy to use but time-consuming, and the results showed large variations. Relatively large natural variability in strawberry firmness was found; therefore, the sample size should be at least 25 fruits. Strawberry fruit firmness was tested in 18 cultivars by two methods. Considerable cultivar differences were found and the ranking of the cultivars was different by the two methods. Large variations in fruit firmness and in soluble solids between individual fruits within samples were observed, especially for cv. 'Korona'. The correlations between firmness and soluble solids content were not significant. Small fruits tended to be firmer than large ones. Ripe fruits were softer than pink ones, but the difference was reduced during storage. The fruits were softer when the internal fruit temperature was 20°;C than at lower temperatures. Fruit colour measured by a Minolta Colorimeter showed smaller variations than in firmness and soluble solids. Only low correlations between colour and other fruit characteristics were found.     

慢生型大豆根瘤菌nfeC基因的定位

在前期工作中 ,从慢生型大豆根瘤菌菌株 GX2 0 1的基因文库中筛选到一个含 nfe C基因同源片段的重组质粒 p GXN2 0 1。在本工作中 ,将 nfe C基因同源片段定位在 4 kb Cla I Xba I片段上。对该片段的部分序列分析表明与已报道的 nfe C基因具有 95%的同源性     

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.     

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.     

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。     

慢生型大豆根瘤菌菌株GX201的脯氨酸脱氢酶基因(putA)的分子克隆

根据已报道的脯氨酸脱氢酶基因（ｐｕｔＡ）序列,通过ＰＣＲ方法,从慢生型大豆根瘤菌菌株ＧＸ２０１的总ＤＮＡ中扩增到—ＰＣＲ产物。序列分析表明,该ＰＣＲ产物的长度为４１８ｂｐ,与已报道的ｐｕｔＡ基因具有９３．５％的同源性。以广谱寄主范围质粒ｐＬＡＦＲ３为载体,在大肠杆菌ＤＨ５α中构建了ＧＸ２０１的基因文库,并以该ＰＣＲ产物为探针,从基因文库中筛选到一重组质粒ｐＧＸＮ３００。     

Survival of inoculant Bradyrhizobium japonicum in an Andosol

The survival of an antibiotic-resistant mutant of a commercial inoculant Bradyrhizobium japonicum strain, A1017ks, was studied in a volcanic ash soil (Andosol) in comparison with a non-volcanic ash soil (Fluvisol) over a period of 84 days. In a non-sterile soil system, the population decline in the Andosol (15% or 1.2 log units) was larger than in the Fluvisol (6% or 0.54 log units). In both soils, however, the inoculant bradyrhizobium survived at fairly high population levels after the period of incubation [10⁶ and 10⁷ colony-forming units (CFU) g^-1 dry soil in the Andosol and Fluvisol, respectively]. In sterile control soil, viable bradyrhizobium cells could not be detected after 1 week of incubation in the Andosol, whereas in the Fluvisol population of introduced bradyrhizobium was maintained throughout the period of incubation. Overall changes in the population of indigenous bacteria and fungi were also monitored. However, no clear pattern of interaction between the inoculant Bradyrhizobium japonicum and the indigenous microbes could be identified. The antibiotic-resistant mutant maintained its resistance in the Fluvisol throughout the 3-month period of incubation, making it a useful model for conducting ecological studies in the soil.     

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.     

慢生型大豆根瘤菌nfe基因的分子克隆

ｎｆｅＣ基因是从慢生型大豆根瘤菌中克隆到的,与竞争结瘤有关的基因。本研究从慢生型大豆根瘤菌菌株ＧＸ２０１的ｐＬＡＦＲ３为载体的基因文库中,筛选出与ｎｆｅ同源基因克隆。以转座子Ｔｎ５ｇｕｓＡ５诱变获得了ｇｕｓ基因表达的Ｔｎ５ｇｕｓＡ５插入突变质粒。     

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     

Yield Response of Intercropped Soybean and Maize Under Rhizobia (Bradyrhizobium japonicum) Inoculation and P and K Fertilization

