Biological nitrification inhibition (BNI)—is it a widespread phenomenon?

Regulating nitrification could be a key strategy in improving nitrogen (N) recovery and agronomic N-use efficiency in situations where the loss of N following nitrification is significant. A highly sensitive bioassay using recombinant luminescent Nitrosomonas europaea, has been developed that can detect and quantify the amount of nitrification inhibitors produced by plants (hereafter referred to as BNI activity). A number of species including tropical and temperate pastures, cereals and legumes were tested for BNI in their root exudate. There was a wide range in BNI capacity among the 18 species tested; specific BNI (AT units activity g⁻¹ root dry wt) ranged from 0 (i.e. no detectable activity) to 18.3 AT units. Among the tested cereal and legume crops, sorghum [Sorghum bicolor (L.)], pearl millet [Pennisetum glaucum (L.) R. Br.], and groundnut [Arachis hypogaea (L.)] showed detectable BNI in root exudate. Among pasture grasses, Brachiaria humidicola (Rendle) Schweick, B. decumbens Stapf showed the highest BNI capacity. Several high- and low-BNI genotypes were identified within the B. humidicola species. Soil collected from field plots of 10 year-old high-BNI genotypes of B. humidicola, showed a near total suppression (>90%) of nitrification; most of the soil inorganic N remained in the NH₄⁺ form after 30 days of incubation. In contrast, soils collected from low-BNI genotypes did not show any inhibitory effect; most of the soil inorganic N was converted to NO₃^– after 30 days of incubation. In both the high- and low-BNI genotypes, BNI was detected in root exudate only when plants were grown with NH₄⁺, but not when grown with NO₃^– as the sole source of N. BNI compounds when added to the soil inhibited nitrification and the relationship was linear (r ² = 0.92^**; n = 12). The BNI from high- and low-BNI types when added to N. europaea in pure culture, blocked both the ammonia monooxygenase (AMO) and the hydroxylamine oxidoreductase (HAO) pathways. Our results indicated that BNI capacity varies widely among and within species; and that some degree of BNI capacity is likely a widespread phenomenon in tropical pasture grasses. We suggest that the BNI capacity could either be managed and/or introduced into pastures/crops with an expression of this phenomenon, via genetic improvement approaches that combine high productivity along with some capacity to regulate soil nitrification process.     

Field decomposition of transgenic Bt maize residue and the impact on non-target soil invertebrates

The release of chemical compounds from plant roots that suppress soil nitrification is termed biological nitrification inhibition (BNI). Determining the environmental factors that control the synthesis and release of BNI-compounds from Brachiaria humidicola (Rendle) Schweick, a tropical pasture grass that thrives on acid soils, is the focus of this investigation. Because the BNI trait is related to the N status of the plant, we investigated the possibility that the expression of this trait would be related to the forms of N found in the root environment. Plants were grown with two sources of N, NH₄⁺ or NO₃⁻ for 60 days and the release of BNI-compounds monitored. Only plants grown with NH₄⁺ released BNI-compounds from roots. The presence of NH₄⁺ and possibly the secondary effect of its uptake (i.e., acidic pH) in the root environment significantly enhanced the release of BNI-compounds. Both the NH₄⁺ and NO₃⁻ grown plants responded to the stimulus from NH₄⁺ in the root environment. BNI-compounds found in root tissue and their release were nearly three times greater in NH₄⁺ grown than from NO₃⁻ grown plants. The BNI-compounds released from roots composed of at least three active components—Type-I (stable to pH changes from 3.0 to 10), Type-II (temporarily loses its inhibitory effect at a pH higher than a threshold pH of 4.5 and the inhibitory effect is reestablished when the root exudate pH is adjusted to <4.5) and Type-III (inhibitory effect is irreversibly lost if the pH of the root exudate reaches 10.0 or above). A major portion of BNI-compounds released in the presence of NH₄⁺ is of Type-I. In the absence of NH₄⁺, mostly Type-II and Type-III BNI-compounds were released. The BNI-compounds inhibited the function of Nitrosomonas europaea through the blocking of both ammonia monooxygenase and hydroxylamino oxidoreductase pathways. These results indicate that the release of BNI-compounds from B. humidicola roots is a regulated function and that presence of NH₄⁺ in the root environment is necessary for the sustained synthesis and release of BNI.     

Can biological nitrification inhibition (BNI) genes from perennial Leymus racemosus (Triticeae) combat nitrification in wheat farming?

