Influence of oxygen on denitrification and aerobic respiration in soil

Summary The influence of the partial pressure of oxygen on denitrification and aerobic respiration was investigated at defined P02 values in a mull rendzina soil. The highest denitrification and respiration rates obtained in remoistened, glucose- and nitrate-amended soil were 43 1 N₂0 h^–1g^–1 soil and 130 1 O₂ h^–1g^–1 soil, respectively. At -55 kPa matric water potential, corresponding to 40% water saturation, N₂0 was produced only below P0₂ 40 hPa. The K _m, for O₂ was 3.0 x 10^–6 M. Formation of N₂O and consumption of O₂ occurred simultaneously with half maximum rates at P0₂ 6.7–13.3 hPa. Nitrite accumulated in soil below 40 hPa and increased with decreasing pO₂. The upper threshold for N₂0 formation in amended soil was P0₂ 33–40 hPa (39-47 M O₂).     

Molybdenum reserves of seed,and growth and N2 fixation by Phaseolus vulgaris L.

Summary Plants grown from seed with high (1.5–7.3 g Mo seed^-1) and low (0.07–1.4 g Mo seed^-1) Mo contents were grown in the presence and absence of Mo in growth media (perlite) or in a flowing-solution culture, in a controlled environment. Neither the high (1.5 g Mo seed^-1) nor the low (0.1 g Mo seed^-1) Mo content in seed from a small-seeded genotype (BAT 1297) was able to prevent Mo deficiency (reduced shoot, root and nodule dry weight, N₂ fixation and seed production) in growth media without an external supply of Mo, whereas both the high (7.3 g Mo seed^-1) and the low (0.07 g Mo seed^-1) contents in seed were able to prevent Mo deficiency in a large-seeded genotype (Canadian Wonder). Responses to Mo treatment by the Two genotypes were inconsistent between the growth media and solution culture experiments. Seed with a large Mo content (3.5 g Mo seed^-1) from the Canadian Wonder genotype was unable to prevent Mo deficiency (reduced shoot and nodule dry weight and N₂-fixation) in a solution culture without an external source of Mo, whereas both the large (1.7 g Mo seed^-1) and the small (0.13 g Mo seed^-1) contents in seed prevented a deficiency in BAT 1297. Growing plants from seed with a small Mo content, without additional Mo, reduced the seed Mo content by 83–85% and seed production by up to 38% in both genotypes. Changes in seed size and increases in shoot, root and nodule dry weight occurred, but varied with the genotype and growth conditions. These effects were also observed in some cases where plants were grown with additional Mo, demonstrating that the amount of Mo in the seed sown can influence plant nutrition irrespective of the external Mo supply. Nodule dry weight, total N content of shoots and seed production were improved by using seed with a small Mo content (1.64–3.57 g Mo seed^-1) on acid tropical soils in Northern Zambia. Plants of both the large- and small-seeded genotypes grown from seed with a small Mo content (<1.41 g Mo seed^-1) had a smaller nodule weight, accumulated less N and produced less seed. The viability of seed with a small Mo content was lower (germination up to 50% less) than that of seed with a large Mo content.     

Short-term measurements of uptake of nitrogen fixed in the rhizospheres of sorghum (Sorghum bicolor) and millet (Pennisetum americanum)

Summary In a series of short-term experiments root systems of young sorghum and millet plants inoculated with N₂-fixing bacteria were exposed to ¹⁵N₂-enriched atmospheres for 72 h. The plants were grown in a normal atmosphere for up to 22 days after the end of the exposure to allow them to take up the fixed N₂. Environmental conditions and genotypes of sorghum and millet were selected to maximise N₂-fixation in the rhizosphere. Detectable amounts of fixed N (> 16 g/plant) were rapidly incorporated into sorghum plants grown in a sand/farmyard manure medium, but measurable fixation was found on only one occasion in plants grown in soil. N₂ fixation was detectable in some experiments with soil-grown millet plants but the amounts were small (2–4 g/plant) and represented less than 1 % of plant N accumulated over the same period. In many cases there was no detectable ¹⁵N₂ incorporation despite measurable increases in ethylene concentration found during an acetylene reduction assay.Published as ICRISAT Journal Article No. JA 740     

