The evidence for in support of allelopathic inhibition of the nitrogen cycle, in particular nitrification, by monoterpenes is reviewed. The suggested processes for the determination of an allelopathic mechanism is outlined. The nitrogen cycle is briefly reviewed, as is the early evidence in support of allelopathic inhibition of the nitrogen cycle. There is supportive evidence for allelopathic inhibition of the nitrogen cycle by monoterpenes from both laboratory bioassays and field experiments. A mechanism for the non-competitive inhibition of ammonia oxidation (the first step in nitrification) has also been proposed and supported. However, much remains to be done in determining the ecological significance of this interaction. The mechanism of this potential allelopathic inhibition as it occurs (or does not occur) in nature has yet to be elucidated.
Allelopathy was first defined in 1937 by H. Molisch who originated the term to refer to inhibitory and stimulatory interactions between all types of plants including microorganisms. To separate this interaction from resource competition, recent work defines allelopathy as the "direct or indirect harmful or beneficial effects of one plant on another through the production and release of chemical compounds" (Rice 1984). Using this definition, allelopathy refers to both autotoxic and heterotoxic effects (Miller 1996). Allelopathic interactions therefore represent chemical competition between plants (Harborne 1993).
Allelopathic chemicals are generally considered to be secondary plant products which are released directly from living plants into the environment via leaching, root exudation, volatilization, or the decomposition of plant residues (Miller 1996, Rice 1984). Most chemicals that have been identified in allelopathic interactions have been identified as either terpenes or phenolic compounds (Harborne 1993).
The complexity of plant-soil systems does not lend itself to a clear cause and effect relationship. Proving the existence of allelopathic interactions has therefore proven to be difficult at best. Much criticism of allelopathy stems from the wealth of circumstantial evidence and anecdotal information in ecological and agricultural literature and the lack of experimental verification of allelopathic interactions (Fischer et al. 1994). As recently as 1990, the assertion was made that no published field study had yet demonstrated direct interference by allelopathy in soil while excluding competitive factors such as resource availability or natural enemies (Weidenhamer 1996). The identification of allelopathic compounds (or their derivatives) and documentation of these chemicals entering the environment and surviving to travel to target plants, is difficult (Seigler 1996). Some of the processes that may confound allelopathy include: resource competition (sunlight, nutrients, or water), degradation of chemicals once they leave the plant, the maze of sources and sinks in the plant-soil system, the concentration and flux rates of a suspected allelochemical, and environmental stresses (Weidenhamer 1996, Einhelig 1996, Seigler 1996).
In order to overcome these obstacles, a series of criteria similar to Koch's postulates have been suggested: (i) the identification and quantification of an interference; (ii) isolation, identification, and synthesis of the compound causing the interference using bioassay driven isolation; (iii) simulation of the interaction using natural application rates; and (iv) the quantification of the amount of compound released into the environment and taken up by the target plant (Seigler 1996, Weidenhamer 1996).
Development of a laboratory bioassay should include testing mixtures of potential allelochemicals for synergistic activity, consideration of the age at which a plant begins to synthesize and release allelochemicals, the role of soil microbes, consideration of the fact that the introduction of allelochemicals cannot be a one-time inoculation, but must be a continual application in the exact dosage of the ecological interaction (commonly a chronic, low exposure (Weidenhamer 1996, Einhelig 1996, Inderjit and Dakshini 1995). It has also been suggested that the exact mechanism of inhibition be determined. This paper will attempt to apply the above suggestions and postulates to the work done on the inhibition of the nitrogen cycle by monoterpenes.
