G. L. Kerven

A rapid method for the measurement of Leucaena spp proanthocyanidins by the Proanthocyanidin (Butanol/HCl) assay

Proanthocyanidin (PA) extraction, sample preparation and proanthocyanidin assay (butanol/HCl) reaction conditions were evaluated for measuring PA in Leucaena spp leaf material. The optimal conditions for extracting PA from leaf tissue are described, with short sequential sonications in 70% aqueous acetone being as efficient as prolonged sequential mechanical agitation. In methanol–based extracts, after back extraction to remove pigments, increasing the water content of the reagent/sample matrix suppressed colour development. The addition of low concentrations of Fe3+ to the butanol/HCl reagent enhanced colour yield, but higher Fe3+ concentrations suppressed colour development. The presence of ascorbic acid in the sample extract was shown to increase colour development. Varying the reagent: sample extract ratio from 4:1 to 6:1 significantly decreased colour yield, but neither ratio was different from 5:1. Optimum conditions for the PA assay were as follows: a water content of 8%, the omission of Fe3+, a reagent: sample extract ratio of 5:1 and the addition of ascorbic acid to the stock PA standard solution to match that contributed by the extract in the final mixture. Sample preparation procedures, using back extraction to remove pigments and non-PA phenolics with diethyl ether and ethyl acetate, respectively, were time-consuming and subject to PA losses. The measurement of PA directly in the 70% aqueous acetone extract eliminated these PA losses, but the PA assay required additional optimisation for direct analysis of crude acetone extracts. In the final optimised procedure, PA was extracted by sequential sonication with 70% aqueous acetone containing 5·26 mM sodium metabisulphite as the antioxidant. These extracts were directly analysed by the butanol/HCl reaction using a reagent: sample extract ratio of 5:1, the omission of Fe3+ from the butanol/HCl reagent and the addition of sodium metabisulphite to match that contributed by the extract. This produced consistent linear calibration curves over the range 25–1000 μg PA with an average recovery of 101%.

In vitro evidence of aluminum effects on solution movement through root cell walls

Abstract Little is known about the primary effects of aluminum (Al) in reducing root growth. However, the sorption of Al by the root cell wall, particularly by calcium (Ca) pectate, has been suggested as being important in the expression of Al toxicity in plants. To overcome problems arising from the close proximity of root cell components that may react with Al, a synthetic Ca pectate membrane was prepared as a model system for Al studies. Solution containing 1 mM Ca (as CaCl2) was passed through the membrane, and the flow rate measured. Solution containing 29 μM Al (as AlCl3) and 1 mM Ca reduced solution flow rate by > 80% from c. 3.5 to c. 0.6 mL/min within 2 min, with a further slight decline over the next 4 min. The Al concentration in solution proximate to the inlet side of the membrane decreased to 15 μM within 10 min, and only 3 μM Al was measured in solution that had passed through the membrane. These results suggest that an important primary effect of toxic Al is a reduction in water movement in...

Factors affecting aluminium sorption by calcium pectate

Extracellular processes, particularly the adsorption of aluminium (Al) by pectate in the cell wall, have been proposed as important in the expression of Al toxicity to plant roots. In vitro studies were conducted on the effects of Al concentration (generally ≤ 32 μM), calcium (Ca) concentration (0.05 to 10 mM) and pH (3.2 to 5.4) on Al sorption by Ca pectate. There was a rapid reaction between Al and Ca pectate, there being no difference in Al remaining in solution after reaction times of 1 to 16 min, and only a slight decrease after 24 h. Increased Al concentration in solution increased linearly the sorption of Al by Ca pectate, with 70 to 84% of the Al originally in solution sorbed with ≤32 μM Al. In contrast, Al sorption decreased with increased Ca concentration in solution, and as pH decreased from 5.4 to 3.2. Only ≤30% of the sorbed Al was desorbed after 1 h by 1 mM CaCl2, 10 mM CaCl2 or 1 mM HCl. The amount of Al desorbed increased with a desorption period of 5 h, particularly with 1 mM HCl. These studies suggest that Al sorbed by Ca pectate in root cell walls is in equilibrium with Al in solution, and that Al toxicity is associated with the strong binding between Al and Ca pectate external to the cytoplasm.

