Characteristics of initial growth of hypernodulation soybean mutants,NOD1-3, NOD2-4 and NOD3-7, affected by inoculation of bradyrhizobia and nitrate supply

Abstract

The characteristics of plant growth of hypernodulation soybean mutant lines NOD1-3, NOD2-4 and NOD3-7 were compared with their parent cv. Williams. The plants were grown using hydroponics in an illuminated growth chamber with or without seed inoculation of bradyrhizobia, and in the absence or presence of nitrate in the culture solution. When the plants were grown without inoculation, the total dry weight of all mutant lines was not different statistically from Williams, both in the absence and presence of nitrate. When they were grown with inoculation of bradyrhizobia, however, the total dry weight of each mutant line was significantly lower than that of Williams, both in the absence and presence of nitrate. These results indicate that the reduced accumulation of total dry matter of hypernodulation mutant lines compared with the wild type may be a secondary effect passively resulting from the large number of nodules, while the hypernodulation trait is a primary effect of the mutated gene. When the plants were grown with inoculation, the nodule number was decreased by the presence of nitrate in Williams, NOD1-3 and NOD2-4, but not in NOD3-7. NOD3-7 may be the most tolerant to nitrate inhibition of nodulation among the NOD mutant lines. In contrast, leaf growth of NOD3-7 and NOD1-3 was different from the wild type; the expanded leaf was smaller, but the leaf emergence rate was faster compared with Williams under all conditions. This indicates that NOD3-7 and NOD1-3 might decrease the ability for leaf expansion or may have a faster leaf emergence rate.     

Determination of leghemoglobin components and xylem sap composition by capillary electrophoresis in hypernodulation soybean mutants cultivated in the field

The hypernodulation soybean mutant lines (NOD1-3, NOD2-4, NOD3-7) and their parent Williams, and the mutant En6500 and its parent Enrei were cultivated in a sandy dune field in Niigata, and the nodules and root bleeding xylem sap were sampled at 50, 70, 90, and 120 d after planting (DAP). The nodule size distribution patterns and concentration of leghemoglobin components were determined. The number of nodules of the hypernodulation mutant lines was about two to three times higher than that of the parent lines irrespective of the sampling date. At 50 DAP the nodule size was relatively smaller in the hypernodulation mutant lines, and the total dry weight of the nodules was almost the same in the mutant lines and their parents. At 70 DAP and 90 DAP, the size distribution of the hypernodulation mutant nodules became .almost the same as that of the parent lines, and both the number and total dry weight of the nodules were higher than those of the parent lines. The concentration of four Lb components was separately measured by capillary electrophoresis. The concentration of the Lb components in the hypernodulation mutant lines tended to be lower than in the parents, but the component ratios were not different between the hypernodulation mutants and their parents. Under field conditions, plant growth and nodulation characteristics were more similar between mutants and parents than in the hydroponic culture reported previously, although the mutants did exhibit hypernodulation traits. These findings suggest that the decrease in the Lb concentration and the different Lb components ratios in the mutants may be caused by secondary effects of excess nodulation, such as photosynthate deficiency, rather than by a genetic defect in mutation. The concentration of major nitrogenous compounds (allantoic acid, allantoin, asparagine, aspartic acid, and nitrate) in the xylem sap was also measured by capillary electrophoresis. The concentration of ureides and nitrate in xylem sap decreased with the plant age, but the asparagine concentration increased during the same period. The concentrations of ureides and asparagine were higher, and the nitrate concentration was lower in the mutant lines than in their parents, possibly due to the higher dependence on N₂ fixation than N0₃ ^- utilization. In the xylem sap, nitrate was the major inorganic anion followed by phosphate, sulfate, and chloride, and potassium was the major cation followed by calcium or magnesium and sodium.     