The amount of available nitrogen (N) is often the limiting factor in terrestrial ecosystem production (Schlesinger 1997). Soil inputs of N are from precipitation, dry deposition, and gaseous N fixation. N fixation is performed by free-living organisms and symbiotic plant and root-associated microorganisms. The supply of N in ecosystems is determined, to a large extent, by the rate at which organic-bound soil N is mineralized or converted from ammonia to ammonium. Ammonium is converted into nitrate via ammonium oxidation (nitrification). Microorganisms oxidize ammonium to provide energy for the fixation of carbon dioxide via the Calvin cycle (Bedard and Knowles 1989). The first step of nitrification involves the oxidation of ammonia by the enzyme ammonia monooxygenase (AMO) to NH2OH, which is later converted to nitrite and then nitrate (Bedard and Knowles 1989). A portion of the N can be lost to the atmosphere as nitrous oxide during this process.
Both nitrate and ammonium are assimilated into plants and microorganisms. Nitrate can be used by microorganisms as a terminal electron acceptor, which results in the production of nitrous oxide or dinitrogen gas (denitrification) (White 1994). When plants and microorganisms once again become part of the organic matter pool, the N undergoes mineralization and continues the cycle.
Using the above definition of allelopathy, it is evident that allelopathy could affect all phases of the nitrogen cycle that involve plants or microorganisms (Rice 1984). Allelopathy could therefore affect biological N fixation, mineralization, and/or nitrification. It has been suggested that the differential fitness among certain forest species could be related to their N uptake abilities in the presence of a predominance of ammonium-N or nitrate-N (Lodhi and Killingbeck 1980, Killham 1990). When plants take up nitrate, they must use energy to convert it to ammonium before it can be used (Rice 1984). The conservation of energy due to the inhibition of nitrification is therefore one argument for inhibition of nitrification. Bigg and Daniel (1978) found that lodgepole pine and Engelmann spruce grew poorly when their only source of N was nitrate, but Douglas fir had much higher production with a nitrate source than an ammonium-N source. Krajia et al. (1973) also found ammonium to be the preferred source for lodgepole pine. A species with the ability to efficiently utilize ammonium and inhibit nitrification may be able to reduce interspecies competition by increasing the ammonium:nitrate ratio in the soil (Lodhi and Killingbeck 1980). This could result in the exclusion of species that require high nitrate concentrations, are inefficient ammonium users, or have a low tolerance for ammonium concentrations (Lodhi and Killingbeck 1980).
In 1974, E.L. Rice introduced a plethora of observational data supporting the inhibition of various stages of the nitrogen cycle. Evidence of allelopathic effects on biological N2 fixation, mineralization, nitrification, (Rice 1984, 1992) and micorrhizae (Rice 1974, 1984, Nilsson et al. 1992, Boufalis and Pellissier 1994) has been presented. It is generally considered doubtful that allelopathy could significantly affect mineralization as it is carried out by a very diverse group of soil organisms (Rice 1984, 1992). In contrast, the process of nitrification is carried out primarily by two genera of bacteria: Nitrosomonas, which oxidize ammonium to nitrite, and Nitrobacter, which oxidizes nitrite to nitrate (Rice 1992). Most studies on the inhibition of the nitrogen cycle have therefore focused on the inhibition of nitrification.
Most work on the inhibition of nitrification traditionally focused on phenolics because they were known to be soluble in water (Rice 1974, 1984). In 1993, Weidenhamer et al. found that many monoterpenes are phytotoxic in concentrations below their solubilities in water. In other words, dilute unsaturated solutions of monterpenes could act as potent biological inhibitors (Weidenhamer et al. 1993). Monoterpenes have also been found in detectable levels in fjords and river water that border forested areas (Ward et al. 1997). Monoterpenes were introduced as viable allelochemicals. Much recent research has therefore focused on monoterpenes as inhibitors of nitrification and the nitrogen cycle.
