Characterization of local rhizobia in Thailand and distribution of malic enzymes

TWenty-six isolates were obtained from nodules of various legume plants (Glycine max, Vigna sinensis, Arachis hypogaea, Desmanthus virgatus, Acacia mangium, Centrosema pascuorum, Pterocarpus indicus, Xylia xylocarpa, and Sesbania rostrata) in Thailand. After confirming their nodulation and nitrogen-fixing abilities, they were identified by 16S rRNA gene analysis as Bradyrhizobium japonicum, Bradyrhizobium elkanii, Rhizobium leguminosarum, Rhizobium gallicum, and Rhizobium galegae. Using these local isolates, the distribution of the activities of both NAD⁺-dependent (DME: EC 1.1.1.39) and NADP⁺-dependent (TME: EC 1.1.1.40) malic enzymes was surveyed. The malic enzyme activities were present in all the isolated rhizobia and in other 17 local Bradyrhizobium strains in Thailand. In almost all the rhizobia, the DME activity predominated whereas the TME activity predominated only in the Rhizobium gallicum strains that were major symbionts of Sesbania rostrata. Southern hybridization analysis was performed to survey the distribution of the malic enzyme genes among the local rhizobia, which are similar to those of B. japonicum. DNA probes (ME1 for DME and ME2 for TME) were prepared by polymerase chain reaction (PCR) using degenerated primers from conserved regions of the protein sequences of bacterial malic enzymes. Southern blot analysis with ME1 as a probe showed a single band in about half of the isolates, especially in B. japonicum and R. leguminosarum strains, suggesting the wide distribution of such DME genes among local rhizobia. In contrast, Southern blot analysis with ME2 as a probe detected a single band only in five B. japonicum strains, suggesting that the TME genes, which are similar to those of B. japonicum, would be unique in a group of B. japonicum.     

Effects of symbiosis with Rhizobium fredii on transport of fixed nitrogen in the xylem of soybean plants

An experiment was conducted to identify the main nitrogenous compound transported in the xylem sap of soybean plants nodulated with Rhizobium fredii. Soybean (Glycine max L. Merr.) cultivars, wild type Bragg (nod⁺, fix⁺) and its nitrate tolerant, hypernodulating mutant ntsll16 (nod⁺⁺, fix⁺) were used for this experiment. These soybean plants were inoculated with a slowgrowing rhizobium, Bradyrhizobium japonicum USDAllO or fast-growing rhizobia consisting of a mixture of R. fredii USDA191, USDA193, and USDA-194 and grown in a phytotron under natural light and controlled temperature conditions. Xylem sap was collected from Bragg and ntsll16 plants at the flowering and pod elongation stages. Acetylene reduction activity per plant or per nodule weight was not different between soybean lines and inoculums. The composition of the nitrogenous compounds in the xylem sap was compared between the symbionts, with B. japonicum and R. fredii. At the flowering stage, ureide-N and amide-N accounted for 53 to 70% and 20 to 27% respectively of the total N in the sap collected from the plants inoculated either with B. japonicum or R. fredii. At the pod elongation stage, ureide-N and amide-N accounted for 74 to 85%, and 7 to 19% of total sap N. With the growth of the soybean plants, the ratio of ureide-N in the xylem sap increased. These results suggest that in the case of wild soybean and the hypernodulating mutant line nodulated by R. fredii, ureide is transported as the main nitrogenous compound of fixed nitrogen in the xylem sap in the same way as in plants nodulated with B. japonicum.     

Relative efficiency,ureide transport and harvest index in soybeans inoculated with isogenic HUP mutants of Bradyrhizobium japonicum

Summary Differences between isogenic uptake hydrogenase (HUP) mutants of Bradyrhizobium japonicum in terms of nodule efficiency, N₂ fixation and N incorporation into various plant parts were studied in a monoxenic greenhouse experiment in order to confirm previous results with soybeans and beans inoculated with various HUP⁺ and HUP^– strains. The HUP+ revertant PJ17-1 of a HUP^– mutant (PJ17) of strain USDA DES 122 showed a completely restored relative efficiency (100% versus 78±2% for the HUP^– mutant), higher nodule efficiency (N₂ fixed per g nodules), higher ureide-N transport rates, higher N contents in pods and higher N harvest indices. All these observations confirm previous experiments with HUP⁺ and HUP^– strains.     