Using a recombinant luminescent Nitrosomonas europaea assay to quantify biological nitrification inhibition (BNI), we found that a wild relative of wheat (Leymus racemosus (Lam.) Tzvelev) had a high BNI capacity and releases about 20 times more BNI compounds (about 30 ATU g⁻¹ root dry weight 24 h⁻¹) than Triticum aestivum L. (cultivated wheat). The root exudate from cultivated wheat has no inhibitory effect on nitrification when applied to soil; however, the root exudate from L. racemous suppressed formation and kept more than 90% of the soil’s inorganic-N in the -form for 60 days. The high-BNI capacity of L. racemosus is mostly associated with chromosome Lr#n. Two other chromosomes Lr#J, and Lr#I also have an influence on BNI production. Tolerance of L. racemosus to is controlled by chromosome 7Lr#1-1. Sustained release of BNI compounds occurred only in the presence of in the root environment. Given the level of BNI production expressed in DALr#n and assuming normal plant growth, we estimated that nearly 87,500,000 ATU of BNI activity ha⁻¹ day⁻¹ could be released in a field of vigorously growing wheat; this amounts to the equivalent of the inhibitory effect from the application of 52.5 g of the synthetic nitrification inhibitor nitrapyrin (one AT unit of BNI activity is equivalent to 0.6 μg of nitrapyrin). At this rate of BNI production it would take only 19 days for a BNI-enabled wheat crop to produce the inhibitory power of a standard commercial application of nitrapyrin, 1 kg ha⁻¹. The synthetic nitrification inhibitor, dicyandiamide, blocked specifically the AMO (ammonia monooxygenase) pathway, while the BNI from L. racemosus blocked the HAO (hydroxylamine oxidoreductase) pathway in Nitrosomonas. Here we report the first finding of high production of BNI in a wild relative of any cereal and its successful introduction and expression in cultivated wheat. These results demonstrate the potential for empowering the new generation of wheat cultivars with high-BNI capacity to control nitrification in wheat-production systems. Responsible Editor: Hans Lambers.     

Plant-based manipulation of nitrification in soil: a new approach to managing N loss?

Much work has gone into the management of nitrification through applications of chemicals known to inhibit enzyme function in nitrifiers with indifferent outcomes when tested in the field. Much less attention has been focused on the capacity of plants to modify nitrification in situ. Subbarao and coworkers in a series of neat and elegant studies have confirmed that a tropical grass species, Brachiaria humidicola, produces chemicals that inhibit nitrification in soil. Critical to the work was the use of a Nitrosomonas europaea strain (nitrifying bacteria) that had been specifically constructed to produce bioluminescence due to the expression of “luxAB’ genes during nitrification. This application led to the development of an assay that enabled the suppression of nitrification to be assessed directly. They produce evidence that the production of chemicals by Brachiaria humidicola roots, described as biological nitrification inhibitors (BNIs), is under plant control. However, the triggers or molecular controls for BNI production have yet to be ascertained. Examination of the capacity of major crops to produce BNIs, including wheat (Triticum aestivum), barley (Hordeum vulgare), rice (Oryza sativa) and maize (Zea mays) indicate that these do not have this capacity. Work is needed on wild relatives of these crops and the major temperate grass species such as Lolium perenne to determine whether these have the capacity to produce BNIs with an aim to introduce this capacity into domesticated lines. The work of Subbarao et al. highlights how molecular biology can be used to introduce traits into micro-organisms responsible for key soil N transformations in a way that facilitates analysis of the interaction between plants and the soil environment so crucial to their growth and survival.     

Free fatty acids from the pasture grass Brachiaria humidicola and one of their methyl esters as inhibitors of nitrification

The tropical pasture grass, Brachiaria humidicola (Rendle) Schweick, produces nitrification inhibitory compounds (termed biological nitrification inhibitors or BNIs) in its shoot and root tissues and releases BNIs from its roots. In the present study, two BNI compounds were isolated and identified from the shoot tissue of B. humidicola using activity-guided fractionation. The recombinant Nitrosomonas europaea containing luxAB genes derived from the bioluminescent marine gram-negative bacterium Vibrio harveyi, were used to determine BNI activity. The BNI compounds in the shoot tissue were identified as linoleic acid (LA) and linolenic acid (LN) using authentic-chemicals obtained from ©Sigma (ED₈₀ 16.0 μg ml^?1 for both LA and LN) for verification. None of the other tested free fatty acids namely stearic acid, oleic acid, arachidonic acid, and cis-vaccenic acid showed any inhibitory effect on nitrification. Among the fatty acid methyl esters (FAME) evaluated [methyl oleate, methyl linoleate (LA-ME) and methyl linoleneate (LN-ME)], only LA-ME showed an inhibitory effect (ED₈₀ 8.0 μg ml^?1). The inhibitory effect of LA, LN and LA-ME in the soil was stable for 120 days at 20°C. Soil treated with LA, LN and LA-ME showed a very low accumulation of NO₃ ^? and the maintenance of soil inorganic N in the NH₄ ⁺ form. The inhibitory effect of LA-ME on soil nitrification was greater than that of LA or LN. In addition to BNI activity, both LA and LA-ME showed a suppressive effect on urea hydrolysis in soil. Both LA and LN blocked the AMO (ammonia monooxygenase) and HAO (hydroxylamino oxidoreductase) enzymatic pathways in Nitrosomonas. Since LA and LN can be produced from vegetable oils such as soybean, flax or sunflower, they have the potential for use as nitrification inhibitors in production agriculture.     