Monoterpenes
Monoterpenes are major constituents of many pine resin oils. They are 10 carbon molecules formed by the polymerization of two isoprene units, and may be acyclic, monocyclic, bicyclic, or tricyclic. They exist in both hydrocarbon and oxygenated forms (Wood 1996). Potential sources of monoterpenes include leachate from leaf litter, canopy leaves, root exudation, and deposition of volatilized monoterpenes from litter, canopy and roots. Leachate from leaf litter is thought to be the largest source (Wood 1996). Fischer et al. (1994) found that most terpenoids accumulate in secretory structures and in plant surface wax. Monoterpenes have been found to have antimicrobial activities, disrupt electron transport, and uncouple oxidative phosphorylation (Ward et al. 1997). As allelopathic agents, they are also thought to inhibit plant growth and germination in several plant communities (Wood 1996).
Evidence for allelopathic inhibition of the N cycle by monoterpenes
In 1986, White began research on monoterpene inhibition of the N cycle. He hypothesized that vegetation in ponderosa pine forests inhibited nitrification by releasing volatile terpenoids that retarded the oxidation of ammonium. He found alpha-pinene, beta-pinene, and limonene in soils under ponderosa pines ranging from concentrations of 0.0135 ug/g to 0.0454 ug/g. White used "trapped vapor" experiments to assay the effects of vapors on nitrification in soils from burned plots. Soil from unburned plots placed in sealed jars containing soil from burned plots (no direct contact) reduced nitrification significantly (87%). A single water extraction of unburned forest floor reduced nitrification by 17%. Vapors from a mixture of five major monoterpenes found in the pine resin completely inhibited nitrification.
Bremner and McCarty (1988) provided some of the only real evidence published against the inhibition of nitrification by monoterpenes. In a lab bioassay, they applied terpenes to agricultural soils as vapors and directly, in concentrations of 10, 100, and 250 ug/g soil. They found that none of the terpenes had a significant effect on nitrification at any concentration. They did, however, find that organic carbon in the form of terpenoids was as effective as glucose at stimulating microbial immobilization of ammonium-N.
The major criticisms of this study were that the 200 ug/g of ammonium-N added to the bioassay soils were not sufficient to invalidate the hypothesis, and that aeration of the soils without reapplication could have resulted in the loss of monoterpenes and no apparent inhibition of nitrification (White 1991). It has also been suggested that monoterpenes exert control over nitrification only in N-limited/C-deficient soils (similar to agricultural soils) (White 1994).
In 1988 and 1990 White proposed a mechanism for the inhibition of nitrification based on the competitive inhibition of the enzyme ammonia monooxygenase (AMO) by the commercially available nitrification inhibitor, Nitrapyrin. He predicted that inhibition by monoterpenes was competitive and that monoterpenes should vary in degree of inhibition with differences in molecular structure.
In a lab bioassay, White (1991) applied concentrations (from 0.6 ul to 24 ul) ponderosa pine monoterpenes to ponderosa pine soils mixed with Douglas fir soils (to increase nitrification rates), ponderosa pine soils, soils from a desert grassland with no previous exposure to monoterpenes, and fertilized soil from desert grasslands. In the Douglas fir-ponderosa pine soils, 3.0 ul of limonene, alpha-phellandrene, and myrcene significantly inhibited nitrification and N mineralization. All additions of 30 ul immobilized the initial inorganic N. In the ponderosa pine soils, all 3 ul additions inhibited nitrification and mineralization without immobilization. Increasing concentrations resulted in increased inhibition. Immobilization was significant for all 24 ul additions. Limonene and a-pinene additions to the unfertilized grassland soils inhibited net mineralization and nitrification in direct proportion to the amount added. No additions resulted in immobilization. Limonene inhibited mineralization in all additions to the fertilized grassland soil and inhibited nitrification in the highest addition. No immobilization occurred. These results supported White's hypothesis that monoterpenes inhibited nitrification. Lower concentrations inhibited net mineralization and nitrification with no immobilization. High concentrations did appear to contribute to immobilization. The results also implied that the degree of inhibition of nitrification was inversely proportional to the inherent rate of nitrification in the soil. Low rates of nitrification resulted in higher rates of inhibition.