Hydrogen metabolism and nitrogen fixation of soybean (Glycine max. L.) inoculated with different strains ofRhizobium spp.

Summary Hydrogenase activities and N₂-fixing capacities of soybean nodules (Glycine max. cv. Hodgson), inoculated with strains ofBradyrhizobium japonicum andRhizobium fredii from different geographical regions, were measured after 35 days of culture under controlled conditions. Of the strains tested, 47% induced nodules with bacteroids which recycled H₂. The data obtained suggest that H₂-recycling ability is not a major factor influencing early N₂-fixation which depends essentially on the precocity and intensity of the initial nodulation.     

Involvement of Bradyrhizobium japonicum denitrification in symbiotic nitrogen fixation by soybean plants subjected to flooding

Denitrification by Bradyrhizobium japonicum bacteroids contributes to nitric oxide (NO) production within soybean nodules in response to flooding conditions. However, the physiological relevance of NO production by denitrification in B. japonicum-Glycine max symbiosis is still unclear. In this work, soybean plants were inoculated with B. japonicum strains lacking the nirK or norC genes which encode the copper-containing nitrite reductase and the c-type nitric oxide reductase enzymes, respectively. 14 days flooding increased nodule number of plants inoculated with the WT and norC strains, but not of plants inoculated with the nirK mutant. However, nodule dry weight was not affected by 14 days flooding regardless of the strain used for inoculation. Supporting this observation, individual nodule growth was significantly higher in plants inoculated with nirK than those inoculated with WT or norC after 14 days flooding. Nodule functioning was strongly inhibited by flooding since leghemoglobin content of the nodules induced by any of the strains was significantly decreased after 7 or 14 days flooding compared to control plants. However, this effect was more relevant in nodules of plants inoculated with the WT or norC mutant than in those inoculated with the nirK mutant. Nitrogen fixation was also estimated by analyzing nitrogen content derived from biological nitrogen fixation in shoots, using the ¹⁵N isotope dilution technique. By using this approach, we observed that the negative effect of 14 days flooding on nitrogen fixation was more pronounced in plants inoculated with the norC mutant. However, nitrogen fixation of plants inoculated with nirK showed the highest tolerance to 14 days flooding. These findings allowed us to demonstrate the previously proposed hypothesis which suggests that NO formed by copper-containing nitrite reductase in soybean nodules, in response to flooding, has a negative effect on nitrogenase activity. We propose that inoculation of soybeans with a B. japonicum nirK mutant, which does not produce NO from nitrate, increases the tolerance of symbiotic nitrogen fixation to flooding.     

Comparison of 15N natural abundance and difference methods for estimating N2 fixation of soybeans grown in a tropical soil

Abstract

A study was carried out to compare the difference or N-yield method with the ¹⁵N natural abundance method for the estimation of the fractional contribution of biological N₂ fixation in the different plant parts of nodulating and non-nodulating isolines of soybeans. The results indicated that the δ¹⁵N values of most plant parts of soybeans were significantly lower (p<0.05) in the nodulating than in the non-nodulating isoline. However, in the case of the root+nodule component, the δ¹⁵N value was higher in the nodulating than in the non-nodulating isoline possibly due to isotopic discrimination of ¹⁵N over ¹⁴N which may have occurred in the nodules. Inoculation of soybeans with the Bradyrhizobium japonicum strain CB 1809 increased significantly (p<0.05) the δ¹⁵N value of the root+nodule component implying that the effectiveness of the soybean-rhizobium symbiosis had increased by inoculation.

Percentage of plant N derived from atmospheric N₂ fixation (%Ndfa) estimated by the ¹⁵N natural abundance method was highly correlated (r=0.762, p<0.01) with that by the difference or N-yield method and the differences between the two methods were not statistically significant. The agreement between the two methods was closer at maturity than at the early reproductive stage.

The %Ndfa obtained by the difference method ranged from 48.4 to 92.6% whereas the %Ndfa obtained by the ¹⁵N natural abundance method ranged from 43.2 to 92.4% in the different plant parts. Based on the ¹⁵N natural abundance method, approximately 15% of the N in pod, shoot, grain, and shell was derived from the soil but in the case of stover, this fraction was about 55%.     