Biological nitrification inhibition (BNI) activity in sorghum and its characterization

Aims

The ability to suppress soil nitrification through the release of nitrification inhibitors from plant roots is termed ‘biological nitrification inhibition’ (BNI). Here, we aimed at the quantification and characterization of the BNI function in sorghum that includes inhibitor production, their chemical identity, functionality and factors regulating their release.

Methods

Sorghum was grown in solution culture and root exudate was collected using aerated NH₄Cl solutions. A bioluminescence assay using recombinant Nitrosomonas europaea was employed to determine the BNI activity. Activity-guided chromatographic fractionation was used to isolate biological nitrification inhibitors (BNIs). The chemical structure was analyzed using NMR and mass spectrometry; pH-stat systems were deployed to analyze the role of rhizosphere pH on BNIs release.

Results

Sorghum roots released two categories of BNIs: hydrophilic- and hydrophobic-BNIs. The release rates for hydrophilic- and hydrophobic- BNIs ranged from 10 to 25 ATU?g^?1 root dwt. d^?1. Addition of hydrophilic BNIs (10 ATU?g^?1 soil) significantly inhibited soil nitrification (40 % inhibition) during a 30-d incubation test. Two BNI compounds isolated are: sakuranetin (ED₈₀ 0.6 μM; isolated from hydrophilic-BNIs fraction) and sorgoleone (ED₈₀ 13.0 μM; isolated from hydrophobic-BNIs fraction), which inhibited Nitrosomonas by blocking AMO and HAO enzymatic pathways. The BNIs release required the presence of NH ₄ ⁺ in the root environment and the stimulatory effect of NH ₄ ⁺ lasted 24 h. Unlike the hydrophobic-BNIs, the release of hydrophilic-BNIs declined at a rhizosphere pH >5.0; nearly 80 % of hydrophilic-BNI release was suppressed at pH ≥7.0. The released hydrophilic-BNIs were functionally stable within a pH range of 5.0 to 9.0. Sakuranetin showed a stronger inhibitory activity (ED₅₀ 0.2 μM) than methyl 3-(4-hydroxyphenyl) propionate (MHPP) (ED₅₀ 100 μM) (isolated from hydrophilic-BNIs fraction) in the in vitro culture-bioassay, but the activity was non-functional and ineffective in the soil-assay.

Conclusions

There is an urgent need to identify sorghum genetic stocks with high potential to release functional-BNIs for suppressing nitrification and to improve nitrogen use efficiency in sorghum-based production systems.     

A colourimetric microplate assay for simple,high throughput assessment of synthetic and biological nitrification inhibitors

Aim

A simple, rapid, colourimetric method for screening biological nitrification inhibitors in plants is presented.

Methods

Our approach combines the use of the Griess assay to track the rate of nitrite (NO₂ ^?) production by pure cultures of ammonia oxidising bacteria in the presence and absence of nitrification inhibitors with a simple method for collecting root exudates from plants. NO₂ ^? formation was tracked colourimetically on a microplate reader over 9 h of incubation. The advantage of this method is that it provides a simple, high throughput means of measuring biological nitrification inhibition in root exudates, using wild-type bacterial cultures.

Results

NO₂ ^? formation rates and inhibition levels measured using the high through-put method were highly correlated with those measured by tracking NO₂ ^? formation using a segmented flow analyser. The method was able to quantify inhibition of Nitrosomonas europaea by the synthetic nitrification inhibitors allythiourea (AT), dicyandiamide (DCD) and 3,4,-dimethylpyrazole phosphate (DMPP) with IC50 values similar to those reported in the literature. The method detected biological nitrification inhibition (BNI) in root exudates from Brachiaria humidicola and the lack of BNI in root exudates from wheat cv. Janz with minimal alteration of the exudates prior to testing. The results also showed that the more common soil ammonia oxidising bacterium (AOB), Nitrosospira multiformis, was much less sensitive to AT and DCD than N. europaea but had similar sensitivity to DMPP.

