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BULLETIN NO.: 13

ING Bulletins onRegional Assessment of Reactive Nitrogen

Series Editor: Y.P. ABROLAssociate Editor: SUSMITA CHATTERJEE

Nitrifi cation – IsIt a Strategic Point of Intervention for Limiting Nitrogen

Losses from Agricultural Systems? – The Concept of Biological Nitrifi cation

Inhibition (BNI) GV SUBBARAO, K NAKAHARA, T ISHIKAWA, M KISHII, N KUDO, IM RAO, M ISHITANI, KL SAHRAWAT, CT HASH, TS

GEORGE, W BERRY, JC LATA and O ITO

EDITOR: N. RAGHURAM

Published ByINDIAN NITROGEN GROUP (ING)

SOCIETY FOR CONSERVATION OF NATURE (SCON)

In Association WithSOUTH ASIAN NITROGEN CENTRE (SANC)

INTERNATIONAL NITROGEN INITIATIVE (INI)

NITRIFICATION – ISIT A STRATEGIC POINT OF INTERVENTION FOR LIMITING NITROGEN LOSSES FROM AGRICULTURAL SYSTEMS? – THE CONCEPT OF BIOLOGICAL NITRIFICATION INHIBITION (BNI)

First Published in 2010

© Society for Conservation of Nature (SCON)

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ISBN: 81-85992-26-6

Suggested Citation:

Subbarao G.V,. Nakahara K., Ishikawa T., Kishii M., Kudo N,. Rao I.M., Ishitani M., Sahrawat K.L., Hash C.T., George T.S., Berry W., Lata J.C. and Ito O (2010) Nitrifi cation – IsIt a Strategic Point of Intervention for Limiting Nitrogen Losses from Agricultural Systems? – The Concept of Biological Nitrifi cation Inhibition (BNI) , In ING Bulletins on Regional Assessment of Reactive Nitrogen, Bulletin No. 3, (Ed. N. Raghuram), SCON-ING, New Delhi, pp i-iv & 1-35.

For copies write to:

Professor Y.P. AbrolPresident, Society for Conservation of NatureG-4,CGIAR Block, NASC Complex, Dev Prakash Shastri Marg New Delhi-110012E-mail: [email protected]

Published and Printed by ING-SCON in collaboration with Angkor Publishers Pvt. Ltd., B-66, Sector 6, Noida-201301. E-mail: [email protected]; Mobile: 9910161199

CONTENTS

1. INTRODUCTION 2

1.1 Why do we Need to Control Nitrifi cation in Agricultural Systems? 3

1.2 Is Modern Agriculture Moving towards High-Nitrifying Systems? 4

1.3 Consequences of High-Nitrifying Systems on the 4 Global Environment

1.4 Options for Regulating Nitrifi cation in Agricultural Systems 5

1.5 Can we Learn from Natural Ecosystems to Control 6 Nitrifi cation in Agricultural Systems?

2. BIOLOGICAL NITRIFICATION INHIBITION (BNI) 8

2.1 The Concept of BNI Function in Plants 8

2.2 Methodology to Detect BNIs in Plant-Soil Systems 10

2.3 Variation in the BNI-Capacity of Major Crops and Forage Grasses 10

2.4 Regulatory Nature of the BNI Function 12

2.5 Stability of BNIs in the Soil Systems 13

2.6 Biological Molecules with BNI Potential and the 13 Mode of Inhibitory Action on Nitrosomonas

2.7 Evidence for the BNI Function under Field Conditions 17

2.8 Potential for Genetic Improvement of BNI Capacity in 20 Forage Grasses and Crops

3. CONCLUDING REMARKS 24

4. FUTURE OUTLOOK 26

Nitrifi cation – is it a Strategic Point of Intervention for Limiting Nitrogen Losses from Agricultural Systems? – The Concept of Biological Nitrifi cation Inhibition (BNI) -GV Subbarao1*, K Nakahara1, T Ishikawa1, M Kishii3, N Kudo1, IM Rao4, M Ishitani4, KL Sahrawat5, CT Hash5, TS George6, W Berry7, JC Lata8 and O Ito1

1 Japan International Research Center for Agricultural Sciences (JIRCAS), 1–1 Ohwashi, Ibaraki 305–8686, Japan

3 Yokohama City University, Kihara Biological Research Institute, 641–12 Maioka, Totsuka, Yokohama, Kanagawa 244–0813, Japan

4 Centro Internacional de Agricultura Tropical (CIAT), A.A. 6713, Cali, Colombia5 International Crops Research Institute for the Semi-Arid Tropics (ICRISAT), Patancheru,

Hyderabad 502 324, A.P., India6 Scottish Crop Research Institute (SCRI), Invergowrie, Dundee, DD2, 5DA, UK7 Department of Ecology and Evolutionary Biology, University of California, Los Angeles,

CA 90024, USA8 UPMC-Paris6, Laboratoire “Biogéochimie et écologie des milieux continentaux”

(UMR7618) BIOEMCO, 46 Rue d’Ulm, 75230 Paris Cedex 05, France

ABSTRACT

Human activity has had by far the single largest infl uence on the global N cycle by introducing massive amounts of reactive N into the ecosystems. A major portion of this reactive N applied as fertilizer, leaks into the environment and has a cascading effect on human health; ecosystem diversity, functions and services; and global warming with negative impacts on the environment. Natural ecosystems use multiple-pathways to regulate the fl ow of N in the N-cycle. In contrast, the massive amounts of N applied to the agricultural systems primarily goes through a single pathway, i.e. nitrifi cation, an ineffi cient pathway that allows reactive N to leak out of the agricultural systems. The present agricultural systems do not channel the reactive N through alternate pathways of the N cycle; this is largely due to uncontrolled soil nitrifi er activity creating a rapid nitrifying soil environments. Biological nitrifi cation inhibition (BNI) is a plant function, where nitrifi cation inhibitors are released from plant roots to suppress nitrifi cation in the soil thereby forcing N into other pathways. This bulletin illustrates the existence, detection, occurrence, physiological regulation of BNI function, and the feasibility for genetic exploitation of BNI-capacity as a trait using conventional breeding approaches. Further research should focus on developing practical genetic tools to facilitate development of the next-generation fi eld crops and pastures with a built-in genetic BNI-capacity for managing N effectively in agricultural production systems.

Key words: Genetic strategies, Global warming, Greenhouse gas emissions, Nitrification control, Nitrogen pollution, Nitrogen use effi ciency.

*Corresponding Author’s E-mail: [email protected]

2 Subbarao et al.

1. INTRODUCTIONOrganic nitrogen mineralization, nitrifi cation and denitrifi cation are important components of the N cycle in soil (Figure 1). Plants have the ability to utilize various forms of N – organic-N, NH4

+-N and NO3–-N (Salsac et al., 1987; Northup

et al., 1995; Nasholm et al., 1998; Kielland, 2001; Yamagata et al., 2001; Nishizawa and Mori, 2001), although NH4

+-N and NO3–-N are the primary N forms utilized

by fi eld crops (Haynes and Goh, 1978). In agricultural systems, nitrifi cation is the dominant pathway of N fl ow; this is refl ected in typical agricultural systems (i.e. neutral upland aerobic soils) where NO3

– accounts for >95% of the total N uptake (Figure 1). This makes the N cycle prone to leaks of reactive N to the environment, making agricultural systems the worst N polluter of the environment (Galloway et al., 2008; Schlesinger, 2009).

