In Situ extraction of rhizosphere organic compounds from contrasting plant communities

Abstract

Currently there is no effective method for capture, identification and quantification of root exudates and rhizosphere secondary metabolites in situ. The purpose of the work reported was to assess if capsules containing non‐ionic carbonaceous resins could be used to non‐destructively sample and compare rhizosphere organic compounds associated with contrasting plant communities. Polyester capsules (Unibest, Inc., Bozeman, MT) containing non‐ionic carbonaceous resins, Ambersorb 563 or XAD‐7 (Rohm and Haas, Inc.), were placed within the rhizosphere of spotted knapweed (Centaurea maculosa), and the native grass, Idaho fescue (Festuca idahoensis), as well as a bare‐soil control in both greenhouse and field studies. At the end a 14‐day period, resins were removed and extracted with sequential elution by water, 50% methanol, and 100% methanol. The eluent fractions were then analyzed for total organic carbon (TOC), total hexose sugars as anthrone reactive carbon (ARC), and total phenols. Samples were then concentrated by rotary evaporating to dryness and analyzed on HPLC equipped with a C‐18 column and tunable ultra‐violet (UV) detector or a photodiode array (PDA) detector. Ambersorb 563 resin extracts from greenhouse and field trials consistently showed 2 times more soluble C and 3–7 times more total soluble sugars in the rhizosphere of knapweed compared to Idaho fescue during the 1996 and 1997 field seasons. This difference was not observed using the XAD‐7 resins during the rather wet field season of 1998. In these studies fescue was found to release higher levels of sugars than knapweed, but not significantly different than control soils. Compounds sorbed to the resins from the knapweed rhizosphere were more effectively eluted by methanol than water and demonstrated both the presence of carbohydrate groups and UV absorption. The XAD‐7 resins allow for sorption of phenolic compounds similar to that of Ambersorb 563, but allow for far greater desorption of these compounds. Non‐ionic resins may provide an effective means of capturing rhizosphere organic compounds in situ, but the low concentrations of sorbed compounds may limit their utility.     

利用高能液相色谱测定根际分泌物中的有机酸: Ⅲ.干扰因子的影响

Methods for determining niene low molecular-weight oragnic acids in root exudates were developed by using reversed-phase high performance liquied chromatography with UV (ultraviolet) detection at 214 nm. The mobile phase was 18 mmol L^-1 kH2PO4 adjusted to pH 2.25 with phosporic acid nd the flow rate was 0.3 mL min^-1,The analytical column was a reversed-phase silica based C-18 column( shim-pack CLC-ODS).The root exudates were collected through submerging the whole root system into aerated deionized water for 2 hours ,The filtered exudate solutions were concentrated to dryness by rotary evaporation at 40℃,dissolved in 10 mL mobile phase.The chromatoraphic conditions of organic acid determination were analyzed.The results showed that there was a high selectivity and sensitivity in the organic acid determination by reversed-phase high performance liquid chromatography.Coefficients of variation for organic acied determination were lower than 10% except lactic acid .The recoveries were consistently between 80.1% to 108.3% .Detection limits were approximately 0.05 to 4.5 mg L^-1 for organic acids except succinic acid with the detection limit of 7.0 mg L^-1 .Phosphorus deficiency may contribute to the release of organic acids in soybean root exudates especially malic,lactic and citric acids.     

Fractionation of selenium and detection of selenomethionine in a soil extract

Abstract

A procedure is described to fractionate, identify and quantify organic selenium compounds at μg/kg levels in soil extracts. The fractionation is based first on the different solubility of humates and fulvates at low pH, followed by adsorption chromatography with XAD‐8 resin. The latter uses a pH gradient to elute the acidic and basic hydrophobic fulvates as well as hydrophilic fulvates. The neutral hydrophobic fulvates were retained on the column and determined by difference from the total extractable selenium. A 0.1MNaOH/0.1 M Na₄P₂O₇solution extracted 33% of the total soil selenium. In the soil studied, organic selenium was 20% of total extractable selenium. One of the hydrophilic fulvates has been identified as selenomethionine by gas chromatography‐mass spectrometry. The quantity of organic selenium and the presence of selenomethionine suggests that organoselenium compounds could be an important source of plant available selenium     