Photoassimilate partitioning in hypernodulation mutant of soybean (Glycine max (L.) Merr.) NOD1-3 and its parent williams in relation to nitrate inhibition of nodule growth

Nodule growth of a hypernodulating soybean (Glycine max (L.) Merr.) mutant line NOD1-3 was compared to that of its wild-type parent cv. Williams from 14 to 18 days after planting (DAP) in the absence of nitrate treatment (hereafter referred to as “0 mM treatment”) or with 5 mM nitrate treatment. The growth rate determined by increase in the diameter of the nodules was relatively lower in the mutant NOD1-3 than that of the parent Williams under nitrogen-free conditions (0 mM nitrate). The inhibition of nodule growth by 5 mM nitrate started at 1 d after the onset of the nitrate treatment in Williams, while the inhibition did not occur before the application of the nitrate treatment for 2 d in NOD1-3. The nodule growth was completely inhibited after 2 d in Williams and after 3 d in NOD1-3 during the 5 mM nitrate treatment period. After 4 d of 5 mM nitrate treatment, the nodule dry weight decreased by 22% in NOD1-3 and by 58% in Williams, respectively. The treatment with 5 mM nitrate decreased the acetylene reduction activity (ARA) in NOD1-3 by 60% per plant and by 50% per nodule g DW and these parameters were less sensitive to the treatment than those in Williams in which the inhibition rate was 90% per plant and 80% per nodule g DW. These results indicate that NOD1-3 is partially nitrate-tolerant in terms of individual nodule growth as well as total nodule dry weight and Nz fixation activity. A whole shoot of Williams and NOD1-3 plants was exposed to ¹⁴CO₂ for 120 min followed by 0 or 5 mM nitrate treatment for 2 d, and the partitioning of the photoassimilates among the organs was analyzed. Under 0 mM nitrate treatment, the percentages of the distribution of ¹⁴C radioactivity between the nodules and roots were 63 and 37% in Williams and 89 and 11% in NOD1-3. Under the 5 mM nitrate conditions, the percentages of the distribution of ¹⁴C between the nodules and roots changed to 14 and 86% in Williams and 39 and 61% in NOD1-3, respectively. These results indicated that the hypernodulating mutant NOD1-3 supplied a larger amount of photoassimilates to the nodules than to the roots under nitrogen-free conditions, and that the nitrate depression of photoassimilate transport to the nodules was less sensitive than that of the parent line.     

Effects of Rhizobium inoculation and magnesium application on growth and nodulation of soybean (Glycine max L.)

Abstract

Magnesium (Mg) deficiency is one of the major nutritional problems in tropic and subtropic areas, where the most soils are acidic. In this study, the effects of Mg application and Bradyrhizobium inoculation on growth, nodulation, symbiotic nitrogen (N) fixation as well as N nutrition status in soybean (Glycine max L.) were investigated in hydroponics under greenhouse conditions. With the increase of Mg up to 0.75?mM at low N and up to 0.5?mM at high N solutions, the dry weights of shoots, roots, and pod grain yield in soybean were increased, while further increase in Mg supply inhibited soybean growth. The availability of Mg was found to entail an improved uptake of N by plants and nodulation process in the root by Bradyrhizobium. Inoculation with rhizobial inoculants not only formed many nodules, but also increased soybean shoot, root biomass and yield, as well as plant N nutrient status.     

Rapid and reversible nitrate inhibition of nodule growth and N2 fixation activity in soybean (Glycine max (L.) Merr.)