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

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

Effects of phosphate fertilizer on biomass production and N2(C2H2) fixation by pot-grown Ulex gallii Planchon in a forest soil

Summary N₂(C₂H₂) fixation by Ulex gallii Planchon (dwarf or autumn flowering gorse/furze) seedlings was determined following 8 months of growth (December-August) in the glasshouse in a very acid, N- and P-deficient forest soil. Application of Na₂HPO₄·12H₂O or North African ground rock phosphate fertilizer was essential for growth, nodulation and C₂H₂ reduction activity. Overall, both the sodium phosphate and the rock phosphate were equally effective P sources and the maximum acetylene reduction by intact roots was measured as 4.09 and 4.69 mol C₂H₄g^-1 fresh weight nodule h^-1, respectively. Applied NH₄Cl severely inhibited nodulation and restricted acetylene reduction activity but not seedling growth. The results are discussed in relation to the spread of U. gallii in the south of Ireland and its potential as a leguminous nurse crop for Sitka spruce on the very impoverished forest soils of the region.     

Quantification and potential availability of non-symbiotically fixed 15N in soil

Summary Non-symbiotic N₂ fixation was studied under laboratory conditions in two soils from Pakistan (Hafizabad silt loam and Khurrarianwala silt loam) and one from Illinois, USA (Drummer silty clay loam) incubated in a ¹⁵N-enriched atmosphere. N₂ fixation was greatest with the Drummer soil (18–122 g g^–1 soil, depending upon the soil treatment) and lowest with the Khurrarianwala soil (4–81 g g^–1 soil). Fixation was increased by the addition of glucose, a close correlation being observed between the amount of glucose added and the amount of N₂ fixed in the three soils (r = 0.96). Efficiency of N₂ fixation varied with soil type and treatment and was greatest in the presence of added inorganic P. Application of Mo apparently had a negative effect on the amount and efficiency of N₂ fixation in all the soils. The percentage of non-symbiotically fixed ¹⁵N in potentially mineralizable form (NH ₄ ⁺ -N released in soil after a 15-day incubation period under anaerobic conditions) was low (2%–18%, depending upon the soil treatment), although most of the fixed N (up to 90%) was recovered as forms hydrolysable with 6N HCl. Recovery in hydrolysable forms was much greater for the fixed N than for the native soil N, indicating that the former was more available for uptake by plants.     

Competition among strains of Bradyrhizobium and vesicular-arbuscular mycorrhizae for groundnut (Arachis hypogaea L.) root infection and their effect on plant growth and yield

Summary Strains of Bradyrhizobium influenced root colonization by a species of vesicular-arbuscular mycorrhizae (VAM), and species of VAM influenced root nodulation by strains of Bradyrhizobium in pot experiments. In a field experiment, the effects of VAM on competition amongst inoculated bradyrhizobia were less evident, but inoculation with Bradyrhizobium strains increased root colonization by VAM. Certain VAM/Bradyrhizobium inoculum strain combinations produced higher nodule numbers. Plants grown without Bradyrhizobium and VAM, but supplied with ammonium nitrate (300 g ml^–1) and potassium phosphate (16 g ml^–1), produced higher dry-matter yields than those inoculated with both symbionts in the pot experiment. Inoculation with either symbiont in the field did not result in higher pod and haulm yields at harvest.ICRISAT Journal Article No. 886     

Physiological and symbiotic characteristics of Rhizobium fredii isolated from subtropical-tropical soils

Summary Physiological and symbiotic characteristics were identified in Rhizobium fredii isolated from subtropical-tropical soils. The generation times of R. fredii Taiwan isolated-SB 357 and -SB 682 were 1.7 and 2.5 h, respectively. These strains were associated with acid production in yeast-extract mannitol medium. They were able to use hexoses, pentose, sucrose, trehalose and raffinose. Strain SB 357 can resist a high concentration of kanamycin (100 g ml^–1 and penicillin (400 g ml^–1). It can tolerate up to 2.34% NaCl and 1031.3 mosmol kg^–1 (23.4 bars). The growth rate of R. fredii SB 357 under the concentration of approximately 450 mosmol kg^–1 (10.2 bars) was not affected by salinity, but responded to osmotic pressure. Both strains (SB 357 and SB 682) isolated from subtropical-tropical soils were able to form an effective N₂-fixing symbiosis with the US soybean cv Clark lanceolate leaflet.     