Conclusions

This method provides a potentially useful way of screening large numbers of root exudate samples allowing for phenotyping of the BNI trait in crop and pasture populations which will be required for the trait to be introduced into commercial varieties.

     

Suppression of nitrification and nitrous oxide emission by the tropical grass Brachiaria humidicola

Nitrification by soil nitrifiers may result in substantial losses of applied nitrogen through NO₃ ^– leaching and N₂O emission. The biological inhibition of nitrification by crop plants or pasture species is not well known. This study was conducted to evaluate the ability of three pasture species, Brachiaria humidicola, B. decumbens and Melinis minutiflora to inhibit nitrification. Plants were grown in a growth chamber for sixty days, fertilized with (NH₄)₂SO₄. After harvesting, the soil was incubated with (NH₄)₂SO₄ for 24 days. Ammonium oxidizing bacteria (AOB), NH₄-N levels, and N₂O emission were monitored at 4 d intervals. Among the species studied, B. humidicola inhibited nitrification and maintained NH₄-N in soil to a much greater extent than the other two species. This nitrification inhibition lasted for 12 days after initiation of soil incubation study (i.e. from 60 DAS when the plants were harvested). The AOB populations and N₂O emission from the soil were significantly lower in the soils where B. humidicola has been grown compared to the other two species. Root exudates and soil extracts of B. humidicola suppressed AOB populations, whereas those of B. decumbens and M. minutiflora did not. The results are in consistence with the hypothesis that B. humidicola suppressed nitrification and N₂O emissions through an inhibitory effect on the AOB population.     

Seasonal variation in the nitrogenase activity of aPanicum maximum var.trichoglume pasture and identification of associated bacteria

Summary The influence of seasonal variation on nitrogenase (N₂-ase) activity of undisturbed soil-plant cores ofPanicum maximum var.trichoglume was measured using the C₂H₂ reduction assay. The largest N₂-ase activity in the field, 14.7 g N ha⁻¹ day⁻¹, occurred in spring when soil moisture was high, soil temperature was low and nitrogenous fertiliser influence was at a minimum. The potential N₂-ase activity of the cores, measured under controlled conditions, reached a maximum of 27.2 g N ha⁻¹ day⁻¹ and averaged 26.3 g N ha⁻¹ day⁻¹ over the 14 month sampling period. N₂-ase activity was positively correlated (P=0.05) with field soil moisture and negatively correlated with field soil temperature (r=0.59 and −0.78 respectively). Multiple regression showed that 69% of the variation of N₂-ase activity in the field was associated with the combined effects of soil moisture and soil temperature. Nitrogen fixing bacteria were isolated from the roots ofP. maximum and based upon morphology, biochemical tests and fluorescent antibody reaction, were found to be closely related toAzospirillum lipoferum.     

Biofortification of rice grain with zinc through zinc fertilization in different countries

Aims and background

The ability to suppress soil nitrification through the release of nitrification inhibitors from plant roots is termed ‘biological nitrification inhibition’ (BNI). Earlier, we reported that sorghum roots release higher BNI-activity when grown with NH ₄ ⁺ , but not with NO ₃ ^- as N source. Also for BNI release, rhizosphere pH of <5.0 is needed; beyond this, a negative effect on BNI release was observed with nearly 80% loss of BNI activity at pH >7.0. This study is aimed at understanding the inter-functional relationships associated with NH ₄ ⁺ uptake, rhizosphere-pH and plasma membrane H⁺-ATPase (PM H⁺-ATPase) activity in regulating the release of BNIs (biological nitrification inhibitors) from sorghum roots.

Methods

Sorghum was grown hydroponically and root exudates were collected from intact plants using a pH-stat system to separate the secondary acidification effects by NH ₄ ⁺ uptake on BNIs release. A recombinant luminescent Nitrosomonas europaea bioassay was used to determine BNI-activity. Root plasma membrane was isolated using a two-phase partitioning system. Hydrolytic H⁺-ATPase activity was determined. Split-root system setup was deployed to understand the localized responses to NH ₄ ⁺ , H⁺-ATPase-stimulator (fusicoccin) or H⁺-ATPase-inhibitor (vanadates) on BNI release by sorghum.