利用高能液相色谱测定根际分泌物中的有机酸: Ⅲ.干扰因子的影响

A solution culture experiment was conducted to investigate the effects of collection time and interfering ions on separation and determination of low-molecular-weight organic acids in root exudates of soybean using the method for directly collecting root exudates.The suitable cooection time of root exudates and the interferiung ions affecting organic acid determination were determined.The method for removing the interfering ions was established and analyzed.The release amount of root exudates increased with the increase of collection time from 0 to 120min but decreased with increasing of collection time from 120to 240min.The maximum exuding amounts of organic acids were observed in root exudates at the collection time of 120min.There was a significant difference of organic acid components between the treatments of collection time of 120min and 240min,Citric acid was founnd only in the treatment of 120min collection time,NO3^- was the main interfering ion in organic acid determination and had the same retention time as oxalic acid.Anion exchange resin(SAX)properly treated by HPLC(high performance liquid chromatography)solvent could remove NO3^- anion in sample solution of root exudates,thus enhancing the recoveries of organic acids in root exudates.There was no significant effect of the chemicals added into sample solution such as H3PO4,SAX and KNO3 on the retention time of organic acids.     

Factors Affecting Stability and Efficiency of Ion Exchange Resins in Studies of Soil Nitrogen Transformation

Abstract

Mixtures of cation and anion exchange resins are used as part of the resin core technique to determine nitrogen transformation in forest soils as they adsorb the NH₄‐N and NO₃‐N from soil solution percolating through the incubated soil cores. In the field, the exchange resins may be subjected to a variety of conditions, involving drying, rehydration, freezing, and thawing. This paper examines how these processes affect adsorption of NH₄‐N and NO₃‐N and the stability of the resins. Lab tests were performed on the anion resin Amberlite IRA‐93, the cation resin Amberlite IR‐120, a mixture of IRA‐93 and IR‐120, and the commercially‐mixed bed resin Amberlite MB1. The background content of NO₃‐N and NH₄‐N on the resins was large and highly variable between different batches of resins in spite of a 2 M NaCl pre‐rinse. The IR‐120 cation resin that was subjected to 48 hours air‐drying contained significantly less NH₄‐N than the moist resins, while the drying of the IRA‐93 anion resin caused a significant release of NO₃‐N from resins with no N addition. Although the variation was large, the mixed bed resin MB1 indicated a release of NH₄‐N, which supports results from long term in situ deployments. A reduced adsorption of NO₃‐N was found on the IRA‐93 anion resins and the MB1 mixed bed resins that were dried prior to N addition while the dry IR‐120 cation resins adsorbec significantly less NH₄‐N than the control resin. No effect of freezing and thawing efficiency was observed on resin stability or N adsorption efficiency. Sufficient blanks that have been subjected to similar moisture changes are necessary in N limited systems with low levels of available NH₄‐N and NO₃‐N.     

An evaluation of anion exchange resin used to measure nitrate movement through soil

Abstract

The attribute that ion‐exchange resins remove ions from solutions moving through them can be used to measure nitrate transport through soils. The characteristics of nitrate adsorption by resins must be known to interpret nitrate accumulation on ion‐exchange resins embedded in soil. The extent to which anion exchange resins retain NO₃‐ from soil leachate was measured in 15.9 cm diam.by 60 cm long intact cores of Nolin (fine silty mixed mesic Dystric Fluventic Eutrochroept) soil. A NC₃ ^‐‐selective resin and a non‐selective resin were tested. Columns were fertilized at a rate of 300 kg N/ha and 150 kg Br/ha and leached with 50 cm of water. Under these conditions, both resins retained approximately 80% of the NO₃‐ and Br^‐ leached through the soil. This compared with greater than 95% retention in laboratory columns containing only resin. The difference in retention was attributed to different flow through the resin associated with the method of resin emplacement.     