White (1991) also completed field experiments to determine the concentrations of monoterpenes in fresh litter, litter (L), duff (F-H), and mineral soil (0-10cm). Extracts were analyzed and found to contain a mixture of monoterpenes including myrcene, alpha-phellandrene, limonene, alpha-pinene, beta-pinene, and delta-carene. In ug/g, total monoterpene concentrations were as follows: fresh litter, 1360; L, 88.7 (L+F-H) to 172; F-H, 88.7 (L+F-H) to 41.8; and mineral soil, 0.03 to 0.23 (White 1991). Further analysis of these soils demonstrated an inverse relationship between net mineralization and monoterpene content of a horizon and immobilization of N in the L horizons (White 1990). White found little evidence in this research to support his hypothesis of a competitive inhibition of AMO (White 1991).
Courtney et al. (1991) also reported that relatively low concentrations of monoterpenes abundant in coastal redwood forests inhibit the production of nitrite by N. europaea. Other whole-cell experiments with a species of Nitrobacter found that redwood monoterpenes did not inhibit the conversion of nitrite to nitrate (White 1994). This supported the hypothesis of inhibition of nitrification via ammonia oxidation by AMO.
In 1993, Keener and Arp gathered data which suggested that, while some inhibitors of AMO bind directly to the active site for ammonia (competitive inhibition), with increasing molecule size, inhibitors might bind to a nonpolar site away from the active site (non-competitive inhibition). They proposed an active site model for AMO which consisted of an ammonia binding site to which competitive inhibitors bind and an alternate site to which non-competitive inhibitors bind. These results implied that nonpolar monoterpenes (which are large enough to be less effective at the active site for ammonia) could be non- competitive inhibitors of AMO.
White (1994) proposed that monoterpenes exert control over nitrification in N limited soils (with low nitrification rates) as supported by his 1991 study. DeLuca and Keeney (1993) demonstrated that agricultural soils are generally low in available organic substrate carbon and high in inorganic N. Bremner and McCarty's (1988) study could then possibly be explained by the differential effects of monoterpenes added to a carbon-limited/nitrogen-rich soil as compared to a carbon-rich/nitrogen-limited soil. He also incorporated Keener and Arp's results and stated that the mechanism of AMO inhibition is more likely to be non-competitive.
Wood (1996) performed a series of extensive experiments to determine the effects of monoterpenes from the leaf litter of Umbellularia californica. Wood first studied the rate of monoterpene loss from leaf litter in field and lab settings. He found that hydrocarbon monoterpenes remained in the litter layer longer than oxygenated monoterpenes and that both heat and water affected the loss of monoterpenes with the greatest loss during the rainy seasons. He suggested that monoterpene loss was also be influenced by secretory structure or other leaf characteristics such as permeability to water or lignin content. Soil nitrification potential in the field was not correlated with soil monoterpene yield, soil root density, or the mass of recent or old litter above soil cores. It was, however, strongly correlated with the loss of monoterpenes from U. californica litter (Wood 1996). Soil nitrification potential followed soil moisture in the dry and early wet season, but decreased nearly 50% well before the soil dried out. This decrease was strongly correlated with monoterpene loss from leaf litter (r-squared=0.92) (Wood 1996).
Wood's (1996) bioassay used soils collected from beneath U. californica trees. All hydrocarbon monoterpenes and oxygenated monoterpenes significantly reduced nitrification potentials relative to controls in additions of 0.24 mg/g soil and 0.59 mg/g soil, respectively. Hydrocarbon monoterpenes were therefore found to be twice as inhibitory as oxygenated monoterpenes. Wood found that mixtures of monoterpene hydrocarbons or oxygenated monoterpenes appeared to act additively in their inhibition of soil nitrification potential.