Soil carbon determination by high temperature combustion - A comparison with dichromate oxidation procedures and the influence of charcoal and carbonate carbon on the measured value

The measurement of organic carbon in soils has traditionally used dichromate oxidation procedures including the Wakley and Black and the Heanes methods. The measurement of carbon in soils by high temperature combustion is now widely used providing a rapid automated procedure without the use of toxic chemicals. This procedure however measures total carbon thus requiring some means of correction for soil samples containing carbonate and charcoal forms of carbon. This paper examines the effects of known additions of charcoal to a range of soil types on the results obtained by the Walkley and Black, Heanes and combustion methods. The results show, that while the charcoal carbon does not react under Walkley and Black conditions, some proportion does so with the Heanes method. A comparison of six Australian Soil and Plant Analysis Council reference soil samples by the three methods showed good agreement between the Heanes method, the combustion method and only slightly lower recoveries by the Walkley and Black procedure. Carbonate carbon will cause an overestimation of soil organic carbon by the combustion method thus requiring a separate determination of carbonate carbon to be applied as a correction. This work shows that a suitable acid pre-treatment of alkaline soils in the sample boats followed by a drying step eliminates the carbonate carbon prior to combustion and the need for an additional measurement. The measurement of carbon in soils by high temperature combustion in an oxygen atmosphere has been shown to be a rapid and reliable method capable of producing results in good agreement with one of the established dichromate oxidation procedures.Abstract The measurement of organic carbon in soils has traditionally used dichromate oxidation procedures including the Wakley and Black and the Heanes methods. The measurement of carbon in soils by high temperature combustion is now widely used providing a rapid automated procedure without the use of toxic chemicals. This procedure however measures total carbon thus requiring some means of correction for soil samples containing carbonate and charcoal forms of carbon. This paper examines the effects of known additions of charcoal to a range of soil types on the results obtained by the Walkley and Black, Heanes and combustion methods. The results show, that while the charcoal carbon does not react under Walkley and Black conditions, some proportion does so with the Heanes method. A comparison of six Australian Soil and Plant Analysis Council reference soil samples by the three methods showed good agreement between the Heanes method, the combustion method and only slightly lower recoveries by the Walkley and Black procedure. Carbonate carbon will cause an overestimation of soil organic carbon by the combustion method thus requiring a separate determination of carbonate carbon to be applied as a correction. This work shows that a suitable acid pre‐treatment of alkaline soils in the sample boats followed by a drying step eliminates the carbonate carbon prior to combustion and the need for an additional measurement. The measurement of carbon in soils by high temperature combustion in an oxygen atmosphere has been shown to be a rapid and reliable method capable of producing results in good agreement with one of the established dichromate oxidation procedures.

Quantitative 27Al NMR spectroscopic studies of Al (III) complexes with organic acid ligands and their comparison with GEOCHEM predicted values

The toxic inorganic monomeric forms of aluminium (Al) that limit plant growth have been shown to be effectively detoxified by complexation with organic acid ligands released by breakdown of added organic materials. The binding capacity of these acids is dependent on the degree of dissociation of their carboxyl groups and their ability to form bonds with Al. 27Al NMR spectroscopy provides a non-invasive technique to study the bonding of Al with potential ligands without disturbing the equilibrium of the system. In single ligand systems containing oxalic acid, three 27Al resonance peaks were observed at 6.4, 11.4 and 16.0 ppm downfield from the Al3+ reference peak at 0 ppm. These were assigned to Alox, Alox2 and Alox3 complexes respectively and were observable at pH values down to 3.5. In the presence of the citrate ligand, two 27Al resonance peaks at 6.1 and 11.3 ppm, assigned respectively to the Alcit and Alcit2 complexes, were observed at pH 3.4. At pH 4.3 and an Al:citrate molar ratio of 1:2, the 6.1 ppm peak was not visible, and the second peak further downfield was split into two unresolved peaks at 10.8 and 12.4 ppm indicating the presence of two forms of the Alcit2 complex. Distribution of Al between the various species, based on integration of the resonance peaks and equilibrium calculations carried out using GEOCHEM, is discussed in light of the stability constants present in the database of GEOCHEM version (v.) 1.23 and GEOCHEM-PC v. 2.0. Large discrepancies between the computed values and the NMR measured values indicate the need to incorporate more recent literature values in the database for realistic equilibrium calculations in systems containing organic acid ligands. The potential of using quantitative 27Al NMR measurements to calculate stability constants is discussed.

Determination of total soluble aluminum in soil solution using pyrocatechol violet, lanthanum and iron to discriminate against micro-particulates and organic ligands

The determination of the Al concentration in soil solution is an important diagnostic tool in studies of Al toxicity in acid soils. Where organic residues have been applied as ameliorants of Al toxicity, it is necessary to measure both the total soluble and monomeric Al concentrations in order to estimate the degree of Al complexation. Determination by ICPAES provides a measure of total Al without discrimination between chemical forms, and thus may include solid-phase Al present as micro-particulates that have passed through a 0.22 pm filter. Colorimetric methods, which do not include an acidification step, discriminate against micro-particulates, but do not measure all Al present as soluble organic forms and thus underestimate total soluble Al. Acidification of soil solutions to decomplex organically bound Al, and thus permit its measurement by colorimetric methods, results in dissolution of micro-particulates and overestimation of total soluble Al. Filtering to 0.025 pm eliminated microparticulates, thereby removing a source of error. However, this process is time consuming and thus unsuitable for routine use.