The biological oxidation of ammonium to nitrate via nitrite is termed “nitrifi cation”, and is carried out primarily by two groups of chemo-lithotrophic bacteria (Nitrosomonas sp. and Nitrobacter spp), ubiquitous components of the soil microbial population (Norton et al., 2002). In addition, the archaea group of soil bacteria is believed to be capable of nitrifi cation as they possess the AMO gene as does the Nitrosomonas spp. The presence of archaea has been reported in most soils and appears to be widespread; however their relative contribution to the soil nitrifi cation is unknown as most archaea cannot be cultured at present (Leninger et al., 2006). Nitrifi cation and denitrifi cation are the components of

Figure 1. Nitrogen cycle in typical agricultural systems (i.e. neutral upland aerobic soils) that is currently dominated by nitrifi cation pathway where >95% of the N fl ows through and NO3

– remains the dominant inorganic form absorbed and assimilated.

Biological Nitrifi cation Inhibition 3

the N cycle critical to the removal of N from organic waste systems (i.e. sewage treatment). However, in agricultural systems, rapid and unchecked nitrifi cation results in ineffi cient N use by crops, leading to N leakage and environmental pollution (Clark, 1962; Subbarao et al., 2006a, 2009a; Schlesinger, 2009). Most plants have the ability to utilize NH4

+ or NO3– as their N source, thus plants need

not depend on NO3– as their sole N source. Minimizing the role of nitrifi cation in

agricultural systems should not limit the availability of N to plants, but will make it more available to plants as it reduces N loss via leaching and denitrifi cation.

1.1 Why do we Need to Control Nitrifi cation in Agricultural Systems?Nearly 90% of the application of N-fertilizer worldwide is in NH4

+ form (or converted into NH4

+ from urea hydrolysis) which is rapidly converted to NO3– in

the soil by nitrifi er activity (Sahrawat, 1980; Mason, 1992; Strong and Cooper, 1992). Being cationic in nature, NH4

+ is held to the soil by the electrostatic forces of the negatively charged clay surfaces and the functional groups of soil organic matter (SOM) (Sahrawat, 1989; Amberger, 1993). This binding is suffi ciently strong to limit NH4

+-N loss from leaching (Amberger, 1993). In contrast, NO3–,

with its negative charge, does not bind to the soil, thus it is mobile in the soil and susceptible to leaching out of the root-zone (Amberger, 1993). Several heterotrophic soil bacteria denitrify NO3

– [i.e. convert NO3– into gaseous N forms:

N2O (a greenhouse gas), NO and N2] under anaerobic or partially anaerobic conditions (i.e. this often coincides with temporary water-logging from heavy rainfall or irrigation and/or improper drainage of fi elds) (Bremner and Blackmer, 1978; Mosier et al., 1996). The loss of N during and following nitrifi cation thus reduces the effectiveness of N fertilization and at the same time cause serious N pollution problems (Clark 1962; Jarvis, 1996). In alkaline soils, however, NH4

+ can be lost via volatilization, thus reducing the advantage of nitrifi cation inhibition (Sahrawat, 1989).

A rapid conversion of NH4+ to NO3

– in the soil results in an ineffi cient use of both soil and applied N. Soil organic N also goes through the nitrifi cation process, making it liable to N loss by the same pathways as the fertilizer N does (Dinnes et al., 2002; Subbarao et al., 2006a, 2009a, b). Nitrifi cation is the single most important process in the N cycle that leads to N losses (Clark, 1962; Barker and Mills, 1980) (Figure 1). In addition, the assimilation of NO3

– by plants requires higher metabolic energy than that required for the assimilation of NH4

+ (20 moles of ATP per mole of NO3

– vs 5 moles of ATP per mole of NH4+) (Salsac et al.,

1987); thus NH4+ assimilation is energetically more effi cient than NO3

– for plants. The assimilation of NO3

–, but not NH4+, results in the direct emission of N2O

from crop canopies reducing further its N-use effi ciency (Smart and Bloom, 2001). Maintaining N in NH4

+ form thus has a number of advantages for improving N uptake and utilization in agricultural systems, even if it should be taken into account the potential negative effect of the rhizosphere acidifi cation from NH4

+ uptake, N-preference in plants and its plasticity. Many of these advantages have been demonstrated using various chemical nitrifi cation inhibitors (Slangen and Kerkhoff, 1984; Sahrawat, 1989; Subbarao et al., 2006a).

4 Subbarao et al.

1.2 Is Modern Agriculture Moving towards High-Nitrifying Systems?Nitrifi cation plays a relatively minor role in many natural climax communities where only a small portion of the N goes through the nitrifi cation process in the nitrogen cycle. In contrast, nitrifi cation plays a dominant role in most agricultural systems (Figure 1) (Vitousek et al., 1997; Nasholm et al., 1998; Smolander et al., 1998; Subbarao et al., 2006a). Modern agricultural systems rely primarily on large inputs of external N (through mineral N fertilizer) to maintain high productivity as naturally fi xed-N is seldom adequate (Dinnes et al., 2002). During the 20th century, several changes took place in agricultural practices have led to increased nitrifi cation in agricultural systems (Rabalais et al., 1996; Poudel et al., 2002). These include a) reduced use of diversifi ed crop rotations, b) separation of crop production systems from animal enterprises, c) increased soil tillage, d) irrigation and drainage of agricultural fi elds and e) increased use of N fertilizers.

Just a case in example – we have noticed an increase in soil pH from 5.5 in the mid 1970’s to about 8.5 at present when Alfi sols were intensively cultivated by raising two crops annually with full irrigation and fertilization at the ICRISAT farm in Patancheru, India. This is largely due to the accumulation of salts (such as calcium, magnesium and sodium) from irrigation water because of high evaporative demand in this SAT region (K.L. Sahrawat, unpublished data). Soil pH greatly infl uences nitrifi cation and nitrifi cation rates in soils often reach highest at pH 8.0 to 9.0 (Sahrawat, 2008). This increase in soil pH thus will accelerate the nitrifi cation rates, which is associated with the intensifi cation of these production systems.

Current production systems that depend heavily on industrially produced inorganic N have replaced earlier N production systems that relied primarily on legumes and/or animals for their N inputs (Dinnes et al., 2002). In addition, the separation of crop and animal production enterprises has led to an even greater dependence on mineral N fertilizers (i.e. bypassing agricultural systems for organic matter recycling); this has also resulted in the reduction of soil organic matter (SOM) levels in croplands worldwide (Tiessen et al., 1994; Neff et al., 2002; van Wesemael et al., 2010). This heavy dependence on mineral fertilizers has contributed further to the stimulation of nitrifi er activity and the subsequent development of high-nitrifying soil environments (McGill et al., 1981; Poudel et al., 2002; Lal, 2003; Bellamy et al., 2005). This dependence on higher levels of N fertilizer coupled with the installation of sub-surface drainage systems in many developed parts of the world has resulted in the acceleration of NO3

– leaching and denitrifi cation, leading to reduced N-use effi ciency in agricultural systems (Clark, 1962; Pratt and Adriano, 1973; Sahrawat, 1989; Dinnes et al., 2002).

1.3 Consequences of High-Nitrifying Systems on the Global EnvironmentThe green revolution which is largely based on the application of industrially-fi xed N to semi-dwarf rice and wheat varieties, doubled global food grain production and reduced food shortages, but at a relatively high environmental cost (Tilman et al., 2001; Hungate et al., 2003). The rapid and unrestricted nitrifi cation found in modern production systems, results in the loss of nearly 70% of N-fertilizer inputs


to agricultural systems (Peterjohn and Schlesinger, 1990; Vitousek and Howarath, 1991; Raun and Johnson, 1999). With the worldwide N-fertilizer applications reaching 150 Tg y–1 (Galloway et al., 2008) and the cost of urea-N reaching US$ 0.45 kg–1.N, this amounts to a direct annual economic loss of nearly 40.5 billion US$. In addition, there are other costs related to environmental problems (without counting the political and economical problems linked to the natural gas market) which are hard to quantify in economic terms that have not yet been addressed (Viets, 1975; Ryden et al., 1984; Tilman et al., 2001; Schlesinger, 2009).