Nodulated soybean (Glycine max. (L) Merr. cv. Williams) plants were hydroponically cultured, and various combinations of 1-week culture with 5 or 0 mm nitrate were applied using 13-d-old soybean seedlings during three successive weeks. The treatments were designated as 0-0-0, 5-5-5, 5-5-0, 5-0-0, 5-0-5, 0-5-5, and 0-0-5, where the three sequential numbers denote the nitrate concentration (mm) applied in the first-second-third weeks. The size of the individual nodule was measured periodically using a slide caliper. All the plants were harvested after measurement of the acetylene reduction activity (ARA) at the end of the treatments. In the 0-0-0 treatment, the nodules grew continuously during the treatment period. Individual nodule growth was immediately suppressed after 5 mm nitrate supply. However, the nodule growth rapidly recovered by changing the 5 mm nitrate solution to a 0 mm nitrate solution in the 5-0-0 and 5-5-0 treatments. In the 5-0-5 treatment, nodule growth was completely inhibited in the first and the third weeks with 5 mm nitrate, but the nodule growth was enhanced in the second week with 0 mm nitrate. The nodule growth response to 5 mm nitrate was similar between small and large size nodules. After the 5-5-5, 5-0-5, 0-0-5, and 0-5-5 treatments, where the plants were cultured with 5 mm nitrate in the last third week, the ARA per plant was significantly lower compared with the 0-0-0 treatment. On the other hand, the ARA after the 5-0-0 and 5-5-0 treatments was relatively higher than that after the 0-0-0 treatment, possibly due to the higher photosynthate supply associated with the vigorous vegetative growth of the plants supplemented with nitrate nitrogen. It is concluded that both soybean nodule growth and N₂ fixation activity sensitively responded to the external nitrate level, and that these parameters were reversibly regulated by the current status of nitrate in the culture solution, possibly through sensing of the nitrate concentration in roots and / or nodules.     

增硝营养对不同基因型水稻苗期硝酸还原酶活性及其表达量的影响

利用控制条件下的溶液培养方法,研究了增硝营养(NH4+∶NO3-比例为100∶0和50∶50)对两种不同的基因型水稻南光和云粳苗期生长和硝酸还原酶(NR)活性及基因表达量的影响。结果表明,不同基因型水稻在增NO3-营养下生物量、氮素含量、氮积累量的增幅南光大于云粳。NO3-的存在增强了水稻硝酸还原酶的活力和NR基因OsNia1、OsNia2的表达。不同基因在水稻幼苗中,两个品种OsNia2的相对表达量均高于OsNia1。就品种而言,无论叶片还是根系,增硝后南光OsNia2mRNA表达量都高于云粳;南光叶片OsNia1mRNA表达量也较云粳叶片高。增硝营养提高了水稻NR基因的表达,增加了NR活性,促进了水稻NO3-的同化利用,从而增加了氮素在植株地上部的积累同化。南光和云粳相比,前者对NO3-的响应更为强烈。     

Effect of varied soil nitrogen supply on growth and nutrient uptake of young Norway spruce plants grown in a shaded environment

To understand the effect of increased soil N supply on tree growth and nutrient uptake, three-year-old Norway spruce seedlings were grown in pots on low-nutrient mineral forest soil supplemented with N in mineral or organic form. Outdoor shaded growth conditions were used, to test the hypothesis that shaded plants are particularly susceptible to high soil N supply. Plants were harvested eleven months after planting. Shoot growth was not affected by the N supply, but N concentrations in needles and roots were increased in plants supplied with mineral N (150 or 300 mg N [kg soil]^—1). Root growth was drastically reduced and root/shoot ratios were decreased in plants with higher N uptake. A high supply of mineral N to soil also decreased the concentrations of other essential elements (P, K) in the needles and thus had effects on plant growth which may impair the stress resistance of trees. Organic N in the form of keratin (150 mg N [kg soil]^—1) did not influence plant growth significantly. The adverse effects of high mineral N supply were particularly pronounced under shaded conditions in comparison to results from other experiments using higher light intensity and temperature conditions.     

Effects of co-inoculation of Bradyrhizobium japonicum SAY3-7 and Streptomyces griseoflavus P4 on plant growth,nodulation, nitrogen fixation,nutrient uptake,and yield of soybean in a field condition