A 2-year trial was carried out at the Tanzania Coffee Research Institute (TaCRI) farm in northern Tanzania. The aim was to assess the response of soybean and maize yield attributes to cropping systems, rhizobia inoculation, and phosphorus (P) and potassium (K) fertilization. The study was laid out in a split-split plot design replicated thrice. The statistical analyses were performed using the 3-way analysis of variance in a factorial arrangement. The computation was performed using the STATISTICA software. The Fisher’s least significant difference (LSD) was used to compare the treatment means at p = 0.05 level of significance. The results showed that both cropping systems, rhizobia inoculation, and P and K fertilization significantly (p = 0.05) influenced most of the soybean yield parameters assessed in this study. Specifically, cropping systems significantly (p = 0.05) improved the number of pods per plant, biological yield, grain yield, and harvest index (HI) (2015 and 2016) of soybean. Rhizobia inoculation also significantly (p = 0.05) improved soybean yield attributes such as the number of pods, 100 seed weight, biological yield and grain yield of soybean (2015 and 2016), and HI in soybean (2015). P and K fertilization also significantly (p = 0.05) improved different yield attributes of soybean over the control. It was noted that increasing potassium from 20 to 40 kg/ha improved most of the soybean yield parameters relative to the control. Doubling of P from 26 kg to 52 kg/ha did not significantly change the soybean yield parameter. The doubled combined fertilizers did not significantly increase the yield parameters of soybean suggesting the use of a lower dose of combined fertilizers. Intercropping maize with rhizobia-inoculated soybean significantly improved maize yield compared with intercrop without inoculation. Monocropped maize gave relatively the same yield as those of maize intercropped with inoculated soybean. Fertilization with P and K also improved the yield attributes of maize over the control.     

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     

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。     

Interactive effects of co-inoculation of Bradyrhizobium japonicum strains and mycorrhiza species on soybean growth and nutrient contents in plant

The main objective of this study was to investigate the effects of co-inoculation with different strains of Bradyrhizobium japonicum (i.e. Helinitro, Rizoking, and Nitragin) and arbuscular mycorrhizal fungi (AMF) species (i.e. Glomus fasciculatum, Glomus versiforme, Glomus intraradices, Glomus mosseae, and Glomus etunicatum) on soybean growth, fungal root colonization, and nutrient uptake of nitrogen (N), phosphorus (P), zinc (Zn), iron (Fe), and copper (Cu). Co-inoculation with various AMF species and rhizobia significantly (p<0.01) increased the soybean biomass production as compared to the non-inoculated controls. Furthermore, AMF colonization of roots of soybean plants increased by 79, 70.1, 67, 63, 57.5, and 50.1% in the presence of G. fasciculatum (GF), G. versiforme (GV), G. intraradices (GI), G. mosseae (GM), and G. etunicatum (GE), and Gmix (a mixed culture of fungi), respectively. Higher nutrient contents were observed in plants co-inoculated with Helinitro and GF. More insight into these results will enable optimization of the effective use of AM fungi in combination with their bacterial partners as a tool for increasing soybean yields in Iran; however, its general analytical framework could be applied to other parts of the world.     

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.     

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.     

Effect of nodulation with Bradyrhizobium japonicum and Shinorhizobium fredii on xylem sap composition of Peking (Glycine max L. Merr )

Five barley cultivars were grown together in complete, low-P·low-pH and high-Al medium containing only NO₃, only NH₄ or both NO₃ and NH₄ as N sources, respectively using an automatic control system of pH for water culture, and the relationship between the differential Al tolerance and the plant-induced pH change of medium among the barley cultivars was investigated.

The pH of the medium containing only NO₃ as N source tended to increase, whereas the pH of the other media containing only NH₄ or both NO₃ and NH₄ as N sources tended to decrease, but the fluctuations of the medium pH could be maintained within the value of 0.2 pH in the complete medium and within the value of 0.1 pH in the high-Al medium.

Barley cultivars still differed in their Al tolerance in the medium which was continuously stirred and circulated at a constant pH. The pattern of Al tolerance was not affected by the N sources in the medium. The plant-induced pH change of medium for each cultivar was influenced by the N sources in the medium, and was not correlated positively with Al tolerance. The contents of Al and Ca or other nutrient cations in roots were positively correlated with Al tolerance and positive correlations were recognized also between the contents of Al and Ca or some other nutrient cations in the roots.

In conclusion, the following mechanisms are proposed. Al tolerant barley cultivars exclude Al actively outside the plasmalemma of the root cells, and the excluded Al may polymerize and or react with P to form Al precipitates. Consequently, in the Al tolerant barley cultivars the Al content may be low in the root protoplasts, high in the whole root tissues and the contents of Ca or other nutrients may be high in the roots. The plant-induced pH change of medium is not considered to be the cause of the differential Al tolerance among barley cultivars.