Influence of natural and synthetic humic substances on the activity of urease

The activity of a purified urease, obtained from Bacillus pasteurii, was inhibited by humic and fulvic acids obtained from an agricultural soil. Enzyme kinetic studies showed that the humic substances affected the affinity of the enzyme for its substrate (K_m) and the maximum velocity of the reaction (V_max). The V_max was inhibited to the same extent by both humic (HA) and fulvic (FA) acids, the precise effect depending on the pH and concentration of humic substance. At pH 4.0, HA concentrations of 25 pg cm^?3 and 10 μg cm^?3 inhibited the V_max by 38.5% and 20% respectively. HA and FA had similar effects on the K_m but in this case the lowering of the affinity of the enzyme for its substrate was not concentration dependent in the range 0–25 μg cm^?3 of humic substance. Typically, the affinity was decreased from a K_M of 50 mM in the control to 67 mM in the presence of HA and FA. The effects were not due primarily to the ash or N contents of the humic substances because de-ashed humic acid and synthetic model humic (made from catechol, guaiacol, pyrogallol, resorcinol and protocatechuic acid) and fulvic acid (made from polymaleic acid), containing virtually no ash or N, were equally as effective. The effect was not related to the phenolic monomers which, before polymerization, had no effect on urease activity.     

Effect of humic acids on the activity of two peroxidases

The activity of two peroxidases-from horseradish roots and Streptococcus faecalis-is inhibited by synthetic and natural humic acids. Kinetic analyses revealed that inhibition of the horseradish enzyme is due to binding of the donor substrate guaiacol to humic acids. Peroxidase from Streptococcus faecalis is inhibited by competitive interactions between the donor substrate NADH and humic acids for the enzyme. Binding of H₂O₂ to any of the enzymes was not influenced. The rate of inhibition by synthetic and natural humic acids was different; hydroquinone humic acid and an acetylated derivative were considerably more effective than humic acids from white peat.     

Effects of humic acid fractions on invertase activities in plant tissues

The effect of some soil organic matter fractions and synthetic humic acids were tested on the invertase activities in tissues of several higher-plant species. The effects varied according to the plant and soil organic matter fraction used. Thus no fraction tested had any effect on the invertase activity of beetroot storage tissue, but several fractions, mainly humic acid and its residues obtained by water or acid boiling, inhibited invertase in carrot storage tissues and wheat roots and coleoptiles. and to a lesser extent mungbean hypocotyls. Soil phenolic acids were without effect in any of the plant tissues tested. Some stimulations of invertase activity occurred with pea root tissue, using the water- and acid-soluble humic-acid fractions. The effects were independent of ash contents, total N or the C:H:N ratio, and could not be ascribed to any particular chemical component.The inhibitory effects on wheat invertase activity produced by soil organic matter were independent of pH in the range 4.5–7.0, and were constant over a 4 h incubation. The inhibitors only slightly affected the K_m but markedly affected the V_max of the reaction, hence they were non-competitive.The effect of a naturally-occurring invertase inhibitor from beetroot storage tissues could be reduced in the presence of humic acid. Although the inhibitor had little effect on wheat root invertase it did reduce the inhibitory effect of the humic acid in this tissue.     

Humic substances and phosphatase activities in plant tissues

Humic acid and its fractions obtained by water- or acid-boiling inhibited phosphatase activity per se in beet, carrot and potato discs, pea roots and epicotyls and wheat roots, coleoptiles and leaves. In wheat roots the order of effectiveness of various humic acid fractions in inhibiting phosphatase activity was acid-boiled soluble > water-boiled soluble > acid-boiled insoluble > water-boiled insoluble > original humic acid. A number of synthetic humic acids were also effective inhibitors of enzyme activity but phenolic acids had no effect.The degree of inhibition was not related to C, H or N contents or to the total ash, carboxyl or phenolic contents of the humic acid samples. Magnesium ions enhanced phosphatase activity and decreased the inhibition of phosphatase activity produced by the humic acid fractions.Humic acid fractions did not affect the maximum temperature for enzyme activity or its pH optimum and had little effect on the Michaelis constant. They did, however, reduce the maximum velocity of the enzyme reaction thus producing a non-competitive inhibition of enzyme activity.It is suggested that the humic acid fractions inhibit phosphatase activity by combining with the enzyme, but not at the most active site of enzyme activity.     