Fertilizer N use is expected to double by 2050 from the current 150 Tg N yr–1 used in agricultural systems (Galloway et al., 2008; Schlesinger, 2009). This will further increase N leakage from agricultural systems, placing much heavier pollution loads on the environment (Vitousek et al., 1997; Tilman et al., 2001; IFA, 2005, Schlesinger, 2009). The leaching of NO3

– from root zone and NO3–

contamination of ground and surface waters is one of the major environmental concerns associated with nitrifi cation (Scheperts et al., 1991; Tilman et al., 2001; Galloway et al., 2008; Schlesinger, 2009). A close link between N-fertilizer usage, increased groundwater NO3

– levels, human health and environmental problems (like severe eutrophization), has been established in several studies (Broadbent and Rauschkolb, 1977; Vitousek et al., 1997; Subbarao et al., 2006a; Schlesinger, 2009). Nitrogen lost from NO3

– leaching from agricultural systems reaches close to 61.5 Tg N yr–1 (Schlesinger, 2009).

In addition to the pollution of terrestrial and marine water bodies, agricultural systems contribute nearly 30% of the current nitric oxide (NO) emissions to the atmosphere (Bremner and Blackmer, 1978; Smith et al., 1997; Hofstra and Bouwman, 2005). In the atmosphere, N2O acts as a powerful greenhouse gas having a global warming potential 300 times that of CO2 (Kroeze, 1994; IPCC, 2001). In addition, the NOs that reach the stratosphere can damage the protective ozone layer (Crutzen and Ehhalt, 1977). During plant growth, the assimilation of NO3

– rather than NH4+ results in N2O emissions from crop canopies; this has been

demonstrated with wheat (Smart and Bloom, 2001). Current estimates indicate that nearly 17 Tg N yr–1 is emitted to the atmosphere as N2O (Galloway et al., 2008; Schlesinger, 2009). By 2100, the global N2O emissions are projected to be four times greater than the current emissions, due largely to increase in N-fertilizer use (Hofstra and Bouwman, 2005; Galloway et al., 2008; Burney et al., 2010).

1.4 Options for Regulating Nitrifi cation in Agricultural SystemsSeveral N-management strategies that utilize rate and/or timing of fertilizer applications such as ‘fall’ vs ‘spring’, basal vs split applications, banding of N fertilizers vs broadcasting, deep placement of N fertilizer vs surface application, point-injection placement of solutions, and foliar applications of urea have been used to enhance the N-use effi ciency of applied fertilizer. Various strategies have been developed to synchronize the fertilizer application with crop N requirements to facilitate rapid uptake and reduce N residence time in the soil to limit losses by denitrifi cation and/or NO3

– leaching (Newbould, 1989; Dinnes et al., 2002). Most agronomic strategies mentioned above have limitations, often associated with additional labor costs and other practical diffi culties (Dinnes et al., 2002).

6 Subbarao et al.

Synthetic Chemical InhibitorsNitrifi cation inhibitors are compounds that delay bacterial oxidation of NH4

+ by depressing activities of soil nitrifi ers. In theory, reducing nitrifi cation under conditions where there is a high risk of N loss from NO3

– leaching or denitrifi cation, should improve N-use effi ciency (NUE) (Hughes and Welch, 1970; Hendrickson et al., 1978; Ranney, 1978; Bremner et al., 1981; Rodgers, 1986). Reducing nitrifi cation rate until the primary crop is in its log phase of growth, would give plants a better opportunity to absorb N while it still remains in the root zone. In addition, rapidly growing crops will absorb more water from precipitation/irrigation, which also lowers the risk of NO3

– being leached out of the root zone (Dinnes et al. 2002; Liao et al., 2004).

Numerous compounds have been proposed and patented as nitrifi cation inhibitors (Malzer, 1979; McCarty, 1999; Subbarao et al., 2006a). Only a few nitrifi cation inhibitors, nitrapyrin, DCD (dicyandiamide), and DMPP (3, 4-dimethyl pyrazole phosphate) have been thoroughly evaluated under fi eld conditions (Goring, 1962; Guthrie and Bomke, 1980; Weiske et al., 2001; Zerulla et al., 2001; Di and Cameron, 2002; Subbarao et al., 2006a). However, these synthetic chemical inhibitors have not been widely adopted in production agriculture due to the lack of cost-effectiveness and their inconsistent performance across diverse agro-climatic and soil environments (McCall and Swann, 1978; Gomes and Loynachan, 1984; Subbarao et al., 2006a).

Slow and Controlled-Release Nitrogen FertilizersSlow and controlled-release (SCR) fertilizers are forms of N fertilizer that extend the time of N availability for plant uptake (Shaviv and Mikkelsen, 1993). SCR fertilizers release N into the soil solution at a reduced rate, which is achieved through special chemical and physical characteristics. SCR fertilizers are produced by providing a protective coating (water-insoluble, semi-permeable or impermeable with pores) or encapsulating the conventional soluble fertilizer materials to control water entry and rate of dissolution, thus nutrient release and availability can be synchronized with plant’s N requirements (Fujita et al., 1992). Because of the slow release of N, the availability of NH4

+ to the nitrifi ers is limited and N loss during and following nitrifi cation is reduced. Field evaluation of polymer-coated urea (POCU) indicates that N losses associated with nitrifi cation can be substantially reduced, along with concurrent improvement in N recovery (Shoji and Kanno, 1994). Because of the reduced N losses, the crop N application rates for POCU is about 40% less than the recommended level for normal N fertilizers (Zvomuya et al., 2003). However, POCU is 4 to 8 times more expensive than normal urea, thus the adoption of POCU in production agriculture is limited and confi ned mostly to niche areas such as horticultural and fl oricultural systems (Detrick, 1996).

1.5 Can we Learn from Natural Ecosystems to Control Nitrifi cation in Agricultural Systems?Natural ecosystems have evolved a variety of mechanisms allowing multiple pathways for N uptake and conservation including direct uptake of organic N by plants (bypassing the mineralization process, thus minimizing the N losses from the system). In certain pine forest systems, polyphenols from litter forms


a complex with dissolved organic N, making it resistant to mineralization. The direct uptake of this polyphenol-organic-N is facilitated through association with certain mycorrhizae, bypassing the mineralization process, eliminating several pathways of the N cycle that are associated with N leakage; this results in tight N cycling in these pine forest ecosystems (Northup et al., 1995).

Several studies have indicated that soil nitrifi cation potential differs among ecosystems. These differences in nitrifi cation potential do not seem to be primarily associated with soil -physical and -chemical characteristics (Clark et al., 1960; Robertson, 1982a, 1982b; Montagnini et al., 1989; Northup et al., 1995; Schimel et al., 1998; Hattenschwiler and Vitousek, 2000; Laverman et al., 2000; Lata et al., 2004; Lovett et al., 2004). Often in these cases, the ammonium levels exceeded nitrate concentrations by a factor of ten, indicating that ammonium is not a limiting factor for nitrifi cation. The infl uence of vegetation in inhibiting nitrifi cation has long been suspected, but not proven (Donaldson and Henderson, 1990a,b; Steltzer and Bowman, 1998; Lewis and Likens, 2000; Christ et al., 2002; Lovett et al., 2004). Certain forest trees, such as Arbutus unedo, are reported to suppress soil nitrifi cation and nitrous oxide emissions; this is hypothesized to be due to the release of phenolic compounds such as gallocatechin and catechin from the litter to the soil (Castaldi et al., 2009). Several researchers have observed a slow rate of nitrifi cation in soils of certain tropical grassland and forest soils (Sylvester-Bradley et al., 1998). This led to the hypothesis that plant roots may infl uence nitrifi cation by releasing phytochemicals that can affect soil nitrifi er activity (Jones et al., 1994; Subbarao et al., 2006a; Fillery, 2007).