Results

Presence of NH ₄ ⁺ in the rhizosphere stimulated the expression of H⁺-ATPase activity and enhanced the release of BNIs from sorghum roots. Fusicoccin, which stimulates H⁺-ATPase activity, also stimulated BNIs release in the absence of NH ₄ ⁺ ; vanadate, which suppresses H⁺-ATPase activity, also suppressed the release of BNIs. NH ₄ ⁺ levels (in rhizosphere) positively influenced BNIs release and root H⁺-ATPase activity in the concentration range of 0-1.0 mM, indicating a close relationship between BNI release and root H⁺-ATPase activity with a possible involvement of carrier-mediated transport for the release of BNIs in sorghum.

Conclusion

Our results suggest that NH ₄ ⁺ uptake, PM H⁺-ATPase activity, and rhizosphere acidification are functionally inter-connected with BNI release in sorghum. Such knowledge is critical to gain insights into why BNI function is more effective in light-textured, mildly acidic soils compared to other soil types.     

Role of nitrite reductase in the ammonia-oxidizing pathway of <Emphasis Type="Italic">Nitrosomonas europaea</Emphasis>

Metabolism of ammonia (NH₃) and hydroxylamine (NH₂OH) by wild-type and a nitrite reductase (nirK) deficient mutant of Nitrosomonas europaea was investigated to clarify the role of NirK in the NH₃ oxidation pathway. NirK-deficient N. europaea grew more slowly, consumed less NH₃, had a lower rate of nitrite (NO₂ ⁻) production, and a significantly higher rate of nitrous oxide (N₂O) production than the wild-type when incubated with NH₃ under high O₂ tension. In incubations with NH₃ under low O₂ tension, NirK-deficient N. europaea grew more slowly, but had only modest differences in NH₃ oxidation and product formation rates relative to the wild-type. In contrast, the nirK mutant oxidized NH₂OH to NO₂ ⁻ at consistently slower rates than the wild-type, especially under low O₂ tension, and lost a significant pool of NH₂OH–N to products other than NO₂ ⁻ and N₂O. The rate of N₂O production by the nirK mutant was ca. three times higher than the wild-type during hydrazine-dependent NO₂ ⁻ reduction under both high and low O₂ tension. Together, the results indicate that NirK activity supports growth of N. europaea by supporting the oxidation of NH₃ to NO₂ ⁻ via NH₂OH, and stimulation of hydrazine-dependent NO₂ ⁻ reduction by NirK-deficient N. europaea indicated the presence of an alternative, enzymatic pathway for N₂O production.     

Low Nitrification Rates in Acid Scots Pine Forest Soils Are Due to pH-Related Factors

In a previous study, ammonia-oxidizing bacteria (AOB)-like sequences were detected in the fragmentation layer of acid Scots pine (Pinus sylvestris L.) forest soils (pH 2.9–3.4) with high nitrification rates (>11.0 μg g⁻¹ dry soil week⁻¹), but were not detected in soils with low nitrification rates (<0.5 μg g⁻¹ dry soil week⁻¹). In the present study, we investigated whether this low nitrification rate has a biotic cause (complete absence of AOB) or an abiotic cause (unfavorable environmental conditions). Therefore, two soils strongly differing in net nitrification were compared: one soil with a low nitrification rate (location Schoorl) and another soil with a high nitrification rate (location Wekerom) were subjected to liming and/or ammonium amendment treatments. Nitrification was assessed by analysis of dynamics in NH₄ ⁺-N and NO₃ ⁻-N concentrations, whereas the presence and composition of AOB communities were assessed by polymerase chain reaction–denaturing gradient gel electrophoresis and sequencing of the ammonia monooxygenase (amoA) gene. Liming, rather than ammonium amendment, stimulated the growth of AOB and their nitrifying activity in Schoorl soil. The retrieved amoA sequences from limed (without and with N amendment) Schoorl and Wekerom soils exclusively belong to Nitrosospira cluster 2. Our study suggests that low nitrification rates in acidic Scots pine forest soils are due to pH-related factors. Nitrosospira cluster 2 detected in these soils is presumably a urease-positive cluster type of AOB.     