Colorimetric interference and recovery of adsorbed ions from ion exchange resins

Abstract

Analytical interference in the colorimetric determinations of ammonium and nitrate was examined in various KCl extracts of several ion exchange resins. No analytical interference was found in the colorimetric NO₃ ^‐‐N determination in any extract of any resin. However, a mixed‐bed (cation + anion) exchange resin extract substantially affected the colorimetric determination of NH₄ ^‐ ‐N. Recovery of adsorbed ammonium and nitrate from ion exchange resins was also studied as a function of KCl extractant strength and number of extractions. The recovery of adsorbed NO₃ ^‐‐N in the first extraction increased with increasing KCl concentration, with a 2 M solution recovering about 80%. However, a 1 M KCl solution gave the greatest recovery of ammonium‐N, recovering about 75–80% of the adsorbed ammonium. The second extraction with the same concentration of KCl solution was greater with the 0.5 and 2.0 M than with the 1 M solution so that total NH₄ ⁺‐N recovery after two extractions was about the same for all three KCl concentrations. The recovery of resin‐adsorbed NH₄ ⁺‐N and NO₃ ^‐‐N appeared independent of their concentrations on the resins.     

Root exudation,organic acids,and element distribution in roots of Norway spruce seedlings treated with aluminum in hydroponics

Seedlings of Norway spruce (Picea abies [L.] Karst.), which had been grown under sterile conditions for three months, were treated for one week in a hydroculture system with either 500 μM AlCl₃ or 750 μM CaCl₂ solutions at pH 4. Organic acids were determined in hot‐water extracts of ground root tissue. Oxalate (3.3—6.6 μmol (g root dry weight)^—1) was most abundant. Malate, citrate, formate, acetate, and lactate concentrations ranged between 1—2 μmol (g root dry weight)^—1. Organic substances and phosphate found in the treatment solutions at the end of the experimental period were considered to be root exudates. Total root exudation within a 2‐day period ranged from 20—40 μmol C (g root weight)^—1. In root exudates, organic acids, and total carbohydrates, total amino acids, and total phenolic substances were quantified. Citrate and malate, although present in hot‐water extracts of root tissue, were not detected in root exudates. Phosphate was released from Ca‐treated plants. In Al treatments, there was indication of Al phosphate precipitation at the root surface. Oxalate and phenolics present in the exudates of Norway spruce seedlings are ligands that can form stable complexes with Al. However, concentrations of these substances in the treatment solutions were at micromolar levels. Their importance for the protection of the sensitive root apex under natural conditions is discussed.     

A novel method for separating root‐derived organic compounds from root respiration in non‐sterilized soils

A novel method of separating exudates from root respiration in non‐sterilized soils has been developed. The method is based on a simultaneous elution of exudates from rhizosphere and the blowout of CO₂ originating from root respiration. The innovation of the method lies in the function of a membrane pump to drive the movement of air and simultaneously the circulation of water according to the Siphon principle. The separation method was tested by means of ¹⁴C pulse labeling of Lolium perenne to track the C dynamics in the production of rhizosphere CO₂ and of exudates, which were eluted. The total ¹⁴C activity of rhizosphere CO₂ and of eluted exudates was found to be 8.5 % and 2.3 % of total assimilated ¹⁴C, respectively. Thus, at least 19 % of root‐derived C can be accounted to root exudation. However, the suggested Siphon method underestimates the amount of exudates and shows only a minimum of organic substances exuded by roots. The diurnal dynamics of exudation was detected, but no significant day‐night changes were measured in root and microbial respiration. Tight coupling of assimilation with exudation, but not with root and microbial respiration, was observed. The advantages, shortcomings, and possible applications of the Siphon method are discussed.     

Determination of ammonium,amides, and soluble carboxylates in plant tissues

Methods for extraction and determination of ammonium, amides and soluble carboxylates in plant tissues are compared and discussed. The procedure recommended involves extraction of finely ground plant tissues with 0.2 M formic acid, determination of ammonium and amides in the extract by overnight alkaline hydrolysis and distillation in Conway microdiffusion dishes, measurement of extracted ammonium using Carlson's rapid diffusion‐conductivity instrument, and estimation of soluble organic acids by titration after separation and purification with ion exchange resins. The method described is reasonably rapid, and gives complete recovery and highly reproducible results. The final solution of organic acids is suitable for direct chromatographic analysis.     