It has been hypothesized that mature grassland ecosystems have the ability to inhibit soil nitrifi cation (Boughey et al., 1964; Lata et al., 1999). In the natural grasslands dominated by Andropogon spp., Brachiaria humidicola and Hyparrhenia diplandra, most of the inorganic soil N is in the NH4

+ form, which is considered to be an indicator of the ecosystem’s maturity (Meiklejohn, 1968; Lodhi, 1979; Sylvester-Bradley et al., 1988; Lata et al., 1999; Subbarao et al., 2006a; Castaldi et al., 2009). Historically, there have been a number of attempts to test this nitrifi cation inhibition hypothesis, but they met with little success, due mainly to the lack of availability of a suitable methodology to collect, detect and quantify the amount inhibitors released from the roots (Robinson, 1963; Munro, 1966a,b; Moore and Waid, 1971; Purchase, 1974, Rice and Pancholy, 1974; Arslan et al., 2010).

Unlike most agricultural systems, selected climax natural ecosystems are known to retain large amounts of N through incorporation into the soil organic matter; but the underlying mechanisms remain poorly understood (Magill et al., 2000). The hypothesis, that plants can suppress or stimulate nitrifi cation has been debated for long time, but without good evidence from in situ studies (Stienstra et al., 1994; Knops et al., 2002; Lata et al., 1999; 2004; Ishikawa et al., 2003; Lovett et al., 2004; Fillery, 2007).

Plant species that dominate some of the climax ecosystems with relatively low nitrifi cation were shown to produce organic compounds that inhibit nitrifi er activity (Basaraba, 1964; Likens et al., 1969; Jordan et al., 1979; Donaldson and

8 Subbarao et al.

Henderson, 1990a,b; Courtney et al., 1991). These inhibitory compounds when added to the soil, suppressed nitrifi cation in the rhizosphere (Jordan et al., 1979). The degree of nitrifi cation inhibition appears to increase with the ecosystem’s maturity with little or no nitrifi cation occurring in some mature ecosystems (Rice and Pancholy, 1972, 1973, 1974; Lodhi, 1982; Thibault et al., 1982; Baldwin et al., 1983; Cooper, 1986; Howard and Howard, 1991; White, 1991; Northup et al., 1995; Schimel et al., 1996; Paavolainen et al., 1998; Ste Marie and Pare, 1999; Erickson et al., 2000).

Since NH4+ assimilation by plants requires four times less metabolic energy

than that needed for NO3–, it is hypothesized that inhibition of nitrifi cation could

be an ecological driving force for the development of low NO3– climax ecosystems

(Rice and Pancholy, 1972; Salsac et al., 1987; Lata et al., 2004). Among the nitrifi cation inhibitory compounds proposed, phenolics, alkaloids, isothiocyanates, and terpenoids have received some attention (Lewis and Papavizas, 1970; Zucker, 1983; Putnam, 1988; Choesin and Boerner, 1991; Bending and Lincoln, 2000; Bertin et al., 2003; Gopalakrishnan et al., 2007; Subbarao et al., 2008; Zakir et al., 2008).

2. BIOLOGICAL NITRIFICATION INHIBITION (BNI)

2.1 The Concept of BNI Function in Plants

How do we defi ne BNI Function?BNI is the ability of certain plant species to release organic molecules/compounds from their roots that have a targeted suppressive effect on soil nitrifying bacteria (Subbarao et al., 2006a,b, 2009a,b). A schematic presentation of the BNI concept along with various processes of the soil N-cycle that are potentially infl uenced by this plant function or BNI is presented in Figure 2.

Nitrogen-use effi ciency (NUEagronomic = dry matter produced per unit of applied N) is a function of both intrinsic N-use effi ciency (NUEintrinsic) and total N uptake. Intrinsic N-use effi ciency (NUEintrinsic i.e. dry matter produced per unit N uptake) of a plant is a physiologically conservative function (Glass, 2003), thus diffi cult to manipulate genetically. Improvements in NUEagronomic mostly come through an improvement in crop N uptake (Finzi et al., 2007). As discussed earlier, the BNI function can improve N uptake due to its inhibitory effects on nitrifi cation, which in some situations could enhance NUEagronomic in production systems (Subbarao et al., 2006a).

The results of recent modeling studies indicate that by inhibiting nitrifi cation, N recovery, and hence NUE can be improved substantially in situations where the loss of N following nitrifi cation by leaching or denitrifi cation is high. A general theoretical ecosystem model, that considers both nitrate and ammonium as N source, was used to investigate the general conditions under which nitrifi cation inhibition enhances primary production and its quantitative impact on the dynamics and budget of N utilization. Primary productivity was positively impacted in tropical savannas dominated by native African grasses, Hyparrhenia diplandra that appear to have greater ability to suppress nitrifi cation (Boudsocq et al., 2009). For the natural and agro-ecosystems, which are subject to high nitrifying and


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10 Subbarao et al.

denitrifying activities, this model predicts that nitrifi cation inhibition by plants is a process that can lead to better N conservation, and thus increase the primary production as the ammonium pathway is more effi cient (i.e. more conservative) than the nitrate pathway. This would be the case if the considered ecosystem is subjected to higher losses under nitrate form (leaching and denitrifi cation) than under ammonium form (volatilization). This model supports previous in situ measurements in savanna systems (Lata, 1999), which showed that grasses that inhibit nitrifi cation exhibit a 2-fold higher productivity in aboveground biomass than that in the non-nitrifi cation inhibiting grasses (see Section 2.8 and Figure 13 for further discussion).

2.2 Methodology to Detect BNIs in Plant-Soil SystemsA bioluminescence assay using a recombinant strain of Nitrosomonas europaea was adopted to detect nitrifi cation inhibitors released from plant roots (hereafter referred to as BNI activity) (Iizumi et al., 1998; Subbarao et al., 2006b). The recombinant strain of N. europaea carries an expression vector for the Vibrio harveyi luxAB genes (Figure 3), and produces a distinct two-peak luminescence pattern during a 30-s analysis period (Subbarao et al., 2006b). The functional relationship between bioluminescence emission and nitrite production in the assay has been shown to be linear using the synthetic nitrifi cation inhibitor, allylthiourea (AT) (Subbarao et al., 2006b). The inhibition caused by 0.22 μM AT in this assay (about 80% inhibition in bioluminescence and NO2

– production) is defi ned as 1 ATU (allylthiourea unit) (Subbarao et al., 2006b). Using the response to a concentration gradient of AT (i.e. a standard dose-response curve), the inhibitory effect of test samples, e.g. root exudates, soil and plant extracts can be expressed in ATU. With these recent developments in methodology, it is now possible to determine and compare the BNI capacity of different crops and pastures (Subbarao et al., 2006b).

2.3 Variation in the BNI-Capacity of Major Crops and Forage GrassesAn evaluation of tropical forage grasses, cereal and legume crops indicated a wide range in the BNI-capacity (Table 1) (Subbarao et al., 2007b). The highest BNI capacity was found in Brachiaria spp. Substantial genotypic variation was also detected in the BNI capacity of Brachiaria humidicola (Table 2).