ABSTRACT

Co-inoculation of nitrogen-fixing bacteria with plant growth-promoting bacteria has become more popular than single inoculation of rhizobia or plant-growth-promoting bacteria because of the synergy of these bacteria in increasing soybean yield and nitrogen fixation. This study was conducted to investigate the effects of Bradyrhizobium japonicum SAY3-7 and Streptomyces griseoflavus P4 co-inoculation on plant growth, nodulation, nitrogen fixation, nutrient uptake, and seed yield of the ‘Yezin-6’ soybean cultivar. Nitrogen fixation was measured using the acetylene reduction assay and ureide methods. Uptake of major nutrients [nitrogen (N), phosphorus (P), potassium (K), calcium (Ca), and magnesium (Mg)] was also measured. This study showed that single inoculation of SAY3-7 significantly increased shoot biomass; nodulation; Relative Ureide Index (RUI %), percent nitrogen derived from N fixation (% Ndfa); N, P, K, Ca, and Mg uptakes; during the later growth stages (R3.5 and R5.5), compared with control. These observations indicate that SAY3-7 is an effective N-fixing bacterium for the plant growth, nodulation, and nitrogen fixation with an ability to compete with native bradyrhizobia. Co-inoculation of SAY3-7 and P4 significantly improved nodule number; nodule dry weight; shoot and root biomass; N fixation; N, P, K, Ca, and Mg uptake; at various growth stages and seed yield in ‘Yezin-6’ soybean cultivar compared with the control, but not the single inoculation treatments. Significant differences in plant growth, nodulation, N fixation, nutrient uptake, and yield between co-inoculation and control, not between single inoculation and control, suggest that there is a synergetic effect due to co-inoculation of SAY3-7 and P4. Therefore, we conclude that Myanmar Bradyrhizobium strain SAY3-7 and P4 will be useful as effective inoculants in biofertilizer production in the future.     

氨基酸部分替代硝态氮对小白菜生长、硝酸盐累积及大量元素含量的影响

A hydroponic experiment was carried out to determine the influence of replacing 20% of nitrate-N in nutrient solutions with 20 individual amino acids on growth, nitrate accumulation, and concentrations of nitrogen （N）, phosphorus （P）, and potassium （K） in pak-choi （Brassica chinensis L.） shoots. When 20% of nitrate-N was replaced with arginine （Arg） compared to the full nitrate treatment, pak-choi shoot fresh and dry weights increased significantly （P ≤ 0.05）, but when 20% of nitrate-N was replaced with alanine （Ala）, valine （Val）, leucine （Leu）, isoleucine （Ile）, proline （Pro）, phenylalanine （Phe）, methionine （Met）, aspartic acid （Asp）, glutamic acid （Glu）, lysine （Lys）, glycine （Gly）, serine （Ser）, threonine （Thr）, cysteine （Cys）, and tyrosine （Tyr）, shoot fresh and dry weights decreased significantly （P ≤ 0.05）. After replacing 20% of nitrate-N with asparagine （Asn） and glutamine （Gin）, shoot fresh and dry weights were unaffected. Compared to the full nitrate treatment, amino acid replacement treatments, except for Cys, Gly, histidine （His）, and Arg, significantly reduced （P ≤0.05） nitrate concentrations in plant shoots. Except for Cys, Leu, Pro, and Met, total N concentrations in plant tissues of the other amino acid treatments significantly increased （P ≤ 0.05）. Amino acids also affected total P and K concentrations, but the effects differed depending on individual amino acids. To improve pak-choi shoot quality, Gln and Asn, due to their insignificant effects on pak-choi growth, their significant reduction in nitrate concentrations, and their increase in macroelement content in plants, may be used to partially replace nitrate-N.     

Relation between soil microbial activity and the effect of seed inoculation with the rhizopseudomonad strain 7NSK2 on plant growth

Summary The relationship between the microbial activity in the soil and the effect of seed inoculation with the rhizopseudomonad strain 7NSK2 was evaluated in a series of pot experiments under greenhouse conditions. The microbial activity in plain soil, as measured by the respiratory activity, was significantly increased by the growth of the plants. Both the respiration rate of the microorganisms and the density of the bacteria and fungi in the bulk soil increased with increasing duration of the plant growth. Upon repeated short-term growth of plants on the same soil, a similar stimulation was noticed.The effect of seed inoculation on the growth of the maize cultivar Beaupré and the barley cultivar Iban was most pronounced in the microbiologically more active soils. The results suggest that the increase of the plant growth by seed inoculation is probably due to the inhibition of deleterious root microorganisms.     