Low molecular weight organic acids released from roots of durum wheat and flax into sterile nutrient solutions

Knowledge of the composition and quantity of organic substances released from roots of different plant species is necessary for understanding the chemical and biological processes in the rhizosphere. The present study was undertaken to quantify low molecular weight organic acids (LMWOAs) released from roots of five cultivars/lines of durum wheat (Triticum turgidum var. durum L.): Kyle, Sceptre, DT618, DT627, and DT637 and four cultivars/ lines of flax (Linum usitatissiumum L.): Somme, Flanders, AC Emerson, and YSED 2. Plants were grown in sterile nutrient solution cultures and amounts of organic acids exuded by roots were analyzed by gas chromatography. The LMWOAs varied significantly among both durum wheat and flax cultivars and oxalic, malonic, fumaric, succinic, acetic, malic, citric and tartaric acids were detected in root exudates of both species. Generally, oxalic and acetic acids were predominant in durum wheat exudates and oxalic, acetic and malic acids were predominant in flax root exudates. High oxalic acid concentrations occurred in root exudates of durum wheat cultivars DT627 and DT637, and flax cultivar YSED 2. Compared with the other durum wheat cultivars, Kyle released the lowest total amount of LMWOAs, whereas among the flax cultivars, YSED 2 had the highest total amount of acids secreted from roots. The data showed that the release of LMWOAs from roots was cultivar dependent. The results provide valuable background information for studying the role of root exudates in soil‐plant relationships.     

一种新型根系分泌物收集装置与收集方法的介绍

根系分泌物在养分活化、改善环境胁迫方面具有重要作用,很多科技工作者对根系分泌物的研究表现出极大兴趣,取得了一系列进展。但土壤栽培条件下,根系分泌物收集是一个难点。本文介绍了一种新型根系分泌物的收集装置与收集方法。该装置由根系生长箱和分泌物收集箱组成,植物在生长箱土壤中生长,通过定向引导作用,根系从生长箱穿过琼脂层进入收集箱中生长,待收集箱内积累一定根系后,通过淋洗收集箱内的介质,实现根系分泌物收集。研究发现,利用该装置收集分泌物,植物总根尖数的90%分布在收集箱。外源有机酸加样回收率可达70%以上。土壤栽培条件下,随生长时期延长,大豆有机酸分泌量逐渐增加,苹果酸分泌量高于柠檬酸。而且土壤栽培条件下大豆柠檬酸和苹果酸分泌量是溶液栽培时的11.4倍和6.7倍。上述研究表明,该装置可以用于土壤栽培条件下根系有机酸的分泌研究。     

Release and activity of allelochemicals from allelopathic rice seedlings

3-Isopropyl-5-acetoxycyclohexene-2-one-1 (1), momilactone B (2), and 5,7,4'-trihydroxy-3',5'-dimethoxyflavone (3) were isolated and identified from an allelopathic rice accession PI312777. These three compounds at low concentrations could inhibit the growth of weeds Echinochloa crusgalli and Cyperus difformis associated with rice, especially mixtures of the compounds had stronger inhibitory activity than did individual compounds. Studies with hydroponic culture, continuous root exudates trapping system (CRETS), and direct resin adsorption methods showed that a total of 7.6 n moles 1, 2, and 3 were exuded from living roots of each seedling into the environment at 10 days after seedlings were transplanted. Furthermore, 1, 2, and 3 were found in the soil growing PI312777 seedlings at day 15 after seedlings emergence and reached a total of 39.5 microg/g soil at day 30. The results indicated that PI 312777 seedlings could release sufficient quantities of 1, 2, and 3 into the environment to act as allelochemicals inhibiting the growth of associated weeds. Investigations on the distribution of 1, 2, and 3 in PI 312777 plant, and its root exudates showed that the levels of 1, 2, and 3 were significantly higher in the shoots and root exudates than in the roots, and only trace 1 was observed in the roots. The results suggest that the roots of rice seedlings are not major site of synthesis or accumulation 1, 2, and 3, but a pathway for their release into the environment. The levels of 1, 2, and 3 in the root exudates were over 2-folds higher under direct resin adsorption than under hydroponic culture and CRETS, and hence, it is the preferred method to collect and identify active allelochemicals in rice exudates in future studies on rice allelopathy.     