Chromatographie et purification des diphenol oxydases du sol

Neutral sterile and lyophilized extracts of fresh soil (NAFS Extract) degraded the following substrates: p-cresol: dl-3(3.4-dihydroxyphenylalanine)(dl DOPA): d(+) catechin and p-phenylcnediamine respectively (19.7); (5.3); (5.7) and (4.4) nmoles O₂ mg C^?1 min^?1 as specific activity (sp. act.). NAFS Extract was fractionated by G100 Sephadex column chromatography into two major peaks (K_D ~ 0 and ~ 1) without an increase in sp. act. DEAE DE 52 cellulose chromatography separated NAFS Extract into three fractions. The first fraction was free from humic acid, relatively homogeneous on polyacrylamide gel electrophoresis and had sp. act.: dl DOPA (17.2): d(+) catechin (8.5): p-phenylenediamine (22.9). The o- and p-diphenol oxidases which accompanied this fraction were well separated as complexes on G100 Sephadex column and were liberated by dialysis against distilled water. Following isolation, we obtained an o-diphenol oxidase on dl DOPA (23.4) and a laccase activity on p-phenylenediamine (33.8) in the free state; these activities being associated with nucleoprotein. Fractions (II) and (III) appeared to be relatively homogeneous in the form of “humic acid—enzyme complexes”. Specific activity were high in fraction (III): dl DOPA (10.8): d(+) catechin (0.7); p-phenylenediamine (5.4). The diphenol oxidase activity extracted from soil (NAFS Extract) was treated by salmine and SP Sephadex C25 to remove humic matter. The EFS Extract obtained had the following sp. act.: dl DOPA (14.0); p-phenylenediamine (6.3). This EFS Extract was separated into three fractions by means of G100 Sephadex column chromatography. The Kn were (I) ~0; (II) ~ 0.52; (III) ~ 1.3 respectively. The first fraction showed an increase of sp. act. only with p-phenylenediamine (9.1) and in the following two fractions the sp. act. were not augmented. The first fraction was further fractionated by means of DEAE cellulose chromatography into four fractions: the-first and the second had no activity on dl DOPA and p-phenylenediamine. The third was an o-diphenol oxidase on dl DOPA (11.0) with traces of laccase: p-phenylenediamine (0.7). The fourth was pure laccase: p-phenylenediamine (18.1). These results suggested that electrostatic, covalent and van der Waals forces contributed to the formation of humic acid enzymes complexes, associated in the tetramer to monomer forms of diphenol oxidases.     

Behavior of phenolic substances in the decaying process of plants

The behavior of phenolic substances in the decaying process of rice straw, ladino clover, and fanen leaves of red oak under moist conditions, and also of rice straw under various conditions were compared in the laboratory. The amounts of phenolic substances, divided into either humic acid and fulvic acid fractions, or ether-extractable, butanol-extractable and organic solvent-unextractable fractions, and the amounts of individual phenolic acids were periodically determined during incubation for 150 days. The following results were obtained.

1) The amounts and behavior of phenolic substances in various fractions differed considerably among the plant materials. The total amount of phenolic substances was remarkably larger in red oak leaves than in the others during the whole period of incubation. The amounts of phenolics in the fulvic acid fraction changed to a larger extent than those in the humic acid fraction during the decaying process of plant materials. The changes in total amount of phenolics in decaying red oak leaves and ladino clover were mainly due to changes in the level of relatively hydrophilic phenolics in the fulvic acid fraction, but the changes in decaying rice straw were mainly due to changes in the level of relatively lipophilic phenolics.