Most recently, Ward et al. (1997) have shown evidence that monoterpenes from coastal redwoods inhibit the enzyme (AMO) in the nitrifier Nitrosomonas europaea. Using the commercial nitrification inhibitor Nitrapyrin as a positive control, Ward et al. used the growth rates of N. europaea in batch culture to determine the degree of inhibition caused by each monoterpene. The five most abundant monoterpenes in the redwood needle resin were used: alpha-pinene, myrcene, limonene, sabinene, gamma-terpinene. Beta- pinene, a structurally similar monoterpene, was also used. Nitrapyrin was used in the recommended dosage of 1ug/ml. All other monoterpenes were added to the batch cultures in concentrations of 1 ug/ml and 10 ug/ml. Daily measurements of nitrate concentrations and growth rates were taken. To evaluate the mode of interaction between these chemicals and nitrification rates, short-term kinetic studies of the two most inhibitory monoterpenes were performed on whole cells. When compared to the controls, the growth rate of N. europaea was significantly inhibited by 1 ug/ml of Nitrapyrin, myrcene, and limonene (Ward et al. 1997). Alpha-pinene and gamma-terpinene significantly inhibited the growth rate at 10 ug/ml only. Sabinene and beta-pinene were not significantly inhibitory at either level (Ward et al. 1997).
Ward et al.'s (1997) kinetic studies and double reciprocal Lineweaver-Burk plots showed evidence for the non-competitive inhibition of AMO by limonene. However, the kinetic data for the inhibition of AMO by alpha-pinene did not clearly distinguish between competitive and non-competitive modes of inhibition. These differing degrees of inhibition may indicate that each compound's ability to inhibit AMO is specific to its molecular structure. Ward et al. asserts that this demonstrates that these compounds are specific inhibitors of N. europaea. This also implies that there may be more than one mode of inhibition. Ward et al. further speculate that the inhibition of limonene and alpha-pinene was most consistent with non-competitive inhibition model, but that a portion of the alpha-pinene molecule may compete with the ammonia substrate for a portion of its binding site.
In summary, evidence from both field and laboratory settings has been presented in support of the monoterpene inhibition of nitrification by the non-competitive inhibition of AMO. Some results also suggest that monoterpenes affect mineralization rates and, in C limited soils with high nitrification rates, act to immobilize nitrogen.
The first step of identification and quantification of this interaction has been documented by many researchers (Rice 1984, White 1991, 1994, Wood 1996). Monoterpenes have been isolated, identified, and shown to inhibit nitrification in laboratory bioassays (White 1986, 1991, Wood 1996, Courtney et al. 1991, Ward et al. 1997). The identification of a potential specific mechanism of inhibition lends further credence to this hypothesis (White 1994, Ward et al. 1997).
However, simulation of the interaction using natural and chronic application rates remains to be done. Steps have been taken to quantify the concentrations of monoterpenes available to inhibit nitrification (Wood 1996, White 1991). Exact measurements of what is actually released over time by monoterpene-producing plants and available to be taken up by the target microorganisms remains elusive.
Finally, creating a bioassay or field experiment that is able to eliminate all other interfering variables, is difficult at best. Density dependent "target- neighbor" studies to eliminate resource competition as suggested by Weidenhamer (1996), would be difficult to apply to this interaction. Many of the above laboratory bioassays show inhibition of nitrification at low monoterpene concentrations and demonstrate increasing inhibition with increasing monoterpene levels (White 1986, 1991, Wood 1996, Courtney et al. 1991, Ward et al. 1997). Field experiments are not as clear. The ability of an alleochemical to create a response in a target plant or microorganism greatly depends on its concentration in the environment, its persistence, and fate in the soil (metabolism or degradation). These factors have not been thoroughly identified or applied to a bioassay or field experiment. Additionally, synergistic or additive effects that occur in nature have not been taken into account by all researchers. Wood does show a correlation between low nitrification rates and monoterpene loss and White (1991) found an inverse relationship between net mineralization and monoterpene content of a horizon. However, this future work remains in the quantification and determination of the mechanism as it may or may not occur in nature.
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