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).     

Estimation of the bacterial community diversity of soybean-nodulating bradyrhizobia isolated from Rj-genotype soybeans

Abstract

The diversities of communities of soybean-nodulating indigenous bradyrhizobia in Japan were estimated using mathematical ecology methods based on the results of polymerase chain reaction–restriction fragment length polymorphism analysis of the 16S–23S rDNA internal-transcribed spacer region. Polar ordination analysis indicated a significant correlation between the compositions of bradyrhizobial communities and northern latitudes in Japan. This result suggests that the composition and the geographical distribution of indigenous soybean-nodulating bradyrhizobia might be affected by soil temperature and the associated diversity of the host plants acclimatized to a particular climate. Furthermore, for estimation of the compositional difference in bacterial communities among three different Rj-genotype soybean cultivars, an analysis of the diversity indexes was conducted and this analysis indicated differences in the composition of the communities isolated from the Rj ₂ Rj ₃-genotype compared with those from the non-Rj- and Rj ₄-genotype cultivars. This result suggests that Rj ₂ Rj ₃-genotype soybeans might affect not only compatibility with particular bradyrhizobia, but also preference to bradyrhizobia for nodulation.     

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.     

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.     

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     

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     

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。     

Microbial inoculation of Rhizobium and phosphate-solubilizing bacteria along with inorganic fertilizers for sustainable yield of soybean [Glycine max (L.) Merrill]

A field experiment was conducted to assess the effect of microbial inoculants and inorganic fertilizers for sustaining the yield of soybean. Application of 100% recommended dose of fertilizer (RDF) gave significantly highest yield (2433 kg ha^?1) over 75% RDF (2317 kg ha^?1) and without RDF (2205 kg ha^?1). Seeds inoculated with Rhizobium (Bradyrhizobium japonicum) and phosphate-solubilizing bacteria (2480 kg ha^?1) gave significantly highest soybean yield over without inoculation (2191 kg ha^?1). Rhizobium and phosphate-solubilizing bacteria with 100% RDF (2674 kg ha^?1) gave significantly highest seed yield than rest of the treatment combinations. Root nodules and their dry weight were remained un-influenced due to fertilizer levels, whereas in bio-fertilizers, it was significantly higher with Rhizobium inoculation (24.3 and 408 mg, respectively) followed by dual inoculation of Rhizobium and PSB. 100% RDF and dual inoculation with Rhizobium and PSB earned Rs. 47916/- and Rs. 51182/- net returns per ha, respectively.     

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.     

Edaphic factors as determinants for the distribution of intrinsic antibiotic resistance in a cowpea rhizobia population

 A large collection of cowpea rhizobia strains was obtained from soil samples collected from either a semi-arid or a tropical rain forest area located at about the same latitude in the north-eastern region of Brazil and evaluated for their intrinsic antibiotic resistance to eight commercial antibiotics. The aim of this study was to correlate antibiotic resistance of native rhizobia strains to edaphic-climatic factors as a way to establish suitable inoculants for specific areas. A large diversity regarding intrinsic antibiotic resistance was found, and 17 clusters were identified as varying from sensitive to gradually resistant up to 500 μg·ml^–1 of the antibiotics tested. Clustering analysis did not show any pattern related to the geographic region where isolates have been obtained. On the other hand, an increase in the antibiotic-resistant rhizobia population was associated with an increase in soil P and Al contents. lsolates which were sensitive to spectinomycin, ampicillin, streptomycin, chloramphenicol and tetracycline were present at higher rates in soils devoid of Al. Rhizobia bacteria producing mucus type I (fluid and capable of spreading over the solid media) were found preferentially in soil with Al concentrations up to 36 mg·kg^–1, diminishing quickly at higher levels. Received: 2 May 1997     

Low pH and aluminum tolerance of Bradyrhizobium strains isolated from acid soils in Indonesia

Sixty-seven strains of Bradyrhizobium isolated from soybean plants growing on acid soils in West Java and Sumatra, Indonesia, were examined for the effect of the pH and aluminum concentration on their growth in nutrient media, compared with 61 strains of Bradyrhizobium from soils in Japan. The results in this study indicated that the indigenous population of Bradyrhizobium in the soils of Indonesia showed a large difference in acid- and Al-tolerance from that of Japan. Eighty-five and 48% of the isolates from Japanese soils and Indonesian soils, respectively, were unable to grow in YEM broth at pH below 4.5. The acid-tolerance was correlated with AI-tolerance of the isolates on YEM agar plates at pH 4.4. Seventy-five percent of the isolates that grew in YEM broth at pH 4.5 were also resistant to 400 µM Al on the YEM plates. Acetylene reduction assay of the root nodules revealed that 3 of the acid- and Al-tolerant isolates from Indonesian soils showed a significantly high nitrogen fixation activity.     