2) Rice straw and ladino clover, especially the former, contained large amounts of p-coumaric and ferulic acids, but these decreased rapidly in the early stage of the decaying process. The amounts in red oak leaves were small, but did not decrease markedly during incubation.

3) The changes in amounts of phenolics in both humic acid and fulvic acid fractions in the decaying process of rice straw were largely influenced by temperature, moisture, and pH, but not to a large extent by C/N ratio and the presence of soil. At higher temperatures under moist conditions, phenolic substances disappeared rapidly. Also, acidification of the system inhibited the degradation process.     

In vitro assessment of the inhibition of humic substances on the growth of two strains of <Emphasis Type="Italic">Fusarium oxysporum</Emphasis>

The effects of four humic substance (HS) samples, a soil humic acid and two humic acids and one fulvic acid isolated from a composting substrate, were evaluated on the mycelial growth of Fusarium oxysporum f. sp. melonis (FOM) and F. oxysporum f. sp. lycopersici (FOL). In general, any HS treatment reduced significantly the radial growth of the FOM mycelium either in normal [potato dextrose agar (PDA) medium] or sub-optimal (water–agar medium) nutritional conditions. Differently, the FOL growth, which was tested only on PDA, was either inhibited or stimulated on dependence of the HS treatment used. The HS fractions isolated from the composting substrate were the most effective inhibitors of mycelial growth of both fungi. Furthermore, any HS treatment was also able to alter the germination process of FOL in aqueous medium, not only by reducing significantly the number of viable germinating conidia but also by generally decreasing the rate of conidial germ-tube elongation. Apparently, the extent of the inhibitory action was related to some chemical and functional properties of HS, such as the COOH group content and elemental composition.     

Oxidative coupling activity in soil extracts

An assay was developed in which 50 mm citrate buffer was used to extract catalysts responsible for oxidative coupling reactions in soil. The assay was based on the formation of the dimerized quinone from the oxidative coupling of 2,6-dimethoxyphenol. Oxidative coupling activity was destroyed by heating the extracts for 15 min at 100°C and was inhibited by H₂O₂ (98% at 10 mm), KCN (96% at 10 mm), dithiothreitol (100% at 0.1 mm) and 2,3-dimercapto-1-propanol (100% at 0.1 mm). In the assay, a pH of 7.0 and a temperature of 55°C were determined as optimal. Freezing the soil extracts provided the best protection against activity loss, whereas storage caused a decline in oxidative coupling activity as a function of increasing temperatures (5–50°C). If soil had been supplemented with sucrose before extraction, activity increased. Soil extracts reacted with syringaldazine, benzidine, o-dianisidine, guaiacol, and p-cresol, substrates previously used to detect phenoloxidase enzymes.     

Enhanced non-extractable residue formation of 14C-metalaxyl catalyzed by an immobilized laccase

We studied the influence of an immobilized laccase from Trametes versicolor on non-extractable residue (NER) formation of the systemic fungicide ¹⁴C-metalaxyl in soil. We added the enzyme (130 mU/g DW) to soil sterilized by gamma irradiation and observed that the amount of NER (6.3 % of applied radioactivity) after 10 days of incubation was enhanced about twofold compared to the sterile soil without laccase addition. Residues formed within samples without enhanced enzyme activity were mainly bound via ester linkages to all fractions of humic matter, i.e., fulvic acids, humic acids, non-humines, and humines, respectively. In contrast, residues formed in presence of immobilized laccase were more strongly bound by covalent linkages such as ether and C-C bonds, especially to humic acids. After chemical degradation of the humic matter, it could be observed that all NER contained the first major transformation product, i.e., metalaxyl acid. The findings underline that the residue formation of metalaxyl in soil may be partly catalyzed by immobilized extracellular oxidative enzymes through oxidative coupling reactions within the humic matter.     