Soil O2 and CO2 effects on root respiration of cacti

Through use of a recently developed technique that can measure CO₂ exchange by individual attached roots, the influences of soil O₂ and CO₂ concentrations on root respiration were determined for two species of shallow-rooted cacti that typically occur in porous, well-drained soils. Although soil O₂ concentrations in the rooting zone in the field were indistinguishable from that in the ambient air (21% by volume), the CO₂ concentrations 10 cm below the soil surface averaged 540 μLL⁻¹ for the barrel cactusFerocactus acanthodes under dry conditions and 2400 μLL⁻¹ under wet conditions in a loamy sand. For the widely cultivated platyopuntiaOpuntia ficus-indica in a sandy clay loam, the CO₂ concentration at 10 cm averaged 1080 μLL⁻¹ under dry conditions and 4170 μLL⁻¹ under wet conditions. For both species, the respiration rate in the laboratory was zero at 0% O₂ and increased to its maximum value at 5% O₂ for rain roots (roots induced by watering) and 16% O₂ for established roots. Established roots ofO. ficus-indica were slightly more tolerant of elevated CO₂ than were those ofF. acanthodes, 5000 μLL⁻¹ inhibiting respiration by 35% and 46%, respectively. For both species, root respiration was reduced to zero at 20,000 μLL⁻¹ (2%) CO₂. In contrast to the reversible effects of 0% O₂, inhibition by 2% CO₂ was irreversible and led to the death of cortical cells in established roots in 6 h. Although the restriction of various cacti and other CAM plants to porous soils has generally been attributed to their requirement for high O₂ concentrations, the present results indicate that susceptibility of root respiration to elevated soil CO₂ concentrations may be more important.     

A paradigm shift towards low-nitrifying production systems: the role of biological nitrification inhibition (BNI)

Background

Agriculture is the single largest geo-engineering initiative that humans have initiated on planet Earth, largely through the introduction of unprecedented amounts of reactive nitrogen (N) into ecosystems. A major portion of this reactive N applied as fertilizer leaks into the environment in massive amounts, with cascading negative effects on ecosystem health and function. Natural ecosystems utilize many of the multiple pathways in the N cycle to regulate N flow. In contrast, the massive amounts of N currently applied to agricultural systems cycle primarily through the nitrification pathway, a single inefficient route that channels much of this reactive N into the environment. This is largely due to the rapid nitrifying soil environment of present-day agricultural systems.

Scope

In this Viewpoint paper, the importance of regulating nitrification as a strategy to minimize N leakage and to improve N-use efficiency (NUE) in agricultural systems is highlighted. The ability to suppress soil nitrification by the release of nitrification inhibitors from plant roots is termed ‘biological nitrification inhibition’ (BNI), an active plant-mediated natural function that can limit the amount of N cycling via the nitrification pathway. The development of a bioassay using luminescent Nitrosomonas to quantify nitrification inhibitory activity from roots has facilitated the characterization of BNI function. Release of BNIs from roots is a tightly regulated physiological process, with extensive genetic variability found in selected crops and pasture grasses. Here, the current status of understanding of the BNI function is reviewed using Brachiaria forage grasses, wheat and sorghum to illustrate how BNI function can be utilized for achieving low-nitrifying agricultural systems. A fundamental shift towards ammonium (NH₄⁺)-dominated agricultural systems could be achieved by using crops and pastures with high BNI capacities. When viewed from an agricultural and environmental perspective, the BNI function in plants could potentially have a large influence on biogeochemical cycling and closure of the N loop in crop–livestock systems.     

Hydrolysis of organic phosphates by corn and soybean roots

Because of the importance of organic phosphates as sources of P for plants, this work was performed to study the hydrolysis of nine organic phosphates by sterile, intact corn (Zea mays L.) and soybean (Glycine max L.) roots. Results showed that the rates of hydrolysis ofp-nitrophenyl phosphate (PNP) in buffered solutions by roots of three varieties of corn and three varieties of soybean ranged from 13 to 22 μmol PO₄−P g⁻¹ root h⁻¹ and from 2.1 to 2.2 μmol PO₄−P 0.1 g⁻¹ root h⁻¹, respectively. The average rate of hydrolysis of PNP in nonbuffered solutions was 2- to 3-fold lower for corn roots and 6- to 10-fold lower for soybean roots as compared with those obtained with buffered solutions. The orthophosphate released from hydrolysis of organic P compounds in buffered solutions during a 48-h incubation of corn roots showed that the maximum rate of hydrolysis of PNP was 4 to 6 times greater than the commonly used substrates: α- and β-glycerophosphates, phenolphthalein diphosphate, and glucose-6-phosphate. The rates of hydrolysis of glucose-6-phosphate and glucose-1-phosphate were similar and about 6- to 12-fold lower than that of PNP. Phosphoethanolamine and phosphocholine were hydrolyzed slightly, ando-carboxyphenyl phosphate was not hydrolyzed. The rates of hydrolysis of organic P compounds in nonbuffered solutions by corn and soybean roots were 1 to 3 and 1 to 10 times lower than those in buffered solutions, respectively. The trends in rates of hydrolysis by soybean roots of buffered organic P substrates were similar to those observed with corn roots, with the exception of glucose-1-phosphate and phosphoethanolamine.     