Effect of increasing oxygen concentration on total denitrification and nitrous oxide release from soil by different bacteria

Summary The effect of increasing oxygen concentrations (0, 5, 10 and 20 Vol% O₂) on total denitrification and N₂0 release was studied in model experiments using a neutral pH loamy soil relatively rich in easily decomposable organic matter and supplied with nitrate (300 g nitrate N/g dry soil). The sterilized soil was inoculated with three different denitrifying bacteria (Bacillus licheniformis,Aeromonas denitrificans andAzospirillum lipoferum) and incubated (80% WHC, 30°C). The gas volume was analysed for O₂, CO₂, N₂O, NO and N₂ by gas chromatography and the soil investigated for changes in ammonium, nitrite, nitrate, pH, total N and C as well as water-extractable C. WithB. licheniformis andAeromonas denitrificans total denitrification increased remarkably with increasing pO₂ as the result of intensified mineralization.Azospirillum lipoferum, however, showed the highest activity at 5 vol% O₂. WithB. licheniformis N₂O was released only in anaerobic conditions and at 5 Vol% O₂ (maximum) or 10 Vol% 02, but not at 20 Vol%, whereasAeromonas denitrificans produced N₂O only in the presence of He gas (maximum) or at 5 Vol% O₂. In contrast to these bacteria, N₂O production withAzospirillum lipoferum was restricted to 10 Vol% O₂ (maximum) and to 20 Vol% 02, with some traces at 5 vol% O₂. With a certain set of conditions, total denitrification and N₂O formation seem to be governed by the mineralization rate of the organisms in question. The increased demand for electron acceptors by a high turnover rate rather than the presence of anaerobic conditions seems to have determined the rate of denitrification.     

Effect of encapsulated calcium carbide on dinitrogen,nitrous oxide,methane, and carbon dioxide emissions from flooded rice

Summary The efficiency of N use in flooded rice is usually low, chiefly due to gaseous losses. Emission of CH₄, a gas implicated in global warming, can also be substantial in flooded rice. In a greenhouse study, the nitrification inhibitor encapsulated calcium carbide (a slow-release source of acetylene) was added with 75, 150, and 225 mg of 75 atom % ¹⁵N urea-N to flooded pots containing 18-day-old rice (Oryza sativa L.) plants. Urea treatments without calcium carbide were included as controls. After the application of encapsulated calcium carbide, 3.6 g N₂, 12.4 g N₂O-N, and 3.6 mg CH₄ were emitted per pot in 30 days. Without calcium carbide, 3.0 mg N₂, 22.8 g N₂O-N, and 39.0 mg CH₄ per pot were emitted during the same period. The rate of N added had a positive effect on N₂ and N₂O emissions, but the effect on CH₄ emissions varied with time. Carbon dioxide emissions were lower with encapsulated calcium carbide than without. The use of encapsulated calcium carbide appears effective in eliminating N₂ losses, and in minimizing emissions of the greenhouse gases N₂O and CH₄ in flooded rice.     

Dual inoculation of Pisum sativum with Rhizobium leguminosarum and Penicillium bilaji

Summary To investigate the effect of single versus dual inoculation of peas (Pisum sativum L.) with Rhizobium leguminosarum biovar viceae and Penicillium bilaji (a soil fungi capable of solubilizing soil P) on N₂ fixation an experiment was carried out under controlled conditions. A sandy loam soil was selected which contained low levels of available N and P. P fertilizer [Ca(H₂PO₄)₂] and P. bilaji significantly increased dry matter production. Peas inoculated with R. leguminosarum showed only a small increase in dry matter, but the additional application of P significantly increased the yield. The total N accumulation was highly dependent on the presence of R. leguminosarum. Using the ¹⁵N method for estimating N₂ fixation, the highest level of N₂-fixing activity was observed in peas inoculated with R. leguminosarum and fertilized with inorganic P. Dual inoculation of peas with P. bilaji and R. leguminosarum significantly decreased the amount of N₂ fixed. Total P uptake was solely dependent on the P fertilizer.     