不同铵钾比对高铵下拟南芥地上部和根系生长的影响

钾在缓解植物铵毒害的过程中起着重要的作用。本文研究了高铵(30 mmol/L)条件下,不同铵钾比(7.5︰1和150︰1)对拟南芥(Col-0)主根、侧根以及地上部生长的影响。结果表明:30 mmol/L NH4+条件下,高铵钾比(150)处理显著加重了拟南芥铵毒害现象,地上部和根系生长所受的抑制作用更为明显并导致更严重的氧化胁迫。相比低铵钾比水平,在高铵处理下,高铵钾比使得拟南芥主根伸长量降低57.4%,侧根数量减少33.3%,而地上部鲜重减轻69.9%。DAB(3,3￠-二氨基联苯胺,3,3￠-diaminobenzidine)叶片染色结果表明,不加铵处理下,外源不同钾水平(0.2和4.0 mmol/L)对拟南芥叶片的氧化胁迫作用没有显著差异;而高铵处理下,相比低铵钾比处理,高铵钾比显著增加了叶片中过氧化氢的含量,加重了其氧化胁迫。伊文思蓝(Evans blue,EB)染色结果表明,不加铵处理下,外源不同钾水平对拟南芥地上部和根部的膜透性没有显著差异,而高铵处理下,高铵钾比显著增强了拟南芥地上部和根部的膜透性,表明其对细胞的伤害程度加重。可见,高铵抑制拟南芥根系和地上部生长,高铵钾比则会加重这种抑制,其原因除了高浓度钾能减少植物对铵的吸收外,可能与高铵钾比条件加剧了植物的氧化胁迫有关。因此,适宜的铵钾比在植物应对铵毒害的过程中发挥重要作用。     

陕西省几种代表性土壤NH4+吸附、解吸动力学特征研究

采用连续液流法测定了五种土壤吸附、解吸ＮＨ＾＋４的动力学性质。研究表明：（１）ＮＨ＾＋４吸附、解吸平衡时间及反应速率，平衡时的吸附、解吸量及吸附平衡常数均随土壤粘粒和ＣＥＣ不同而变化；（２）不同动力学模型及同一模型对不同土壤的拟合性不同。     

Nitrogen fixation and CO2 exchange in soybeans (Glycine max L.) inoculated with mixed cultures of different microorganisms

N₂ fixation, photosynthesis of whole plants and yield increases in soybeans inoculated with mixed cultures of Bradyrhizobium japonicum 110 and Pseudomonas fluorescens 20 or P. fluorescens 21 as well as Glomus mosseae were found in pot experiments in gray forest soil carried out in a growth chamber. The effects of pseudomonads and vesicular-arbuscular (VA) mycorrhizal fungus on these parameters were found to be the same. Dual inoculation of soybeans with mixed cultures of microorganisms stimulated nodulation, nitrogenase activity of nodules and enhanced the amount of biological nitrogen in plants as determined by the ¹⁵N dilution method in comparison to soybeans inoculated with nodule bacteria alone. An increased leaf area in dually infected soybeans was estimated to be the major factor increasing photosynthesis. P. fluorescens and G. mosseae stimulated plant growth, photosynthesis and nodulation probably due to the production of plant growth-promoting substances. Increasing phosphorus fertilizer rates within the range of 5–40 mg P 100 g^-1 1:1 (v/v) soil: sand in a greenhouse experiment led to a subsequent improvement in nodulation, and an enhancement of N₂ fixation and yield in soybeans dually inoculated with B. japonicum 110 and P. fluorescens 21. These indexes were considerably higher in P-treated plants inoculated with mixed bacterial culture than in plants inoculated with nodule bacteria alone.     

不同铵硝配比对弱光下白菜氮素吸收及相关酶的影响

以黑色遮阳网覆盖模仿弱光环境, 使光照强度为自然光的20%左右, 以自然光照为对照, 采用精确控制水培溶液氮素营养, 研究NH₄⁺-N/NO₃^--N 比例分别为0/100、25/75、50/50、75/25、100/0 对弱光下白菜氮代谢及硝酸还原酶和谷氨酰胺合成酶活性的影响。结果表明, 弱光下, 白菜的鲜重及叶片总氮量以NH₄⁺-N/NO₃^--N 比为25/75 时最大, NH₄⁺-N/NO₃^--N 比为100/0 时最低。随弱光处理的进行, 白菜叶片中硝酸还原酶活性及谷氨酰胺合成酶活性均呈下降趋势, 但NH₄⁺-N/NO₃^--N 比为25/75 时, 可维持叶片内较高的硝酸还原酶活性及谷氨酰胺合成酶活性。试验表明, NH₄⁺-N/NO₃^--N 比25/75 是白菜在弱光下生长的较适宜氮素形态配比。     

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.     