Inhibitory action of dissolved humic substances on the growth of soil bacteria degrading DDT

Abstract

Dissolved humic acid (HA) and fulvic acid (FA) prepared from a Dando brown forest soil (Typic Dystrochrept) inhibited the growth of soil bacteria degrading DDT (1,1,1-trichloro-2,2-bis(4-chlorophenyl)ethane) in the culture. The population of DDT-degrading Gram-variable rod Bll6 decreased by the application of both HA and FA, suggesting the presence of bactericidal effect. Such inhibitory effect was stronger for HA and resulted in a lower degrading activity of DDT in the culture of Bll6. No inhibitory effect was observed on the growth of DDT-degrading Bacillus sp. B75. The electron spin resonance spectra showed the presence of organic free radicals in both HA and FA. The relative concentration of the radicals was higher in HA. Storage of HA solution for 3 months at 4°C decreased the concentration of the radicals as well as the inhibitory action. The addition of catalase decreased the inhibitory effect of humic acid. It is suggested that a hydroxy radical, which is derived from free radicals of humic substances, is involved in the inhibition of bacterial growth and degradation of DDT.     

Decomposition and distribution of residual activity of some 13C-microbial polysaccharides and cells,glucose, cellulose and wheat straw in soil

Studies were made to determine the rate of decomposition of some ¹⁴C-labeled microbial polysaccharides, microbial cells, glucose, cellulose and wheat straw in soil, the distribution of the residual ¹⁴C in various humic fractions and the influence of the microbial products on the decomposition of plant residues in soil. During 16 weeks from 32 to 86 per cent of the C of added bacterial polysaccharides had evolved as ¹⁴CO₂. Chromobacterium violaceum polysaccharide was most resistant and Leuconostoc dextranicus polysaccharide least resistant. In general the polysaccharides, microbial cells, and glucose exerted little effect on the decomposition of the plant products. Upon incubation the ¹⁴C-activity was quickly distributed in the humic. fulvic and extracted soil fractions. The pattern of distribution depended upon the amendment and the degree of decomposition. The distribution was most uniform in the highly decomposed amendments. After 16 weeks the bulk of the residual activity from Azotobacter indicus polysaccharide remained in the NaOH extracted soil. From C. violaceum polysaccharide both the extracted soil and the humic acid fraction contained high activity. About 50–80 per cent of the residual activity from the ¹⁴C-glucose, cellulose and wheat straw amended soils could be removed by hydrolysis with 6 n HCl. The greater part of this activity in the humic acid fraction was associated with the amino acids and that from the fulvic acids and residual soils after NaOH extraction with the carbohydrates. About 8 16 per cent of the activity of the humic acid fraction was present in substances (probably aromatic) extracted by ether after reductive or oxidative degradation.     

Characterization of humus-protease complexes extracted from soil

Pyrophosphate (140 mM, pH 7.1) extracts of two arable soils and one pasture soil were ultrafiltrated separating the extracted material into three fractions: A_I with nominal molecular weight (nmw) > 100 kD, A_II with nmw between 10 kD and 100 kD and R with nmw < 10 kD. Protease activity was determined in the fractions by using three different substrates: N-benzoyl-l-argininamide (BAA), specific for trypsin; N-benzyloxy-carbonyl-l-phenylalanyl l-leucine (ZPL), specific for carboxypeptidases; and casein, essentially a non-specific substrate. The derivative fractions were also analysed for their amino acid N and humic (HA) and fulvic (FA) acid contents. The organic matter of extracts and derivative fractions obtained from the pasture soil was analysed by isoelectric focusing (IEF) and that of fractions analysed by pyrolysis gas chromatography (Py-GC). Activities of the extract were monitored for their thermal stability and those of the extract and derivative fractions for their optimal pH.Due to the mechanical disintegrating action of sodium pyrophosphate over the humic substances during the fractionation process the amount of total organic C and FA in the fractions was ranked as R > A_II > A_I. The lowest amino acid N/organic C was found in the R fraction, whereas A_II fraction was rich in humic acids, carbohydrates and amino acid N and A_I fraction showed the lowest carbohydrate content. At least 70% of the total BAA- and ZPL-hydrolysing activity was associated to particles with nmw higher than 10 kD and at least 30% of these activities were present in particles with nmw higher 100 kD. Casein-hydrolysing activity was quite evenly distributed among the three fractions (A_I, A_II and R). The extracted protease-organic complexes were resistant to thermal denaturation and some of them showed optimal activity at pH values higher than 10 as a result of the polyanionic characteristics of the humic material surrounding enzyme molecules and of the presence of alkaline protease. Comparison of data obtained in Py-GC analyses and in protease activity suggests that BAA-hydrolysing activity was associated to a highly condensed humic matter and ZPL-hydrolysing activity to less resistant humic substances, while at least some of the extracted casein-hydrolysing activity was present as glyco-proteins not associated to humus. BAA-hydrolysing activity was probably inhibited by fresh organic matter of carbohydrate origin whereas lignin derived organic matter probably inhibited ZPL- and casein-hydrolysing activity.     