Forage grasses of B. humidicola and B. decumbens, which are highly adapted to the low-N production environments of the South American Savannas (Rao et al.,1996; Miles et al., 2004), showed the greatest BNI capacity among tropical grasses (Subbarao et al., 2007b). In contrast, P. maximum, which is adapted to high N availability environments showed the least BNI capacity (Rao et al., 1996; Subbarao et al., 2007b). Among the cereal crops evaluated, only sorghum showed signifi cant BNI capacity. Other cereal crops including rice, maize, wheat and barley, did not possess suffi cient BNI capacity (Subbarao et al., 2007b; Zakir et al., 2008).

Inhibition of nitrifi cation (i.e. BNI capacity) is most likely part of an adaptation mechanism for the conservation and effi cient use of N in natural systems having low N availability (Lata et al., 2004; Subbarao et al., 2006a). Thus N stress could be a driving force for the evolution of the BNI function (Rice and Pancholy, 1972; Lata et al., 2004). It is not surprising that legumes do not show appreciable BNI capacity. In the case of legumes, it is likely that the BNI attribute would have


Table 1. The BNI released from intact plant roots of various plant species grown in sand-vermiculite (3:1 v/v) culture for 60 days

Serial No.

Plant Species Total BNI released from four plants (ATU d–1)

Specifi c BNI (ATU g–1 root dry wt d–1)

Pasture grasses. 1. Brachiaria humidicola (Rendle) Schweick. 51.1 13.4 2. B. decumbens Stapf 37.3 18.3 3. Melinis minutifl ora Beauv. 21.4 3.8 4. Panicum maximum Jacq. 12.5 3.3 5. Lolium perenne L ssp. Multifl orum (Lam.) Husnot 13.5 2.6 6. Andropogon gayanus Kunth 11.7 7.7 7. B. brizantha (A. rich.) Stapf 6.8 2.0

Cereal crops 8. Sorghum bicolor (L.) Moench var. hybrid sorgo 26.1 5.2 9. Pennisetum glaucum (L.) R. Br. var. CIVT 7.0 1.811. Oryza sativa L. var Sabana 6 0 0

Oryza sativa L. var. Toyo 0 012. Zea mays L. var. Peter no. 610 0 013. Hordeum vulgare L. var. Shunrai 0 014. Triticum aestivum L. var. Norin-61 0 0

Legume crops15. Arachis hypogaea L. var. TMV 2 9.4 2.516. Glycine max (L.) Merr. var. Orinoquia 3 0 0

Glycine max. (L.) Merr var. Natsuroyosooi 0 0Glycine max(L.) Merr non-nodulating type – EN 1282 0 0

17. Vigna unguiculata (L. Walpers ssp. unguiculata var. Caupi

0 0

18. Phaseolus vulgaris (L.) (accession G 21212) 0 0LSD (0.05) 7.1 2.8

Note: ‘0’ activity indicates that the inhibitory effect is possibly below the detection limit of the assay system used.Source: Subbarao et al., 2007b.

Figure 3. Physical map of recombinant luminous Nitrosomonas europaea (pHLUX20) developed to detect and quantify nitrifi cation inhibitors in the plant-soil systems

source: Iizumi et al., 1998.

12 Subbarao et al.

little or no adaptive value due to their ability to fi x N symbiotically. Conserving N may not offer as much of a comparative advantage for legumes as it might attract competition from non-legumes.

Our preliminary studies indicate that soybean root exudates stimulate nitrifi cation in laboratory soil incubation tests (Subbarao et al., 2007c). Several forest systems dominated by leguminous trees (Acacia magnium and A. auriculaeformis) have soils that had no infl uence or even stimulated nitrifi cation. In contrast, forests dominated by non-legume trees such as Eucalyptus citriodora, Pinus elliotti, and Schima superb, showed low nitrifi cation rates (Li et al., 2001). Recent studies indicate that a wild relative of wheat, Leymus racemosus, has BNI capacity similar to that of Brachiaria spp., with BNI-activity release rates ranging from 20 to 30 ATU g–1 root dry wt. d–1 (Subbarao et al., 2007c).

2.4 Regulatory Nature of the BNI Function The synthesis and release of BNIs is a regulated attribute in B. humidicola (Subbarao et al., 2007a). To some extent, the release of BNIs from roots is related to the plant N status (Subbarao et al., 2006b). In particular, the form of N applied (i.e. NH4

+ vs NO3–) has a major infl uence on the synthesis and release of BNIs

from roots in B. humidicola and in wild wheat, L. racemosus (Subbarao et al., 2007a,c). Plants grown with NO3

– as their N source did not release BNIs from roots (Subbarao et al., 2007a). BNIs were released from plants grown with NH4

+ as their N source (Subbarao et al., 2007a,c, 2009a,b). Even for plants grown with NH4

+, the presence of NH4+ in the rhizosphere was critical for the synthesis and

release of BNIs from their roots (Subbarao et al., 2007a,c). Though, high levels of BNIs were detected in the root tissues of NH4

+ grown plants, their release was observed only when their roots were exposed to NH4

+ (Subbarao et al., 2007a,c, 2009a,b).

In addition to the presence of NH4+ in the medium, the rhizosphere pH could

also infl uence the release of BNIs from roots. Recent results indicated that sorghum plants do not release BNIs from roots in the presence of NH4

+, if the rhizosphere pH was maintained at 7. If the pH of the root exudate collection solutions (1 mM NH4Cl) was not controlled and allowed to be dropped to about 4, sorghum plants released substantial amount of the BNI activity from roots (about 15 ATU

Table 2. Genotypic variation in the BNI released from roots of B. humidicola germplasm accessions. Four plants per pot were grown for 180 d before collecting the root exudates

Serial No. Accession No. Total BNI released from four plants (ATU d–1)

Specifi c BNI (ATUg–1 root dry wt. d–1)

1. CIAT 26159 126.2 46.32. CIAT 26427 118.5 31.63. CIAT 26430 151.0 24.14. CIAT 679 68.8 17.55. CIAT 26438 93.5 6.56. CIAT 26149 22.3 7.17. CIAT 682 53.4 7.58. P. maximum 0.6 0.1

LSD (0.05) 21.7 6.0

Source: Subbarao et al., 2007b.


g–1 root dry wt. d–1) (G.V. Subbarao, unpublished data). These results suggest that it is likely that BNI function is better expressed in plants when grown on light-textured soils with pH 6.00 or lower. Such effects of pH on the BNI activity and release in the solution culture studies have not been evaluated in the soil as the plant growing medium, but merits attention in the future.

Further, the release of BNIs from plant roots appears to be a highly regulated physiological function. The presence of NH4

+ in the root environment is necessary not only for the acceleration of synthesis of BNIs and/or precursors of BNI compounds in the roots, but also for their release (Subbarao et al., 2007a, 2009a). The physiological consequences associated with the uptake of NH4

+, such as activation of H+ pumps in the plasmalemma and acidifi cation of rhizosphere appears to facilitate the release of BNIs in sorghum roots (Zhu et al., 2010). Further, the release of BNIs from roots is a localized phenomenon (Subbarao et al., 2009a). The release of BNIs appears to be confi ned to only those roots exposed to NH4

+ in the rhizosphere, not from the entire root system; this ensures that inhibitors are released from roots in suffi ciently high concentrations (Subbarao et al., 2009a). The availability of NH4

+ in the soil either from soil organic N mineralization or through the application of N-fertilizers such as urea or ammonium sulfate can enhance nitrifi er activity (Robinson, 1963; Woldendorp and Laanbroek, 1989). The regulatory role of NH4

+ in the synthesis and release of BNIs suggests a possible adaptive role in protecting NH4

+ from nitrifi ers, a key factor for the successful evolution of BNI capacity as an adaptation mechanism (Subbarao et al., 2007a).