Intrinsic antibiotic resistance and competition in fast- and slow-growing soybean rhizobia on a hybrid of Asian and US cultivars

Summary Six fast-growing soybean rhizobia (Rhizobium fredii) and thirteen slow-growing soybean rhizobia (Bradyrhizobium japonicum) were examined for resistance to 10 antibiotics. Axenic studies were carried out to determine the competitiveness of dual-strain inocula consisting of fast- and slow-growing rhizobia isolated from subtropical-tropical soils for nodule occupancy on a hybrid of Asian and US soybean cultivars. Nodule occupancy was determined by intrinsic resistance to erythromycin and neomycin. The results showed wide variability in resistance to 10 antibiotics for fast- and slow-growing rhizobia. The intrinsic antibiotic resistance of fast- and slow-growing rhizobia was extremely high against nalidixic acid (400 g ml^–1) and penicillin (200 g ml^–1). The competitive ability of inoculant strains for nodule occupancy varied for different combination sets and with the plant growing media. Our results show that fast-growing rhizobia nodulate a hybrid of Asian and US soybean cultivars. Fast-growing soybean rhizobia did not completely exclude nodulation by the slow-growing strains, which formed 0–79% nodules, depending on the strain used in the inoculum.     

Influence of methyl isocyanate on the growth,ATP levels,and nitrogen fixation of Azotobacter chroococcum

Summary Growth, ATP levels, and N₂-fixing activity of Azotobacter chroococcum culture exposed to 500 g ml^-1 of methyl isocyanate were investigated. In methyl isocyanate-exposed cells, the lag phase was extended up to 16 h but the control cells started to multiply immediately. ATP levels and N₂-fixing activity as determined by acetylene reduction assay were markedly reduced in the treated cells.     

Nitrogen-fixing potential of Acacia mangium and Acacia auriculiformis seedlings inoculated with Bradyrhizobium and Rhizobium spp.

Summary Two Australian Acacia species, A. mangium and A. auriculiformis were inoculated in vitro with eight strains of Bradyrhizobium spp. and two strains of Rhizobium spp. On the two plant species, only Bradyrhizobium spp. strains formed effective N₂-fixing nodules. A. mangium, which nodulates effectively with a restricted range of Bradyrhizobium spp. strains, is a specific host compared to A. auriculiformis. A. auriculiformis is assumed to be a promiscuous host because it nodulates effectively with a wide range of Bradyrhizobium spp. strains. Nodule efficiency as expressed by the ratio of N₂ fixed to nodule dry weight appeared to be higher in A. auriculiformis (0.44–0.81) than in A. mangium (0.23–0.55).     

Dinitrogen (C2H2) fixation in relation to nitrogen fertilization of grey alder [Alnus incana (L.) Moench.] plantations in a peat bog

Summary Nitrogenase activity was measured in young grey alder plantations in a peat bog in central Sweden. The stands were treated in three ways: (1) daily irrigation during the growing season with a complete nutrient solution, including N; (2) application of bark ash or wood ash before planting; and (3) fertilization every second year with solid PK fertilizers. Acetylene reduction assays were performed on (1) detached nodules and attached nodules, either on (2) whole enclosed plants or (3) enclosed nodules. The acetylene reduction rate for the enclosed plants showed a maximum in July when mean values of nearly 80 mol C₂H₄ (g nodule dry matter)^-1 h^-1 were reached. No diurnal patterns were observed. The irrigated stands, with an N supply, showed overall nitrogenase activities that corresponded well with those of the other treatments. Only in the case of temporarily increased soil nutrient concentrations in the irrigated stands did the nitrogenase activity fall considerably. In 6-to 7-year-old intensively managed irrigated stands N₂-fixation was estimated as 85–115 kg N ha^-1 year^-1 which was about 55% of the total N uptake of the trees.     