Natural 15N abundance of soil N pools and N2O reflect the nitrogen dynamics of forest soils

Natural ¹⁵N abundance measurements of ecosystem nitrogen (N) pools and ¹⁵N pool dilution assays of gross N transformation rates were applied to investigate the potential of δ¹⁵N signatures of soil N pools to reflect the dynamics in the forest soil N cycle. Intact soil cores were collected from pure spruce (Picea abies (L.) Karst.) and mixed spruce-beech (Fagus sylvatica L.) stands on stagnic gleysol in Austria. Soil δ¹⁵N values of both forest sites increased with depth to 50 cm, but then decreased below this zone. δ¹⁵N values of microbial biomass (mixed stand: 4.7 ± 0.8‰, spruce stand: 5.9 ± 0.9‰) and of dissolved organic N (DON; mixed stand: 5.3 ± 1.7‰, spruce stand: 2.6 ± 3.3‰) were not significantly different; these pools were most enriched in ¹⁵N of all soil N pools. Denitrification represented the main N₂O-producing process in the mixed forest stand as we detected a significant ¹⁵N enrichment of its substrate NO₃⁻ (3.6 ± 4.5‰) compared to NH₄⁺ (−4.6 ± 2.6‰) and its product N₂O (−11.8 ± 3.2‰). In a ¹⁵N-labelling experiment in the spruce stand, nitrification contributed more to N₂O production than denitrification. Moreover, in natural abundance measurements the NH₄⁺ pool was slightly ¹⁵N-enriched (−0.4 ± 2.0 ‰) compared to NO₃⁻ (−3.0 ± 0.6 ‰) and N₂O (−2.1 ± 1.1 ‰) in the spruce stand, indicating nitrification and denitrification operated in parallel to produce N₂O. The more positive δ¹⁵N values of N₂O in the spruce stand than in the mixed stand point to extensive microbial N₂O reduction in the spruce stand. Combining natural ¹⁵N abundance and ¹⁵N tracer experiments provided a more complete picture of soil N dynamics than possible with either measurement done separately.     

Organic matter quality and management effects on enrichment of soil organic matter fractions in contrasting soils in Zimbabwe

Spatial variability of soil total nitrogen (N), available N (KCl extractable NH₄⁺ and NO₃⁻), and spatial patterns of N mineralization and nitrification at a stand scale were characterized with geostatistical and univariate analysis. Two extensive soil spatial samplings were conducted in an evergreen broadleaf forest in Sichuan province, southwestern China in June and August 2000. In a study area of 90 × 105 m², three soil samples were collected from each 5 × 5 m² plot (n = 378) in June and August, and were analyzed for total N and available N contents. Net N mineralization and nitrification were measured by in situ core incubation and the rates were estimated based on the difference of NH₄⁺ and NO₃⁻ contents between the two sampling dates. Total N, NH₄⁺, and NO₃⁻ were all spatially structured with different semivariogram ranges (from high to low: NH₄⁺, NO₃⁻, and total N). The semivariograms of mineralization and nitrification were not as spatially structured as available N. NH₄⁺ was the dominant soil inorganic N form in the system. Both NH₄⁺ and NO₃⁻ affected spatial patterns of soil available N, but their relative importance switched in August, probably due to high nitrification as indicated by greatly increased soil NO₃⁻ content. High spatial auto-correlations (>0.7) were found between available N and NH4⁺, available N and NO₃⁻ on both sampling dates, as well as total N measurements between both sampling dates. Although significant, the spatial auto-correlation between NH₄⁺ and NO₃⁻ were generally low. Topography had significant but low correlations with mineralization (r = −0.16) and nitrification (r = −0.14), while soil moisture did not. The large nugget values of the calculated semivariograms and high-semivariance values, particularly for mineralization and nitrification, indicate that some fine scale (<5 m) variability may lie below the threshold for detection in this study.     

Effects of the invasive plant Mikania micrantha H.B.K. on soil nitrogen availability through allelopathy in South China