THE ACCUMULATION OF SOIL ORGANIC MATTER AND ITS CARBON ISOTOPE CONTENT IN A CHRONOSEQUENCE OF SOILS DEVELOPED ON AEOLIAN SAND IN NEW ZEALAND

Soil organic matter was extracted by a mixture of O.IM Na₄P₂O: O.IM NaOH from a chronosequence of weakly weathered soils developed on aeolian sand, and fractionated into humin (non-extractable), humic acid, and fulvic acid. The mass of total organic carbon in the profiles, the ¹⁴C content and the ¹³C/12C ratios were also determined. The weight of total carbon increased rapidly at first and then gradually without attaining a steady state. This trend was also shown by the humin and fulvic acid fractions, but the humic acid fraction appeared to have reached a maximum after about 3000 years. The order of total weights of the organic fractions was humin > fulvic acid > humic acid. The evidence suggests that the proportions of the humic fractions formed by decomposition are related to soil differences but not to vegetation. The greater part of the plant material found in the soils appears in the humin and fulvic acid fractions.     

Chemical–structural information from solid-state 13C NMR studies of a suite of humic materials from a lower montane forest soil,Colorado, USA

Chemically and physically fractionated samples extracted from the surface horizon of a soil developed under a mix of coniferous and deciduous vegetation in southwestern Colorado were studied. ¹³C NMR data on this soil's organic matter and its HF(aq)-washed residue, as well as the classic acid/base-separated humic fractions (humic acid, fulvic acid, humin), were examined for chemical–structural detail, e.g., the various structural functionalities present (especially lipids, carbohydrates, aromatics, polypeptides and carbonyl/carboxyls). Among the humic fractions, it was found that the lipid concentrations are in the order humic acid>fulvic acid= humin; for carbohydrates the order is fulvic acid>humin>humic acid; for aromatic carbons the order is humic acid>humin>fulvic acid; for polypeptides it is humic acid>fulvic acid>humin and for carbonyl/carboxyl species it is humin>humic acid>fulvic acid, but the differences are small. ¹³C spin–lattice relaxation times indicate that at least two types of “domains” exist in each, corresponding to “higher” and “lower” concentrations of paramagnetic centers, e.g., Fe³⁺.     

Chemical heterogeneity of humic substances: characterization of size fractions obtained by hollow-fibre ultrafiltration

To investigate the chemical heterogeneity of humic substances in relation to molecular size, fulvic and humic acids were extracted and purified from the surface horizon of a Humic Gleysol in northern Switzerland. A fractionation scheme using hollow‐fibre ultrafiltration cartridges was developed and used to obtain four size fractions of the humic acid with nominal molecular weight ranges > 300 kDa, 100–300 kDa, 30–100 kDa, and 10–30 kDa. The fulvic acid and all humic acid fractions were characterized by size exclusion chromatography, elemental analysis (C, H, N, S), as well as spectroscopic techniques including UV‐VIS, CP‐MAS ¹³C‐NMR, FT‐IR, and fluorescence spectroscopy. Clear chemical differences between the humic acid size fractions were observed. Smaller size fractions of the soil humic acid contained more chargeable functional groups and a larger percentage of aromatic carbon than the larger size fractions. Conversely, the percentage of aliphatic carbon increased with increasing apparent molecular weight. The chemical composition of the smallest humic acid fraction differed clearly from the fulvic acid fraction, despite similar apparent molecular size and carboxyl carbon content. Small humic acids contained much more aromatic carbon and less aliphatic carbon than the fulvic acid fraction. Apparently, humic size fractions differ in their chemical composition, which can have important implications for their environmental behaviour.     