2.5 Stability of BNIs in the Soil SystemsThe BNI activity of roots is determined by their inhibitory effects on the biological activity of a recombinant luminescent Nitrosomonas, during a 30-min incubation period (Subbarao et al., 2006b). However, nitrifi cation in the soil may occur over a longer period of time, often taking several weeks for the oxidation of soil ammonium (i.e. nitrifi ed); thus, inhibitory compounds released from roots that persist in the soil for several weeks can be effective in suppressing nitrifi er activity and reducing soil nitrifi cation. This hypothesis was tested by adding the extracted BNI activity from root exudates (of B. humidicola) to the soil at various levels (0 to 20 ATU g–1 soil) along with NH4

+ (200 mg N kg–1) as the N source and incubated for 55 days at 20ºC. These studies indicated that for inhibitory activity in the soil, a threshold level of 5 ATU g–1 soil was needed before the inhibitory effect became evident on soil nitrifi cation; nearly 50% inhibition was observed when the BNI activity levels reached 10 ATU g–1 soil and a nearly complete suppression of soil nitrifi cation was achieved at 20 ATU g–1 soil (Figure 4) (Subbarao et al., 2006b). Further, it was shown that certain BNIs (such as linoleic acid and linolenic acid) partially lose their effectiveness in the soil after 80 days, and the inhibitory effect was lost totally at/after 100 days (Subbarao et al., 2008). In addition, previous preliminary measurements on mixed tropical savanna soils showed that this effect can resist natural air drying and conservation in the dark (Lata, 1999).

2.6 Biological Molecules with BNI Potential and the Mode of Inhibitory

14 Subbarao et al.

Action on NitrosomonasPlants are known to release a wide range of substances that have biological activity (Bremner and McCarty, 1988; Bending and Licoln, 2000; Subbarao et al., 2006a; Raaijmakers et al., 2009). These include molecules released from plant roots that belong to phenolic, fatty acid, isothiocyanate and terpene groups (Choesin and Boerner, 1991; Bennett and Wallsgrove, 1994; Langenheim, 1994; Bending and Lincoln, 2000; Kraus et al., 2003; Subbarao et al., 2006a, 2009a,b). The compounds responsible for the BNI activity were not elucidated until recently despite the phenomenon being fi rst proposed to occur in the early 1960s based on the results from empirical studies in the fi eld (for review see Subbarao et al., 2006a). Several nitrifi cation inhibitors belonging to different chemical groups have been successfully isolated and identifi ed from plant tissues or root exudates using bioassay-guided purifi cation with a recombinant Nitrosomonas europaea assay (Figure 5) (Subbarao et al., 2006b; 2008; 2009; Gopalakrishnan et al., 2007; Zakir et al., 2008).

The compounds with the BNI activity in the aerial parts of B. humidicola were identifi ed as the unsaturated free fatty acids, linoleic acid and α-linolenic acid (Subbarao et al., 2008). They are relatively weak inhibitors of nitrifi cation with IC50 values of 3 × 10–5 M; while the IC50 value of synthetic nitrifi cation inhibitor 1-allyl-2-thiourea is 1 × 10–7M. However, other free fatty acids having varying chain lengths or the numbers of double bonds, e.g. stearic, oleic, arachidonic and cis-vaccenic acid did not show inhibitory activity, indicating that there are specifi c chemical structural requirements to inhibit Nitrosomonas function (Subbarao et al., 2008). BNI compounds linoleic acid and α-linolenic

Figure 4. Effectiveness of BNI activity (released from roots of B. humidicola) in inhibiting nitrate formation in the soil (during 55 days of incubation at 20οC)

source: Subbarao et al., 2006b.


acid might possess the structure and chain length needed to inhibit nitrifi cation. These two BNI compounds possibly inhibit both ammonia monooxygenase (AMO) and hydroxylamine oxidoreductase (HAO) enzymatic pathways, which catalyze essential reactions of the ammonia oxidation process in Nitrosomonas (Subbarao et al., 2008). When linoleic acid and α-linolenic acid were added to the soil, the nitrifi cation rates were substantially suppressed for several months (Subbarao et al., 2008). The BNI activity in the crude extracts of the root exudates of B. humidicola and Leymus racemosus (wild wheat) appear to block both AMO and HAO enzymatic pathways with equal effectiveness (Subbarao et al., 2007a,c). In addition, BNIs could also disrupt the electron transfer pathway/s from HAO to ubiquinone and cytochrome (which needs to be maintained to generate reducing power, i.e., NADPH), that is critical to the metabolic functions of Nitrosomonas (Figure 6); this requires further research to provide information on the mechanisms involved (Subbarao et al., 2009b); in contrast, synthetic nitrifi cation inhibitors AT, nitrapyrin and DCD, which inhibit nitrifi cation by suppressing only the AMO enzymatic pathway in Nitrosomonas (Subbarao et al., 2007a,c) (Figure 6).

From the root exudates of hydroponically-grown Sorghum bicolor, phenylpropanoid, methyl 3-(4-hydroxyphenyl) propionate (MHPP) was identifi ed as the nitrifi cation inhibitor (Figure 5), which contributes partially to the inhibitory activity released from roots (Zakir et al., 2008). The IC50 value for MHPP is approximately 9 x 10–6M (Zakir et al., 2008). In the root tissue of B. humidicola, two phenyl propanoids, methyl-p-coumarate and methyl ferulate (Figure 5) were identifi ed as the major nitrifi cation inhibitors (Gopalakrishnan et al., 2007).

Figure 5. Chemical structures of the compounds reported to possess the BNI activity in plants

16 Subbarao et al.

The IC50 values for methyl-p-coumarate and methyl ferulate are 2 × 10–5 and 4 × 10–6 M respectively (Gopalakrishnan et al., 2007). The corresponding free phenolic acids namely p-coumaric acid and ferulic acid, which are involved in lignin biosynthesis, showed no inhibitory activity at concentrations of less than 1 × 10–2 M (Gopalakrishnan et al., 2007). It is hypothesized that B. humidicola releases methyl-p-coumarate and methyl ferulate or simple metabolites derived from these BNI compounds into the soil environment via turnover of root tissues in the pasture systems (Gopalakrishnan et al., 2007). However, the BNI compounds mentioned above were not detected in root exudates.

Discovery of BrachialactoneThe major nitrifi cation inhibitor released from roots of B. humidicola has been discovered and named ‘brachialactone’, a cyclic diterpene; this compound has a dicyclopenta[a,d]cyclooctane skeleton (5–8–5 ring system) with a γ-lactone ring bridging one of the fi ve-membered rings and the eight-membered ring (Subbarao et al., 2009a) (Figure 7). Similarly, 5–8–5 tricyclic terpenoids (ophiobolanes and fusicoccanes) are found in both fungi and plants (Muromtsev et al., 1994; Toyomasu et al., 2007). However, to our knowledge, any derivative that has a lactone ring is novel. Fusicoccane-type cyclic diterpenes are biologically synthesized from geranylgeranyl diphosphate by a two-step cyclization catalyzed by terpene cyclases (Toyomasu et al., 2007) (Figure 7).

The inhibition of nitrifi cation in an in vitro assay with pure cultures of N. europaea was linearly related to brachialactone concentration in the range of 1.3 to 13.3 μM (Figure 8). Brachialactone, with an ED80 of 10.6 μM, should be considered as a potent nitrifi cation inhibitor when compared with nitrapyrin or dicyandiamide, two of the most widely used synthetic nitrifi cation inhibitors

Figure 6. The mechanisms involved in the inhibitory effects of selected synthetic nitrifi cation inhibitors and the BNIs released from the roots of B. humidicola (based on Iizumi et al., 1998; Subbarao et al., 2007a)


(ED80 of 5.8 μM for nitrapyrin and 2,200 μM for dicyandiamide). Brachialactone inhibits Nitrosomonas by blocking both AMO and HAO enzymatic functions, but appears to have stronger effect on the AMO than on the HAO pathway (Subbarao et al., 2009a). Nearly 60–90% of the inhibitory activity released from the roots of this tropical grass is due to brachialactone (Figure 8).