Allelopathy has been regarded as a mechanism for successful exotic plant invasion. However, it is not clear if and what effects of allelopathic substances may exert on soil nutrient. The exotic plant Mikania micrantha H.B.K. (M. micrantha) has invaded many forests in south China, and recent studies have suggested it has allelopathic potential for other plants and soil microbial community. Thus, we hypothesized that M. micrantha could influence soil nutrients and N transformation through allelopathy. We measured total C and N, NO₃ ⁻, NH₄ ⁺ and pH of the soil beneath M. micrantha and the adjacent open soil, and then measured the above soil properties after treating soil with 3 concentrations of aqueous extracts of M. micrantha (T₁: 0.005 g ml⁻¹; T₂: 0.025 g ml⁻¹; T₃: 0.100 g ml⁻¹). In addition, a bioassay was conducted to determine the allelopathic potential of the soil beneath M. micrantha. The results showed that M. micrantha significantly affected soil nutrients and N transformation. Soil beneath M. micrantha had inhibitory effects on seed germination and seedling growth of test plant, and had significantly higher C, N, ammonia, net nitrification rate than those of open soil. The plant extracts decreased soil pH, and T₁ decreased it the most, and it increased soil C and N, and T₁ represented the greatest increase in both C and N. The extracts also increased both NO₃ ⁻ and NH₄ ⁺ in soil, whereas no significant difference existed among the 3 extract treatments. Compared to the water control, the soil net mineralization rate was higher under T₁, while lower under T₂ and T₃. However, the extracts increased the soil nitrification rates under all the treatments (T₁, T₂ and T₃). Our results suggest that the water soluble allelochemicals of M. micrantha improve soil nutrient availability, through which the invasive plant M. micrantha may successfully invade and establish in new habitats.     

Growth and physiological activity in <Emphasis Type="Italic">Larix kaempferi</Emphasis> seedlings inoculated with ectomycorrhizae as affected by soil acidification

To evaluate the effect of ectomycorrhizal colonization on growth and physiological activity of Larix kaempferi seedlings grown under soil acidification, we grew L. kaempferi seedlings with three types of ectomycorrhizae for 180 days in acidified brown forest soil derived from granite. The soil had been treated with an acid solution (0 (control), 10, 30, 60, and 90 mmol H⁺ kg⁻¹). The water-soluble concentrations of Ca, Mg, K, Al, and Mn increased with increasing amounts of H⁺ added to the soil. Ectomycorrhizal development significantly increased in soil treated with 10 and 30 mmol H⁺ kg⁻¹ but was significantly reduced in soil treated with 60 and 90 mmol H⁺ kg⁻¹. The concentrations of Al and Mn in needles or roots increased with increasing H⁺ added to the soil. The total N in seedlings significantly increased with increasing H⁺ in soil and colonization with ectomycorrhiza. The maximum net photosynthetic rate at light and CO₂ saturation (P _max) was greater in soil treated with 10 mmol H⁺ kg⁻¹ than in controls, and was less is soils treated with greater than with 30 mmol H⁺ kg⁻¹, especially with 60 and 90 mmol H⁺ kg⁻¹. However, colonization with ectomycorrhiza significantly reduced the concentration of Al and Mn in needles or roots and increased the values of P _max and total dry mass (TDM). The relative TDM of L. kaempferi seedlings was approximately 40% at a (BC, base cation)/Al ratio of 1.0. However, ectomycorrhizal seedlings had a 100–120% greater TDM at a BC/Al ratio of 1.0 than non-ectomycorrhizal seedlings, even though the acid treatment reduced their overall growth.     

Phosphatase activity in corn and soybean roots: Conditions for assay and effects of metals

Studies with sterile root materials showed that the optimum pH values of phosphatase activity in three varieties of each of corn (Zea mays L.) and soybean (Glycine max. L.) were 4 and 5, respectively. The activity on either side of the optimum pH fell sharply, and there was no activity at pH 9. Thus, these roots contain acid but no alkaline phosphatase activity. Acid phosphatase activity was not uniformly distributed in roots and root hairs. Studies with 20 metals showed that their effectiveness in inhibiting acid phosphatase activity of roots varied with the type of plant used. When the metals were compared at 250 μM (1.25 μmole. 5 mg⁻¹ of homogenized roots), the inhibition of acid phosphatase of corn and soybean roots showed that Ag(I), Fe(III), Se(IV), V(IV), As(V) and Mo(VI) were the most effective inhibitors of this enzyme in corn roots, with percentage inhibition ≥30%. In addition to these metals, Sn(II), Hg(II), and W(VI) inhibited acid phosphatase in soybean roots by >30%. Other metals and one non-metallic element that inhibited acid phosphatase activity in corn and soybean roots were: Cu(I), Cu(II), Cd(II), Ni(II), Fe(II), Pb(II), Ba(II), Co(II), Mn(II), Zn(II), B(III), As(III), Cr(III), and Al(III); their degrees of effectiveness varied with type of roots used. Generally, the inhibitory effect of the metals was much less when their concentration was decreased by 10-fold. In addition to the effect of these elements, phosphate ion inhibited acid phosphatase activity of corn and soybean roots. Related anions such as NO ₂ ⁻ , NO ₃ ⁻ , Cl⁻, and SO ₄ ²⁻ were not inhibitory.