Caution of intensified spread of antibiotic resistance genes by inadvertent introduction of beneficial bacteria into soil

The use of beneficial bacteria isolated especially from rhizosphere soils called plant growth-promoting rhizobacteria (PGPR) has been attracting particular interests. However, a potential source of antibiotic resistance genes (ARGs) carried by PGPRs and derived biocontrol agents and/or bio-fertilizers is widely forgotten and ignored. It is very urgent to raise the question if large-scale introduction of beneficial bacteria into soils can aggravate the situation in the spread of ARGs in environment. In this article, we conclude and analyze the possibility of a potential ARGs reservoir associated with introduction of PGPR strains into soils, and offer some advice on PGPR isolation, development and application.     

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     

大薸对奶牛场废水中耐药基因的去除效果和驱动因子分析

为探究大薸对奶牛场废水的净化效果,该研究选用温室模拟培养结合高通量测序方法,研究大薸对奶牛场废水中耐药基因（Antibiotic Resistance Genes,ARGs）的去除效果及驱动因子。结果显示：对于奶牛场3种实际废水（原水、厌氧池和氧化塘废水）,大薸整体呈现良好的净化效果,废水中大部分ARGs的绝对丰度呈现正去除效果,在原水、氧化塘废水和低浓度厌氧池废水中,大薸处理后ARGs绝对丰度（lg值）分别下降0.25（ermA 和fexA）～3.66（blaOXA-1）、0.08（blaTEM-1）～3.51（strB）和0.09（fexA）～4.07（strB）,而对高浓度厌氧池废水,则仅有9/16的ARGs呈现正去除。经过大薸处理后,不同废水中的微生物多样性和丰度均升高,废水中优势菌种Proteobacteria和Firmicutes相对丰度下降,而Bacteroidetes 和Actinobacteriota相对丰度上升,相较于无植物处理组中微生物群落差异较大。冗余分析（Redundancy Analysis,RDA）结果表明,优势菌属（Hydrogenophaga、Flavobacterium、Bacillus和Gemmobacter）结合环境因子对ARGs变化的解释率分别为9.6%、6.0%、7.0%和5.1%,网络分析结果亦表明,ARGs变化与微生物和环境因子密切相关。因此,大薸对ARGs的去除是通过微生物变化和环境因子改变等多种作用共同驱动的。研究结果可为畜禽污水中ARGs污染防控提供理论依据,有助于推动植物生态处理技术的绿色可持续发展。     

畜禽粪便厌氧发酵过程抗生素抗性基因归趋及驱动因子分析

针对畜禽养殖业抗生素抗性基因(antibiotic resistance genes, ARGs)污染问题,该文选取厌氧发酵技术,对比不同厌氧发酵体系内ARGs消长与潜在宿主菌,挖掘不同因子与ARGs的相互关系。结果表明,厌氧发酵体系内微生物群落变化是ARGs消长的主要驱动因子,确定ARGs的潜在宿主菌是目前研究的难点;抗生素和重金属也是ARGs消长的重要驱动因子,控制抗生素污染和重金属污染可有效减缓ARGs污染;可移动遗传元件在ARGs水平传播过程中起着重要作用。综合而言,厌氧发酵体系内各个因子直接或间接影响ARGs消长,其中工艺参数是控制整个厌氧发酵体系的先决因素,在特定工艺参数下,微生物群落与体系物化指标相互影响与制约;微生物通过分子内部可移动遗传元件实现ARGs在不同微生物之间的水平传播。综上所述,通过综合协调各类因子实现厌氧发酵体系内ARGs消控是今后研究重点。     

高压脉冲电场对大豆分离蛋白功能性质的影响

该文研究了高压脉冲电场对大豆分离蛋白功能性质的影响。结果表明：随着脉冲强度和脉冲处理时间的延长，大豆分离蛋白的溶解度、乳化性、起泡性及疏水性都增加。当脉冲强度或处理时间大于35 kV/cm或432 μs(溶解度)、30 kV/cm或144 μs(乳化性)、35 kV/cm或432 μs(起泡性)及30 kV/cm或288 μs(疏水性)时，由于蛋白质分子变性程度增加，分子发生聚集，功能性质反而下降。     

Multiphase characterization of wild Vigna associated root nodule bacteria from Japanese subtropical islands unveiled novel high temperature resistant Bradyrhizobium strains having high symbiotic compatibility with soybean and mungbean