Aluminium reactions with polygalacturonate and related organic ligands

Aluminium (Al), in inorganic monomeric forms, has been recognised as a limiting factor for root growth in many acid soils. Plant tolerance to Al may be achieved by the detoxification (complexation) of Al by organic ligands present in the rhizosphere. The Al-complexing ability of seven organic ligands, citric, oxalic, gluconic, glucuronic, mucic, galacturonic and polygalacturonic (pectin) acids, was investigated. The proportion of organically-complexed Al was determined using colorimetric methods based on differences in reaction rate with pyrocatechol violet or aluminon. The colorimetric methods confirmed that citric acid forms a strong complex with Al at pH 4.2. In contrast, pectin and related organic ligands weakly complexed Al in acidic conditions. In an additional study, the Al-binding ability of pectin and Ca-pectate was compared at a biologically significant concentration of 32 µM Al. Only 29% of free Al remained in solution in the presence of Ca-pectate, while 54% remained when pectin was present. This suggests that Ca-pectate, rather than pectin, is responsible for binding Al in root cell walls and consequently plays an important role in Al toxicity to plants. Root growth of mungbean (Vigna radiata (L.) Wilczek) confirmed differences in the ability of citrate, oxalate and galacturonate to complex Al.

Effects of organic acid fractions extracted from Eucalyptus camaldulensis leaves on root elongation of maize (Zea mays) in the presence and absence of aluminium

Complexes of aluminium (Al) with organic ligands are believed to represent an important detoxification mechanism in acid soils. However, relatively little is known about the particular ligands produced by decomposing vegetation or about their effects on plant growth in the presence or absence of toxic Al. This paper reports an experiment on the effects of decomposition products of Eucalyptus camaldulensis leaves on the root elongation of maize (Zea mays) cv. DK687 in the presence or absence of Al. The static solution culture experiment used fulvic acid (FA) and humic acid (HA), extracted from E. camaldulensis leaves, at three nominal concentrations, viz. 40, 120 and 360 mg C L-1, replicated 4 times in the presence and absence of 30 µM Al. In the absence of Al, root elongation was increased by 30% by HA at 40 mg C L-1 and by 36% by FA at 120 mg C L-1. In the presence of 30 µM Al, the effects of toxic Al on root elongation were negated by FA and HA at all concentrations. Aluminium was totally complexed in all treatments except FA at 40 mg C L-1 in which treatment only 2.7 µM Al was present in the monomeric form. The E. camaldulensis FA and HA at concentrations of 40 and 120 mg C L-1, either in the presence or absence of Al, stimulated maize root elongation. Aluminium was strongly complexed by the E. camaldulensis FA and HA. The present results, in which FA and HA alleviated Al toxicity limitations on root elongation of maize, are relevant to the protection afforded to plant growth in acid soils amended with organic materials. They highlight the need to focus more on the role of FA and HA.

Sterile solution culture techniques for aluminum toxicity studies involving organic acids

Microbial degradation of low molecular weight organic acids can be a serious source of error in solution culture experiments concerned with the protection of plant roots by these compounds against aluminum (Al) toxicity. Under normal glasshouse conditions, the problem was not solved by sterilisation of the various components of the solution culture system and surface sterilisation of the soybean and mungbean seeds. Of four water soluble antibiotics tested, streptomycin, rifampicin, and trimethoprim were phytotoxic at concentrations ≥ 50 mg/L; cefotaxime, while not phytotoxic up to 150 mg/L, gave poor control of microbial growth in 2-day root elongation tests with mungbean. However, addition of 30 mg/L cefotaxime plus 20 mg/L trimethoprim at planting and a further 30 mg/L cefotaxime after one day was effective in controlling microbial numbers and preventing significant loss of added citrate to the solutions. At these concentrations the antibiotics were non-phytotoxic and appeared not to cause any complexation of A1. Use of the method showed that citrate, L-malate, and oxalate were highly effective detoxifiers of A1 in solution.

The determination of dissolved organic carbon by inductively coupled plasma atomic emission spectroscopy

Abstract Dissolved organic carbon (DOC) present in soil solutions was measured by inductively coupled plasma atomic emission spectroscopy (ICPAES) using the two C(I) lines at 197.09 nm and 247.856 nm. Carbon standards, prepared from sucrose, in the range 100 to 1000 mg L‐1 produced linear coefficients of determination of r2 ≥ 0.9998 for each of the lines with calculated detection limits of 0.034 and 1.094 mg L‐1 for each line, respectively. Inorganic carbon was eliminated from solution by the addition of concentrated nitric acid and sparging with high purity nitrogen for 30 min. A comparison of the DOC values for soil solutions extracted from nine soil samples by ICPAES and dichromate oxidation showed a high degree of correlation ( r2 ≥ 0.9982).