NO-3胁迫下K+、Ca2+对黄瓜幼苗膜质过氧化及活性氧清除酶系统的影响

通过水培试验探讨了NO^-₃胁迫下K⁺、Ca²⁺对黄瓜幼苗膜质过氧化及活性氧清除酶系统的影响。结果表明，在相同NO^-₃浓度胁迫7d后， Ca²⁺浓度越大，膜脂过氧化产物丙二醛(MDA)含量越高，而K⁺浓度越大，电解质相对渗透率越高，由此说明K⁺、Ca²⁺对细胞膜造成伤害的机理不同。黄瓜幼苗活性氧清除酶系统对K⁺、Ca²⁺的响应亦不同，在一定程度上，K⁺和Ca²⁺ 可提高SOD、POD和CAT活性，保护植物免受自由基伤害，继而可增强植物对逆境的适应能力。     

Survival and potential denitrifying activity of Azospirillum lipoferum and Bradyrhizobium japonicum inoculated into sterilized soil

Summary Potential denitrifying activity and population dynamics of Azospirillum lipoferum (137C) and Bradyrhizobium japonicum (G2sp) inoculated into a -sterilized soil were studied for a period of 3 weeks. The denitrifying enzyme potential of soil inoculated independently with each bacterial species was strongly stimulated by the presence of a plant (Zea mays L.). Simultaneous inoculation of both bacteria also produced a higher denitrifying enzyme potential than simple inoculation. Even with double inoculation, the presence of a plant did not modify the evolution of the activity. The response of the population dynamics to these treatments followed a different pattern. The population dynamics of A. lipoferum was not affected by the presence of the plant or by the presence of B. japonicum. In contrast, the presence of both a plant and of A. lipoferum seemed to promote the growth of B. japonicum.     

Fixed ammonium and potassium release from two soils

Abstract

Release of native and added K⁺ and NH⁺ ₄ from two soils was monitored during a 166 day incubation/leaching experiment. One soil (Brookston) represented a major soil series In Ontario whereas the other (Harriston) was suspected having a relatively large fixation capacity. Treatments were imposed involving addition of 50 μM g^‐1 soil of K⁺(KCl) or NH⁺ ₄ (NH₄Cl) only or one added after the other on successive days. The addition of either K⁺ or NH⁺ ₄ on day 2 tended to inhibit the release of the other added on day I. Also the addition of either K⁺ or NH⁺ ₄ on day 1 tended to inhibit the sorption or fixation of the other on day 2. The release rate of K⁺ during the 10 to 166 day period was almost constant and not affected by the addition of NH⁺ ₄. Alternatively, the addition of K⁺ on day 2 slowed the release rate of NH⁺ ₄ measured by NO^? ₃ appearance from day 10 to 40 but had no effect thereafter. At the end of the experiment considerably more K⁺ than NH⁺ ₄ was retained suggesting that K⁺ was more firmly fixed. However, the continuing nitrification of NH⁺ ₄ must be contrasted with periodic removal of K⁺ by leaching with 0.01 M CaCl₂ solution since the equilibrium between exchangeable and fixed ions was affected. There were no notable differences between the two soils inspite of a considerable difference in clay content.     