2.7 Evidence for the BNI Function under Field ConditionsA conservative estimate of the live root biomass from a long-term grass pastures was 1.5 Mg ha–1 (Fisher et al., 1994), with a BNI capacity of 17–50 ATU per g of root dry wt. per day (Subbarao et al., 2007a), we estimate that BNI activity of 2.6 × 106 to 7.5 × 106 ATU.ha–1 day–1 could potentially be released from B. humidicola roots. This estimate amounts to an inhibitory potential equivalent to the application of 6.2 to 18 kg of nitrapyrin per ha.year–1 (based on 1 ATU being equal to 0.6 μg of nitrapyrin), which is large enough to have a signifi cant infl uence on the function of soil nitrifi er populations and nitrifi cation rates. Brachiaria humidicola pastures develop abundant and highly vigorous root systems that explore deeper soil layers and sequester large amounts of carbon in soil (Figure 9) (Fisher et al., 1994; Rao, 1998).

Field studies made at the Centro Internacional de Agricultura Tropical (CIAT), Palmira, Colombia indicated a 90% decrease in the ammonium oxidation rates in the soil (Figures 10, 11) due to very low populations of nitrifi ers [AO bacteria and AO archaea; determined as amoA genes] in B. humidicola plots within 3 years of establishment (Subbarao et al., 2009a). Two other pasture grasses, Panicum

Figure 7. (a). The chemical structure of brachialactone, the major nitrifi cation inhibitor isolated from the root exudates of B. humidicola

source: Subbarao et al., 2009a); (b) synthesis of fusicocca-2,10(14)-diene from isoprene units by PaFS. This enzyme is a diterpene hydrocarbon synthase possessing both prenyltransferase and terpene cyclase activity

source: Toyomasu et al., 2007).

18 Subbarao et al.

maximum and Brachiaria hybrid cv. Mulato that have a low to moderate level of BNI capacity (3 to 10 ATU g–1 root dwt d–1) showed only an intermediate level of inhibitory effect on soil ammonium oxidation rates (Figure 11). The inhibitory function from the roots of these tropical pasture grasses appears to be primarily targeted towards suppressing soil nitrifi er activity rather than the soil microbial activity in general. Soil nitrifi er activity as estimated from ammonia oxidizing bacteria and ammonia oxidizing archaea populations indicated a 90% decline in fi eld plots planted with B. humidicola within 3 years, but did not have a signifi cant effect on the total soil bacterial population (Subbarao et al., 2009a).

Figure 8. Inhibition of nitrifi cation by brachialactone and the contribution of brachialactone to BNI activity released from roots. (A) Inhibitory effects of brachialactone on N. europaea in an in vitro assay. (B) Contribution of brachialactone to the BNI activity released from roots (i.e. root exudates) of B. humidicola. Root exudates were collected from intact plants using 1 L of aerated solution of 1 mM NH4Cl with 200 μM CaCl2 over 24 h. Each data point represents the effects of the root exudates collected from hydroponically grown plants in a glasshouse during March to May of 2007 and 2008

Source: Subbarao et al., 2009a.


Figure 9. Brachiaria humidicola cv. Llanero with abundant root system grown in a low fertility acid soil of the Llanos in Colombia

Source: I.M. Rao. CIAT, Cali, Colombia.

Figure 10. The proof of concept for the BNI function from selected fi eld experiments. The different tropical pasture grasses (B. humidicola (CIAT 679; CIAT 16888), B. hybrid cv. Mulato; P. maximum, and soybean were grown in the fi elds for 3 years to monitor the changes in soil nitrifi cation potentials by the BNI function and its effects on nitrous oxide emissions

Nitrous oxide emission was also suppressed to the extent of >90% in fi eld plots of B. humidicola, compared to that from the fi eld planted to soybean, which lacked BNI capacity (Figure 12). There appears to be a negative relationship between the BNI capacity of the roots and N2O emissions, based on fi eld monitoring of N2O emissions over a three-year period in tropical pasture grasses that have a wide range in the BNI capacity in their roots (Figure 12).

20 Subbarao et al.

2.8 Potential for Genetic Improvement of BNI Capacity in Forage Grasses and CropsThe existence of genotypic variability is a prerequisite for the genetic improvement of any trait using a conventional breeding program. Signifi cant genetic variability exists for the BNI capacity in the roots of B. humidicola (Table 1). Specifi c BNI activities (ATU g–1 root dry wt d–1) ranged from 7.1 to 46.3 ATU indicating that there may be potential for genetic improvement of the BNI capacity by selection and recombination (Subbarao et al., 2007a).

Using two ecotypes of the tropical grass Hyparrhenia diplandra (high-nitrifi cation ecotype and low-nitrifi cation ecotype), it has been shown that nitrifi cation can be stimulated or suppressed depending on the ecotype, suggesting that the suppression of soil nitrifi cation by these tropical grasses could largely be a genetic attribute (Figure 13) (Lata et al., 2004). These effects (i.e. the ability to infl uence soil nitrifi cation) are refl ected in their growth and biomass production (Lata et al., 2000) and are also variable in the population at the individual level. Attempt of correlating this variability to markers such as plant microsatellites showed a clear pattern (J.C. Lata, unpublished data). Preliminary results indicated a signifi cant genotypic variability for the BNI capacity in barley germplasm (T.S. George and G.V. Subbarao, unpublished data). Sorghum, one of the most promising fi eld crops for the BNI function, showed signifi cant genotypic variation for the BNI capacity in roots (Figure 14) (Subbarao et al., 2009b).

In cultivated wheat, preliminary results suggested a lack of signifi cant BNI capacity (Subbarao et al., 2006b). However, research with wild wheat indicated that the roots of a wild-wheat, L. racemosus, possess a high-BNI capacity (Figure 15)

Figure 11. Soil ammonium oxidation rates (mg NO2– N per kg of soil per day) in fi eld plots planted to

with tropical pasture grasses (differing in BNI capacity) and soybean (lacking BNI capacity in roots) [over 3 years from establishment of pastures (September 2004 to Nov. 2007); for soybean, during planting seasons every year, and after six seasons of cultivation]

Source: Subbarao et al., 2009a.


Figure 12. The relationship between the BNI capacity of plant species to the N2O emissions from fi eld plots. The N2O emissions were monitored over a period of three years, Sept. 2004 to Nov. 2007 (Adapted from Subbarao et al., 2009).

Figure 13. At the experimental site in Lamto Savanna in Cote d’Ivoire, with the ecotypes of Hyparrhenia diplandra, a tropical grass, differing in their ability to infl uence soil nitrifi cation. Pictures were taken on April, 1995. The sites are under similar climatic and pedologic conditions, but exhibit different above ground biomass: from 270 + 55 g. m2 in the low-nitrifying site to 130 + 30 g. m2 in the high-nitrifying site

source: Lata, 1999.

(Subbarao et al., 2007c). Inhibitors released from the roots of wild-wheat effectively suppressed soil nitrifi cation for more than 60 d (Subbarao et al., 2007c). Using chromosome-addition lines derived from the hybridization of this wild-wheat (i.e. L. racemosus) with cultivated wheat (Kishii et al., 2004), it was shown that the genes conferring high-BNI capacity were located in chromosome Lr#n, and could

22 Subbarao et al.

be successfully introduced into and expressed in cultivated wheat (Figure 16) (Subbarao et al., 2007c). These results indicated that there exists a potential for developing the next-generation of wheat cultivars with suffi cient BNI-capacity in the roots to suppress soil nitrifi cation in production systems based on wheat (Subbarao et al., 2007c).