ABSTRACT

Vigna riukiuensis plant – a rare type of vigna, found only in Taiwan and the islands of Okinawa prefecture, Japan – possesses intrinsic property of high level of salt and heat tolerance. To understand the diversity and identify suitable rhizobia, multiphase characterization of root nodule bacteria associated with V. riukiuensis grown in Ishigaki and Iriomote Islands of Okinawa prefecture was performed. Multigene phylogenetic analysis of housekeeping genes based on 16S rRNA gene sequences, 16S-23S rRNA gene internal transcribed spacer (ITS) and 23S rRNA gene sequences identified three main groups closely similar to Bradyrhizobium japonicum, B. elkanii and B. jicamae family. However, analysis of symbiotic nifH and nodD1 genes and their phylogenetic trees showed similar topology, having only few discrepancies in comparison to the housekeeping gene phylogeny. Interestingly, for some of the isolates having similarity with B. elkanii, growth was observed at 40°C, which exceed the highest record for B. elkanii to the best of our knowledge. All the isolates were observed to have the capability of forming root nodules and fix nitrogen in their original host plant V. riukiuensis and two other crops: soybean and mungbean. Most of the isolates showed similar or higher nitrogen-fixing capability in comparison with B. diazoefficiens USDA110 in V. riukiuensis and V. radiata (mungbean), and Iri 5/6 in V. riukiuensis, Iri 5/12 in soybean and Ishi 7/2 in mungbean showed highest acetylene reduction assay (in µmol/h/gm nodule dry weight) activity, which was significantly higher than B. diazoefficiens USDA110. In addition, six isolates attained higher soybean biomass production compared with B. diazoefficiens USDA110, suggesting high symbiotic compatibility with soybean. Among them, Iri 5/7 of B. elkanii group contributed 29% higher soybean biomass production than B. diazoefficiens USDA110 and could grow at 40°C, hence it could be a promising soybean inoculant in the tropics.     

异氰酸酯改性大豆油基木材胶黏剂的制备与性能

环氧大豆油丙烯酸酯（Acrylated Epoxidized Soybean Oil, AESO）具有原料来源丰富、环境友好、耐水性好等优点,以AESO为原料制备新型无醛木材胶黏剂是降低当前甲醛系胶黏剂用量的重要途径。针对AESO同时含双键、羟基和环氧基的结构特点,以六亚甲基二异氰酸酯（Hexamethylene Diisocyanate, HDI）为改性剂、过氧化苯甲酸叔丁酯为自由基引发剂,制备了改性大豆油基木材胶黏剂及其杨木胶合板材,分析了胶黏剂的化学结构、流变性、固化行为、胶合机理及其对胶合板物理力学性能的影响。结果表明：HDI改性后的胶黏剂具有更高的交联密度、内聚力和胶接力,其杨木胶合板的耐水胶合强度可达1.18 MPa;HDI可与AESO的羟基、环氧基反应生成氨基甲酸酯结构,同时HDI的加入降低了AESO树脂的黏度和最大固化温度,提高了胶黏剂的加工性能;HDI改性显著提高胶合板的物理力学性能（P<0.05）,当HDI与AESO的质量比为1∶9时,胶合板的横向静曲强度和弹性模量分别为57.6和5 920 MPa,且板材在120°C下2 h不开裂。该研究为以生物质大豆油为原料制备无醛胶黏剂提供了一种可行路径,有利于木材工业的绿色与可持续发展。     

抗病性不同大豆品种根面及根际微生物区系的变化 Ⅰ.非连作大豆(正茬)根面及根际微生物区系的变化

采用平板计数法测定了3个抗病性不同的大豆品种在生育期内根面和根际微生物区系的变化情况,并应用荧光计数法直接测定了根际细菌和真菌的生物量。结果表明,土体的微生物种类最丰富、根际的次之、根面的较单一。播种后从三叶期到鼓粒初期,根面和根际的可培养细菌总数随生育期逐渐增加,鼓粒初期达最大值,而成熟期则有明显的下降;大豆根际细菌生物量也存在相同的变化规律。抗病性不同的大豆品种其根面、根际可培养细菌总数存在差异;抗病品种大豆的根瘤重明显高于感病品种。种植一季后感病品种根际积累的病原生物(镰孢霉Fusarium.sp.和大豆胞囊线虫Heterodera.glycines的胞囊数)明显高于抗病品种。说明大豆根系分泌物对微生物具有选择性的促进或抑制作用,不同大豆品种以及同一大豆品种在不同生育时期根系分泌物的组成和数量不同,从而使大豆根面及根际形成了特定的微生物区系组成。