Soil Extraction of Readily Soluble Heavy Metals and As with 1 M NH4NO3-Solution - Evaluation of DIN 19730 (6 pp)

Background, Aims and Scope   The German DIN 19730 (1997) describes a method for the extraction of readily available trace elements from soil by shaking the soil with 1 M NH4NO3-solution. Based on this method trigger and action values for the transfer of heavy metals and arsenic from soils to plants have been published in the German Federal Soil Protection and Contaminated Sites Ordinance (BBodSchV 1999). The chemical mechanisms involved in this soil extraction procedure were evaluated in some detail to create requirements to improve environmental risk assessment for soil contaminations.Methods   The chemical mechanisms involved when soil is extracted with 1 M NH4NO3-solution were evaluated. This was followed by a laboratory experiment to quantify the formation of soluble metal ammine complexes during the extraction. Cd, Zn, Ni, Cu, Co and Hg were extracted from 16 soils by 1 M NH4NO3, 1 M KNO3 and water. pH was adjusted in 5 steps between 5.0 to 7.5. The potassium cation (K+) and the ammonium cation (NH4+) behave similarly when cations from soil surfaces are desorbed, because they have almost identical ion radii (e.g. 0.133 and 0.143 nm). K+ does not form ammine complexes with other ions, whereas, due to the increasing formation of NH3 from NH4 by dissociation with rising pH, metal ammine complex formation is an important process in soil extraction when using ammonium salt solutions. A difference in the extraction efficiency of 1 M NH4NO3- and 1 M KNO3-solution for a given soil can therefore be attributed to the formation of soluble metal ammine complexes. Conclusion   Our experiments resulted in considerably higher extraction rates of Cu, Cd and Hg by 1 M NH4NO3-solution as compared to 1 M KNO3-solution. This effect, caused by the formation of soluble metal ammine complexes, was only evident in soils with higher readily soluble heavy metal contents and a soil pH above 6 – 6.5 for Cu and 7 – 7.5 for Cd. Further chemical mechanisms involved when soils are extracted with 1 M NH4NO3 are a moderate decrease in pH and an increase in ionic strength. Most of the colloids and parts of soluble metal-organic complexes are precipitated due to the high ionic strength. High ionic strength also decreases the activity of metal-OH+ species and the electrostatic potential of the particle surfaces, which in turn, increases the desorption of heavy metal cations from negatively charged soil surfaces. In contrast, the adsorption of anions like arsenate is favoured by the decreasing electrostatic potential. The prediction of heavy metal uptake by plants from the results of the 1 M NH4NO3-solution extraction fits well for elements, which are mainly bound by low strength electrostatic forces to the soils. Such conditions are found in acidic soils for Cd and Tl, which have a low tendency for hydrolysis compared to other heavy metals. The correlation between 1 M NH4NO3 soil extraction and plant uptake is less significant for Ni and Zn. Only low positive correlation coefficients have been found for Pb, As, Hg and for the Cu-uptake by wheat. Imprecise prediction of plant uptake of heavy metals by the extraction with 1 M NH4NO3-solution is mainly caused by conditions leading to an overestimation of plant availability such as elements are strongly bound to soils, or low soluble trace element contents in soils. Neutral to alkaline soil pH can also lead to imprecise prediction due to increasing formation of soluble metal-organic (Cu, Pb, Hg) and metal ammine (Hg, Cu, Cd) complexes and less importantly due to the formation of colloids. Therefore, at low 1 M NH4NO3-extractable soil contents usually no high plant contents are to be expected. Recommendation and Outlook   Extraction of soil with 1 M NH4NO3-solution is a suitable method for the determination of readily soluble and plant available trace element contents. The chemical soil extraction process may cause misleading predictions of the transfer of trace elements to plants for some soil properties. This knowledge should be used to improve risk assessment of soil contaminations. It has to be considered, that the processes involved in plant uptake of trace elements are too complex to expect that just one soil extraction method can always guarantee a correct prognosis of toxicological significant element contents in plants. Soil analyses may be used for the preliminary examination of suspicious areas and the demarcation of contaminated areas. The results of soil analyses should be checked additionally by plant analyses especially under conditions with a high probability for misleading results by the 1 M NH4NO3-extraction. Alternatively, extraction with 1 M KNO3-solution can be performed to exclude the effect of metal ammine complex formation.     