Effect of soil humic substances on surface redox activity of oat roots

The effect of a low molecular weight (<5kDa, LMW) and a high molecular weight (>5kDa, HMW) humic fractions on surface redox activities of oat roots was studied. Oxidation of the electron donor NADH and reduction of the artificial electron acceptor ferricyanide [K3Fe(CN)6] exogenously supplied to the roots both alone or in combination, was measured in the presence or absence of soil humic substances. HMW humic fraction inhibited NADH oxidation either in the presence or absence of ferricyanide, while LMW humic fraction inhibited NADH: ferricyanide oxidoreductase activity due to the contemporary addition of the two redox compounds to the solution bathing the roots. NADH: ferricyanide oxido‐reduction was partially due to the release of substances from the roots. However, the presence of soil humic fractions (LMW or HMW) did not significantly modify this behaviour. Rather, the inhibitory effect of soil humic substances was even more evident when the oxidoreduction solely due to the root activity was considered. The results confirm the presence of multiple types of oxidoreductase activities at the surface of oat roots and show that the two humic fractions HMW and LMW may differently affect these activities. Partial inhibition of NADH oxidase activity is interpreted as a possible way of interference of humic substances with metabolic processes involved in cell wall formation. The effects on surface redox activities are discussed in terms of the role of soil humic substances in promotion of plant growth.     

Effect of molecular complexity and acidity of earthworm faeces humic fractions on glutamate dehydrogenase,glutamine synthetase,and phosphoenolpyruvate carboxylase in Daucus carota α II cells

Carrot cells were grown in cultures supplemented with two hormones [2,4-dichlorophenoxyacetic acid (2,4-D) and 6-benzylaminopurine (6BAP)] and two humic fractions extracted from earthworm faeces, one with high acidity and a low apparent molecular size (<3500) and the other with low acidity and a large molecular size. 2,4-D stimulated growth through an effect on cell enlargement, while the strongly acidic humic fraction (0.2 mg l^-1) and the weakly acidic fraction (1 mg l^-1) were both less effective. With 4–16 h of pre-incubation, the highly acid humic fraction, mainly alone, induced the best increase in protein content; the effect of the weakly acid humic fraction and the hormones was generally less important. The two humic fractions also differed in their influence on glutamate dehydrogenase activity. After 2 h of pretreatment, the highly acidic fraction increased glutamate dehydrogenase activity, while the other fraction did not affect it. After 4–16 h of pre-incubation, the activity of this enzyme was still not influenced by these humic fractions. The presence of the two hormones did not interfere with the humic matter effects. Glutamine synthetase activity was not affected by a pre-incubation of up to 4 h with the two humic fractions, but it was stimulated after 8–16 h of pre-incubation. A 2,4-D+6BAP mixture stimulated glutamine synthetase activity (from +12 to +50%). Again, the presence of the hormones did not interfere with the effects induced by the humic fractions. After 16 h of pre-incubation, phosphoenolpyruvate carboxylase activity was increased by the highly acidic humic fraction (+93%) and by both humic fractions together (+34%). An explanation of the different incubation times necessary for the humic fractions to exert stimulatory effects on these enzymes is proposed here. The regulatory properties of the strongly acidic humic fraction appeared to depend on the combination of high acidity (expecially carboxylic C) with low molecular size.     