N natural abundance of biologically fixed N2 in soybean is controlled more by the Bradyrhizobium strain than by the variety of the host plant

The ¹⁵N natural abundance technique is one of those most easily applied ‘on farm’ to evaluate the contribution of biological N₂ fixation (BNF) to legume crops. When proportional BNF inputs are high, the accuracy of this technique is highly dependent on an accurate estimate of the ¹⁵N abundance of the N derived from N₂ fixation (the ‘B’ value). The objective of this study was to determine the influence of soybean variety on ‘B’ value. Plants of five soybean varieties were inoculated separately with two Bradyrhizobium strains (one Bradyrhizobium japonicum and one Bradyrhizobium elkanii) grown in pots of soil virtually free of bradyrhizobia capable of nodulating soybean. The proportion of N derived from BNF (%Ndfa) was estimated in separate pots where a small quantity of enriched ¹⁵N ammonium sulphate was added. The %Ndfa was then used with the ¹⁵N natural abundance data of the nodulated soybean and non-N₂-fixing reference plants, to determine the ‘B’ value for each soybean variety/Bradyrhizobium association. The varieties nodulated by the B. japonicum strain showed significantly greater N content and %Ndfa than those nodulated by the B. elkanii strain, and in all cases the ‘B’ value of the shoot tissue (‘B_s’) was higher. The differences in ‘B_s’ values between varieties nodulated by the same Bradyrhizobium strain were insignificant, indicating that this parameter is influenced much more by the Bradyrhizobium strain than by the variety of the host plant.     

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.     

Nutrient Solution and Nutrient Soil Solution Parameter Evolution in Tomato with Dynamic Fertigation under Saline Conditions

This trial was carried out to study the evolution of the nutrient parameters of the nutrient solution applied to tomato plants (Lycopersicum sculentum Mill. Forteza) cultivated in Mediterranean greenhouse conditions under different fertigation management models. The dynamic model is based on soil water content, which was measured by tensiometers, and on soil solutions obtained with suction cups (porous ceramic cup water samplers). The local traditional method consists of following technical recommendations, and the classical model requires the estimation of Crop Factor (Kc) and knowing the nutrient extraction. Nutrient solution and water applied are functions of the fertigation management criteria. The water used for fertigation was classified as C₄-S₃ according to the Riverside classification system. The cultivation period lasted from 15 August to 20 April. The nutrient parameters studied in nutrient and soil solution were pH, electrical conductivity (EC), nitrate (NO₃ ^?), phosphate (H₂PO₄ ^?), potassium (K⁺), calcium (Ca²⁺), magnesium (Mg²⁺), sodium (Na⁺), and chloride (Cl^?). The pH shows similar trends under the different treatments. Electrical conductivity is in the range of 2.8–4.5 dS m^?1. Chloride, sodium, magnesium, and sulfate are exclusively modified by the salt concentration in the irrigation water, so it can be assumed that the three treatments vary equally. Nitrate, potassium, phosphate, and calcium are modified depending on each fertigation management method. Soil solution is modified by the nutrient solution applied. Dynamic management allows low nutrient concentration in the nutrient solution to be maintained and keeps soil nutrient concentration low, reducing fertilizer losses and therefore aquifer contamination.     

Methionine sulfoximine suppressed the stimulation of dark carbon fixation by ammonium nutrition in wheat roots

To evaluate the role of NH₄ ⁺ assimilates in dark carbon fixation in roots in providing carbon skeletons expended for NH₄ ⁺ assimilation, the rate of dark carbon fixation in roots was measured using NaH¹⁴CO₃. The ¹⁴C-metabolites were analyzed in wheat (Triticum aestivum L.) plants grown in NH₄ ⁺ media for various periods of time with or without methionine sulfoximine (MSX) treatment. The dark carbon fixation rate in the roots of wheat plants that had been grown with NH₄ ⁺ for 1 d was approximately 6-fold higher than the rate in control roots. The stimulation of dark carbon fixation in NH₄ ⁺-grown plants, however, was not observed in MSX-treated roots. In the roots of NH₄ ⁺-grown plants, the concentration and ¹⁴C-Iabeling of acidic metabolites such as citrate and malate considerably decreased whereas those of basic metabolites, especially asparagine, increased noticeably. With MSX treatment, the incorporation of ¹⁴C into basic metabolites was negligible. In response to NH₄ ⁺, phosphoenolpyruvate carboxylase (PEPC) activity increased, and PEPC proteins accumulated in wheat roots. Neither activity nor amounts of PEPC in roots increased in the presence of MSX. These findings suggest that primary assimilation of NH₄ ⁺ in roots is essential for the stimulation of dark carbon fixation, which coincides with the increased activity of root PEPC, to sufficiently replenish carbon skeletons necessary for NH₄ ⁺ assimilation.     

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