Resin extraction of labile, soil organic phosphorus

In order to develop a method for estimating labile, soil organic phosphorus (P_o), macroporous anion-exchange and adsorbent (XAD) resins were tested for their ability to extract P_o from a permanent pasture soil. Experimental variables included: ionic form, soil-water resin ratio, addition of cation-exchange resin, extraction time, extraction temperature, continuous and stepwise extraction and soil pretreatment (chloroform and microwaves). The amounts of extracted P_o and P_i (inorganic phosphorus) showed little variation between the anion-exchange resins, when used in the bicarbonate form. In the chloride form, the amounts extracted were less and more variable. XAD resins extracted much less P_o and very little P_i. The macroporous anion-exchange resin, Lewatit MP500A in the bicarbonate form, was chosen for further studies. It extracted 21 mg P_o kg^?1 from air-dried soil and 8 mg P_o kg^?1 from moist, incubated soil. The specific amounts of P_o and P_i extracted were little affected by variations in the ratio between soil, water and resin, but increased with extraction time and temperature. Chloroform pretreatment of the soil mainly increased extracted P_i, whereas microwave pretreatment only increased extracted P_o. The magnitude of these increases was approximately constant irrespective of extraction temperature, indicating that the increases came from killed micro organisms.     

Indications for passive rather than active release of natural nitrification inhibitors in Brachiaria humidicola root exudates

Plants have the ability to suppress microbial nitrification process through secondary metabolites released from their root exudates or/and leaf litter. For decades, grasses were suggested to control nitrification process, and recently, Brachiaria humidicola accession 26159 (BH) as a tropical and subtropical grass has been shown to reduce nitrification rates under laboratory and soil conditions. In this study, experiments were conducted under controlled conditions in nutrient solution culture to investigate whether the reported release of natural nitrification inhibitors from root exudates of BH is an active or passive phenomenon. So different variables such as N-form (nitrate vs. ammonium), collecting medium (distilled water vs. 1 mM NH₄Cl) and collecting period (6 vs. 24 hrs) were included to study the hypothesis. Results showed when root exudates were collected in distilled water there was no nitrification inhibition activity for all ammonium and nitrate grown plants. However, when collection was done in a medium containing 1 mM NH₄Cl, root exudates showed significant nitrification inhibition activity similar to results obtained by Subbarao et al. The observed nitrification inhibition activity had a positive correlation to ammonium treatment particularly in collection medium, probably due to root cells damage induced by low pH and membrane depolarization under ammonium nutrition. This was more supported by application of shoot homogenates of NH₄⁺, NO₃^? or NH₄NO₃ grown plants that showed significant nitrification inhibition activity compared to distilled water and DMPP controls in a bioassay test, independent of N-form. Potassium concentrations in root exudates (as a result of potassium leakage) were found to increase in root washings of plants, which were grown with ammonium, particularly when root exudates were collected in 1 mM NH₄Cl solution. In addition, higher electric conductivity of root washings after collection of root exudates in ammonium containing medium (low pH) and also in nitrate containing medium which adjusted to pH 3 by applying H₂SO₄, strongly suggest that release of natural nitrification inhibitors from root exudates of B. humidicola may not be an active process, but instead it is rather a passive phenomenon by ammonium induced root physicochemical damages.     

Nitrate and phosphate recovery from anion exchange resins

Abstract

The recovery of nitrate and phosphate from two anion exchange resins was determined. A general purpose and nitrate‐selective resin were extracted with potassium chloride (KCl), acidified ammonium chloride (NH4C1), and sodium sulfate (Na₂SO₄). The recovery of both nitrate and phosphate were significantly affected by the choice of resin and extractant. The combination of Na₂SO₄ extractant and nitrate‐selective resin resulted in less than half as much nitrate recovery as any other combination of resin and extractant. Consequently, if it is important to recover nitrate from the resins, Na₂SO₄ should not be used. Greater quantities of phosphate were recovered from the nitrate selective resin with all extractants. However, phosphate recovery was not significantly affected by choice of extractant. More rigorous extraction was required for the nitrate‐selective resin to achieve a level of nitrate recovery comparable to the general purpose resin. The nitrate selectivity also resulted in less phosphate being adsorbed from solutions high in nitrate. Finally, new batches of resin should be tested because differences between them can be significant.     