Trace gas emissions from soil of the central highlands of Mexico as affected by natural vegetation: a laboratory study

In the central highlands of Mexico, mesquite (Prosopis laevigata) and huisache (Acacia schaffneri), N₂-fixing trees or shrubs, dominate the vegetation and are currently used in a reforestation program to prevent erosion. We investigated how natural vegetation or cultivation of soil affected oxidation of CH₄, and production of N₂O. Soil was sampled under the canopy of mesquite (MES treatment) and huisache trees (HUI treatment), outside their canopy (OUT treatment) and from fields cultivated with maize (ARA treatment) at three different sites while production of CO₂, and dynamics of CH₄, N₂O and inorganic N (NH₄⁺, and NO₃^–) were monitored in an aerobic incubation. The production of CO₂ was 2.3 times higher and significantly greater in the OUT treatment, 3.0 times higher in the MES treatment and 4.0 times higher in the HUI treatment compared to the ARA treatment. There was no significant difference in oxidation of CH₄ between the treatments, which ranged from 0.019 g CH₄–C kg^–1 day^–1 for the HUI treatment to 0.033 CH₄–C kg^–1 day^–1 for the MES treatment. The production of N₂O was 30 g N₂O–N kg^–1 day^–1 in the MES treatment and >8 times higher compared to the other treatments. The average concentration of NO₃^– was 2 times higher and significantly greater in the MES treatment than in the HUI treatment, 3 times greater than in the OUT treatment and 10 times greater than in the ARA treatment. It was found that cultivation of soil decreased soil organic matter content, C and N mineralization, but not oxidation of CH₄ or production of N₂O.     

The effects of feeding by Oniscus asellus on leaf litter sulfur constituents

Summary Feeding by the isopod, Oniscus asellus, produced changes in the sulfur constituents of leaf litter substrates (Acer negundo, A. saccharum, Fagus grandifolia, Picea rubens, and Tsuga canadensis). Isopod consumption of leaf litter generally accelerated the mineralization of carbon-bonded S and increased the formation of ester sulfate in all substrates. After the isopod egestion of A. negundo leaves, fecal decomposition over 6 weeks increased total S concentration from 68 to 120 mol S/g due to the catabolism of organic carbon. During the same period sulfate decreased from 34 to 20 mol S/g and carbon-bonded S increased from 34 to 100 mol S/g. Thus the total S pool in aged feces became enriched with organic S (83% of total S). Macroinvertebrate consumption accelerated the transformation of S constituents and the carbon-bonded S concentration approached that of the Oa organic horizon of a northern hardwood forest.Dedicated to the late Prof. Dr. M.S. Ghilarov     

Influence of arbuscular mycorrhizae and phosphorus fertilization on growth, nodulation and N2 fixation (15N) inMedicago sativa at four salinity levels