FTIR‐spectroscopic characterization of humic acids and humin fractions obtained by advanced NaOH,Na4P2O7, and Na2CO3 extraction procedures

Aim of our study was the development of the methodological basis for the characterization of humic fractions of a long‐term field experiment. Humic acids (HAs) were extracted from three layers of a nontilled soil using three different extractants (1 M NaOH, 0.1 M Na₄P₂O₇, 1 M Na₂CO₃), and the humin fraction was enriched. NaOH as extractant for FTIR analysis of humic substances yields higher resolved IR spectra, especially in the important regions of stretching vibrations including aromatic and aliphatic groups and in the fingerprint area including amides, aliphats, and aromats than the other extractants. The NaOH extraction has lower extraction yields as compared to Na₄P₂O₇ and Na₂CO₃ and represents a different part of the soil organic matter (SOM). This is reflected by lower C : N ratios and higher E₄ : E₆ and fulvic acid–to–humic acid ratios as compared to the other extractants. The FTIR band areas of HA fraction obtained by NaOH showed an increase of the aromatic and carbonyl groups and a decrease of amide groups with increasing soil depth. Aliphatic groups showed contradicting results: The bands of the stretching vibrations increased, and the band of the bending vibrations decreased. We assume that band interactions in the bending vibrations were responsible for that phenomenon under the assumption of an increase of aliphatic groups with increasing soil depth. The IR bands of the enriched humin fraction showed a decreasing trend in case of both aliphatic bands deriving from stretching vibrations and an increase of aromatic characteristics with depth. Our study led to the conclusion that HA fractions obtained by 1 M NaOH represent a small and dynamic fraction indicated by the measured yields in combination with values of N_t, C : N, E₄ : E₆ ratios, and ratios of fulvic acids (FA) to HA. The humin fraction has a high contribution to the total organic C and represents a more stabilized fraction of SOM which still shows changes in its aromatic and aliphatic characteristics with soil depth.     

Separation and preconcentration of Cd(II), Cu(II), Ni(II), and Pb(II) in water and food samples using Amberlite XAD-2 functionalized with 3-(2-nitrophenyl)-1H-1,2,4-triazole-5(4H)-thione and determination by inductively coupled plasma-atomic emission spectrometry

A separation and preconcentration procedure was developed for the determination of trace amounts of Cd(II), Cu(II), Ni(II), and Pb(II) in water and food samples using Amberlite XAD-2 fuctionalized with a new chelating ligand, 3-(2-nitrophenyl)-1H-1,2,4-triazole-5(4H)-thione (Amberlite XAD-2-NPTT). The chelating resin was characterized by Fourier transform infrared spectroscopy (FT-IR) and used as a solid sorbent for enrichment of analytes from samples. The sorbed elements were subsequently eluted with 10 mL of 1.0 M HNO(3), and the eluates were analyzed by inductively coupled plasma-atomic emission spectrometry. The influences of the analytical parameters including pH, amount of adsorbent, eluent type and volume, flow rate of the sample solution, volume of the sample solution, and effect of matrix on the preconcentration of metal ions have been studied. The optimum pH for the sorption of four metal ions was about 6.0. The limits of detection were found to be 0.22, 0.18, 0.20, and 0.16 μg L(-1) for Cd(II), Cu(II), Ni(II), and Pb(II), respectively, with a preconcentration factor 60. The proposed method was applied successfully for the determination of metal ions in water and food samples.