Qualitative and quantitative analysis of water‐soluble root exudates in relation to plant species and development

The composition of root‐derived substances is of great importance for the understanding of processes in the rhizosphere. Therefore, methods allowing a comprehensive collection and chemical analysis of the organic root exudates are necessary. In this study, we compare different methods with regard to their suitability to collect and characterize root exudates. Because the percolation or water logging method failed to quantitatively extract root exudates, a dipping method was developed which allowed an almost complete sampling of coldwater‐soluble root exudates. By ¹⁴CO₂ labeling of the shoots the composition of root exudates was found to be influenced by plant species and growth stage. In comparison to pea plants maize plants had a higher share of carboxylic acids and a lower share of sugars. Younger maize plants exuded considerably higher amounts of ¹⁴C labeled organic substances per g root dry matter than older ones. During plant development the relative amount of sugars decreased at the expense of carboxylic acids. The described methods are well suited for the elucidation of the influence of growth factors on root exudation.     

大孔树脂法纯化红花芸豆色素及初步鉴定

该文通过静态吸附和解吸实验,选择了合适的大孔树脂纯化红花芸豆色素,优化了吸附和解吸条件,并应用纸色谱法和紫外—可见光谱法对纯化后的色素进行了初步的分离鉴定。结果表明,HPD400型大孔树脂对pH值为1的红花芸豆色素酸提液吸附率为88.38%,pH值2的75%乙醇可较好的洗脱色素。纯化后色素有2个主要成分,初步鉴定为天竺葵定-3-葡萄糖苷和芍药定-3-葡萄糖苷。     

不同青菜品种吸锌能力差异及与根系分泌物的关系

青菜 [Brassica campestris var. chinensis(L.)Makino]不同品种对土壤中锌的累积吸收有明显差异[1] ,我们推断根系分泌物在青菜吸收锌的过程中可能起着重要作用。因此在本项研究中 ,对 2个不同锌利用效率的青菜品种的根系分泌物组成、数量及其与锌吸收的关系进行了探讨。结果表明 ,2种锌水平下 ,五月慢根系分泌物中低分子量有机酸及氨基酸的总量均明显高于黑油白菜 ,缺锌时品种间差异达 0.05显著水平 ;缺锌条件下 ,五月慢根系分泌物中草酸、丙氨酸的含量同样显著高于黑油白菜。五月慢在缺锌时有较高的根系分泌物总量及其草酸、丙氨酸含量都显著增加 ,这可能是五月慢具有较高累积吸锌量及锌利用效率的重要原因之一。     

大豆根系分泌物中氨基酸对根腐病菌生长的影响

采用砂培和室内模拟方法,研究了两个抗病性不同的大豆品种水溶性根系分泌物中氨基酸组分随作物生长的变化;同时检测了培养基中添加大豆根系分泌物和纯品氨基酸对大豆根腐病菌菌落生长的影响。结果表明,添加大豆苗期和花荚期根分泌物均显著促进尖镰孢菌菌丝生长,添加成熟期根分泌物显著促进腐皮镰孢菌菌丝生长。易感根腐病大豆品种合丰25号花荚期以后根分泌物中氨基酸种类多于抗根腐病大豆品种绥农10号。感病大豆品种根系分泌的氨基酸总量随生育时期增加,在鼓粒期达到最高;抗病大豆品种根系分泌的氨基酸总量在花荚期最高。感病大豆品种根系分泌的主要氨基酸为精氨酸,抗病大豆品种根系分泌的氨基酸主要为天冬氨酸。氨基酸纯品培养中,添加精氨酸和酪氨酸处理的尖镰孢菌菌落直径显著高于不加氨基酸的对照菌落直径;添加丝氨酸和天冬氨酸的处理菌落直径则显著低于对照处理。同时,添加天冬氨酸的培养基上腐皮镰孢菌菌落直径显著低于不加氨基酸的对照。可见,不同大豆品种根系分泌物中氨基酸组分对病原菌生长起着一定的作用,其表现的作用受根际氨基酸种类和氨基酸浓度影响较大,对于不同病原菌的作用存在差异。