The rose of an isolate of the arbuscular mycorrhizal (AM) fungusGlomus mosseae in the protection ofMedicago sativa (+Rhizobium meliloti) against salt stress induced by the addition of increasing levels of soluble salts was studied. The interactions between soluble P in soil (four levels), mycorrhizal inoculum and degree of salinity in relation to plant growth, nutrition and infective parameters were evaluated. Salt stress was induced by sequential irrigation with saline water having four concentrations of three salts (NaCl, CaCl₂, and MgCl₂).¹⁵N-labelled ammonium sulphate was added to provide a quantitative estimate of N₂ fixation under moderate to high salinity levels. N and P concentration and nodule formation increased with the amount of plant-available P or mycorrhizal inoculum in the soil and generally declined as the salinity in the solution culture increased from a moderate to a high level. The mycorrhizal inoculation protected the plants from salt stress more efficiently than any amount of plant-available P in soil, particularly at the highest salinity level applied (43.5 dS m^–1). Mycorrhizal inoculation matched the effect on dry matter and nutrition of the addition in the soil of 150 mg P kg^–1. Nevertheless the highest saline solution assayed (43.5 dS m^–1) affected more severely plants supplemented with phosphorus than those with the addition of mycorrhizal inoculum. Such a saline-depressing effect was 1.5 (biomass), 1.4 (N) and 1.5 (P) times higher in plants supplied with soluble phosphate than with AM inoculum. Mechanisms beyond those mediated by P must be involved in the AM-protectioe effect against salinity. The¹⁵N methodology used allowed the determination of N₂ fixation as influenced by different P applications compared to mycorrhizal inoculation. A lack of correlation between nodule formation and function (N₂ fixation) was evidenced in mycorrhizal-inoculated plants. In spite of the reduced activity per nodule in mycorrhizal-inoculated In spite of the reduced activity per nodule in mycorrhizal-inoculated plants, the N contents determined indicated the highest acquisition of N occurred in plants with the symbiotic status. Moreover, N and P uptake increased while Ca and Mg decreased in AM-inoculated plants. Thus P/Ca ratios and cation/anion balance in general were altered in mycorrhizal treatments. This study therefore confirms previous findings that AM-colonized plants have optional and alternative mechanisms available to satisfy their nutritive requirements and to maintain their physiological status in stress situations and in disturbed ecosystems.     

Influence of phenamiphos on the vesicular-arbuscular mycorrhizal symbiosis in Leucaena leucocephala

Summary Concentrations of phenamiphos ranging from 0 to 40 rg/g soil were established in a typical Oxisol (Tropeptic Eutrustox), inoculated or uninoculated with Glomus aggregatum. The effect of the nematicide on the development of vesicular-arbuscular mycorrhizal (VAM) symbiosis was evaluated in the greenhouse using Leucaena leucocephala as an indicator host plant. Treatment of soil with phenamiphos did not have a significant influence on the development of mycorrhizal activity measured in terms of subleaflet phosphorus concentrations. Similarly, the nematicide did not have an adverse effect on the level of mycorrhizal colonization or on the P content of shoots, as determined at the time of harvest. However, shoot dry weight was adversely influenced by phenamiphos when the chemical was applied to the uninoculated soil at 20 g/g soil or higher, and when it was applied to the inoculated soil at 40 g/g soil. It is concluded that phenamiphos is not likely to influence the growth of Leucaena or its symbiotic association with VAM fungi if the concentrations applied do not exceed levels known to suppress nematodes.Hawaii Institute of Tropical Agriculture and Human Resources Journal series No. 3146     

Soybean nodulation and nitrogen fixation on soil amended with plant residues

Residues from some tree species may contain allelopathic chemicals that have the potential to inhibit plant growth and symbiotic N₂-fixing microorganisms. Soybean [Glycine max (L.) Merr] was grown in pots to compare nodulation and N₂-fixation responses of the following soil amendments: control soil, leaf compost, red oak (Quercus rubra L.) leaves, sugar maple (Acer saccharum Marsh) leaves, sycamore (Platanus occidentalis L.) leaves, black walnut (Juglans nigra L.) leaves, rye (Secale cereale L.) straw, and corn (Zea mays L.) stover. Freshly fallen leaves were collected from urban shade trees. Soil was amended with 20 g kg^-1 air-dried, ground plant materials. Nodulating and nonnodulating isolines of Clark soybean were grown to the R2 stage to determine N₂-fixation by the difference method. Although nodulation was not adversely affected, soybean grown on leaf-amended soil exhibited temporary N deficiency until nodulation. Nodule number was increased by more than 40% for soybean grown on amended soil, but nodule dry matter per plant generally was not changed compared with control soil. Nonnodulating plants were severely N deficient and stunted as a consequence of N immobilization. Nodulating soybean plants grown on leaf or crop residue amended soil were more dependent on symbiotically fixed N and had lower dry matter yields than the controls. When leaves were composted, the problem of N immobilization was avoided and dry matter yield was not reduced. No indication of an allelopathic inhibition on nodulation or N₂-fixation from heavy application of oak, maple, sycamore, or walnut leaves to soil was observed.