腐殖酸对汞的络合稳定特性及其环境学意义

对3种腐殖酸与Hg的络合稳定特性研究结果表明,腐殖酸与Hg的结合存在松结态与紧结态2种形态,其中富里酸主要以松结态为主,灰色胡敏酸与棕色胡敏酸则以紧结态占绝对优势。3种腐殖酸中富里酸对Hg的络合容量最高,但络合强度最低,其络合汞的环境活性将较高;灰色胡敏酸对Hg的络合容量最低,但络合稳定性最高,故所结合Hg的环境活性最弱。但受容量因素制约,灰色胡敏酸对Hg的络合稳定性将因Hg/灰色胡敏酸相对比例的升高而急剧下降。棕色胡敏酸对Hg络合特性介于富里酸与灰色胡敏酸之间。     

Physiological and Genetic Responses to Pesticide Mixture Treatment of Veronica beccabunga

The effects of a five-pesticide mixture on pesticide accumulation, phytohormone levels (indole-3-acetic acid, gibberellic acid, jasmonic acid, and salicylic acid), pigment contents (total chlorophyll and carotenoid), antioxidant enzyme (catalase and guaiacol peroxidase) activities, lipid peroxidation product (malondialdehyde), and DNA profiles were investigated in the leaves of Veronica beccabunga. Laboratory-acclimatized plants were treated with a mix of five pesticides (atrazine, disulfoton, chlorpyrifos, metalaxyl, and ethion) in doses of 50?ppt, 1?ppb, 100?ppb, and 1?ppm for 1, 3, and 6?days. The accumulation of each pesticide, from highest to lowest, was as follows: chlorpyrifos, atrazine, metalaxyl, disulfoton, and ethion. The amounts of total chlorophyll and protein decreased with increased pesticide concentration. Antioxidant enzyme activities and malondialdehyde amount increased linearly with increasing pesticide exposure. However, the highest pesticide concentration caused decreases in guaiacol peroxidase (POD) activity and malondialdehyde (MDA) content at all treatment times. Both jasmonic and salicylic acid levels increased with pesticide exposure and decreased gradually after. It was also determined that application of the pesticide mixture affected the DNA profiles of V. beccabunga. The most band changes were detected on the sixth day of treatment.     

Studies on the humus forms of forest soils

Samples described in the previous paper were analyzed for humus composition by the method of Kumada el al,, elementary composition of humic acids, nitrogen distribution among humic acid, fulvic acid, and humin, and organic matter composition by the modified Waksman method. The samples obtained by physical fractionation from each horizon of Higashiyama soil were as follows: f₁ and f₂ from the L layer, f₁, f₂ and f₃ from the F layer, f₁ f₂, sand, silt, and clay fractions from the H-A and A horizons.

With the progress of decomposition, the following tendencies were rather clearly observed.

The extraction ratio of soluble humus, amounts of humic acid and fulvic acid, and PQ, value tended to increase with some exceptions. The degree of humification of humic acid proceeded. Most humic acids belonged to the Rp type, but those of the clay fractions belonged to the B type.

As for the elementary composition of humic acid, transitional changes from the Lf₁ to the clay fraction of the A horizon were observed. But differences in elementary composition among humic acids were far less, compared with those among whole fractions.

Nitrogen contents in humic, fulvic, and humin fractions increased with the progress of decomposition and humiliation, and the largest relative increase was found in fulvic acid nitrogen.

According to the modified Waksman's method, the amounts of residues and protein increased, while the total amounts of each extract, except for the HCl extract, and the amounts of sugars and starch, phenolic substances, hemicelluloses and pectin, and cellulose decreased. Sugars and starch comprised only a small portion of the hot water extract, and polyphenols substances comparable to sugars and starch were also found in the extract. Hemicelluloses and pectin accounted for only about one-half of the HCl extract. Several characteristic differences in the elementary composition of extracts and residues were found.

Pheopigments existed in benzene-ethanol extracts and their amounts seemed to decrease from Lf₁ to Ff₂.