Figure 14. The system used to collect the root exudates from sorghum plants; methanol extracts of root exudates from three sorghum genotypes/germplasm lines with differences in the BNI capacity (i.e. the capacity to release inhibitors from roots) (G.V. Subbarao and N. Kudo, unpublished results)

Figure 15. BNIs (biological nitrifi cation inhibitors) released from roots (i.e. root exudates) of two cultivars of wheat and their wild relative L. racemosus; plants were grown with either NH4

+ or NO3-

as the nitrogen source; root exudates was collected from intact roots in aerated collection solutions over a 24 h period; vertical bars represent Fisher LSD (P<0.001) for the interaction term (N source x species)

Source: Subbarao et al., 2007c.


Demonstrating that the high-BNI capacity of L. racemosus can be expressed in chromosome addition line of cultivated wheat provides the opportunity to further explore the introduction of the BNI trait into elite wheat cultivars. As wheat utilizes nearly a third of the global N fertilizer output (Raun and Johnson, 1999), introducing high-BNI capacity into cultivated wheat could have a large impact on reducing N leakage from wheat production systems globally. However, the alien chromosome of this chromosome-addition line also carries many undesirable traits that reduce grain yield potential. This is an example of negative linkage drag, which is commonly observed in products of crosses, including early-generation backcrosses, of elite cultivars with wild relatives or other exotic germplasm. It would thus be necessary to transfer to wheat only small segment/s of this L. racemosus chromosome containing favorable alleles of genes controlling the BNI trait to minimize the negative linkage drag that is associated with this introgression.

During the 1980’s, it was shown that more than 50% of wheat varieties bred by the CIMMYT had the 1RS.1BL translocation involving the short arm of the 1R chromosome from rye (Secale cereal L.), which has provided multiple disease resistance (Singh et al., 2006). However, if the original translocation is accompanied by many undesirable traits, it will be necessary to perform further reduction of the introgressed L. racemosus chromosome segment. This can be achieved by suppressing the effect of the Ph1 gene in wheat (which prevents homoeologous recombination between wheat and alien chromosomes), using the ph1 gene mutant of common wheat (Sears, 1977) as demonstrated in wheat-rye crosses (Lukaszewski, 2000).

Introduction of the BNI trait from L. racemosus to barley would be diffi cult following this strategy, because diploid barley is very sensitive to chromosome

Figure 16. Karyotype analysis of DALr#n, a chromosome-addition line derived from L. racemosus x T. aestivum; A. DAPI staining revealed 44 chromosomes; B. The probe of L. racemosus genomic DNA (green) and TaiI and Afa family repetitive sequences showed the presence of two Lr#n chromosomes; arrows indicate Lr#n chromosomes conferring high-BNI capacity which was successfully expressed in cultivated wheat

Source: Subbarao et al., 2007c.

24 Subbarao et al.

manipulation (compared to tetraploid durum wheat or hexaploid bread wheat). Also, a gene to induce homoeologous recombination like that found in wheat has not been reported for barley. One possible method to introduce the L. racemosus chromosome to barley could be through the use of a tetraploid barley line, which has its chromosome number doubled with colchicine as this would be more tolerant to the addition of alien chromosomes. Utilization of barley-chromosome addition lines of wheat is an alternative. A set of these addition lines has been produced (Islam et al., 1975), and it would be possible to manipulate the homoeologous barley and L. racemosus chromosomes in wheat fi rst (by crossing the corresponding barley and L. racemosus chromosome addition lines and generating the required centromeric translocation) and then transferring the translocation into barley by crossing the tetraploid barley chromosome substitution line with cultivated diploid barley. Selected case studies presented above suggest that the BNI function merits exploitation as a trait in conventional breeding programs to introduce/improve/strengthen the BNI capacity of major food and feed crops (Subbarao et al., 2009b).

3. CONCLUDING REMARKS Modern agricultural systems are dependent on large inputs of mineral N as the primary N source (De Wit et al., 1987; Subbarao et al., 2006a). Concurrently, with this large increase in the use of N there has also been major changes in cropping systems (i.e. moving away from diverse crop rotations to monocultures) and agricultural practices that have resulted in our present highly productive agricultural systems, which are also high-nitrifying systems (Poudel et al., 2002). Many of the high-yielding crop varieties that were bred for these high production environments were also inadvertently selected for their preference for NO3

– over NH4

+. It also appears that most of our staple food crops lack any functional BNI capacity. These factors taken together seem to have contributed signifi cant selective pressure for the development of the current nitrifi cation-dominated N-cycle found in agricultural systems (Figures 1, 17). These systems tend to be ineffi cient in using fertilizer-N and prone to N loss, resulting in serious environmental problems (Tilman et al., 2001; Hungate et al., 2003; Schlesinger, 2009).

Of the several practices used for increasing the effi ciency of N use in agriculture, the introduction of the BNI capacity from the relatives of major crops and pastures could be part of a powerful strategy for limiting nitrifi cation in agricultural systems. A genetic exploitation of the BNI capacity and the preference for NH4

+, found in the wild relatives of some crops (such as wild-wheat, L. racemosus) and forage grasses (e.g. Brachiaria sp.) could provide a biological option to deliver BNI activity to these highly –productive agricultural systems. The next-generation of crops and forage grasses should be developed with this built-in BNI capacity as an integral part of the strategies to improve N-cycling effi ciency in agriculture and to reduce the negative impact of human activities on global environment.

Recent fi ndings indicate that a number of diverse chemical molecules with an inhibitory effect on Nitrosomonas sp. can be produced and released by plant roots. The AMO enzyme (the critical enzyme involved in Nitrosomonas for ammonia oxidation) has a high affi nity for a wide range of substrates in addition to


ammonium as their primary substrate (Hauck, 1980; McCarty, 1999). This unique feature of the AMO enzyme has been exploited during the development of synthetic chemical nitrifi cation inhibitors (Subbarao et al., 2006a). Recent fi ndings indicate that biological molecules with diverse chemical structure inhibit nitrifi er activity possibly by interfering with the functioning of the AMO enzymatic pathway. Beyond this, future research could also relate to the second stage of nitrifi cation concerning the Nitrobacter bacteria. There is thus an enormous potential for the identifi cation and utilization of new biological molecules with novel chemical structures that are powerful nitrifi cation inhibitors. This potential is available for exploitation for the development of a range of biological and chemical strategies to control nitrifi cation in agricultural systems.

The evidence indicates that there are potential differences in N2O emissions among plant species (Figure 12), linked to their varying BNI capacities (Subbarao et al., 2009a). The comparison of inhibiting and non-inhibiting grass ecotypes in savanna ecosystems showed that the denitrifi cation potential was about 10-fold lower in the low nitrifying sites than that in the high-nitrifying sites (Lata et al., 2004). Presently, such differences are not considered by the Intergovernmental Panel on Climate Change in the estimation of the projected N2O emissions from agricultural systems (Stehfest and Bouwman, 2006). For example, there are >250 million ha (Mha) of South American Savannas occupied by native grass or pastures of the introduced grasses such as Brachiaria spp. (Fisher et al., 1994). These pastures are low nitrifying (Subbarao et al., 2007a) and low N2O emitting type systems. However, if these grasslands were brought under agricultural production with crops such as soybean, maize, upland rice or other crops that lack the BNI

Figure 17. The current agricultural production systems largely driven by industrially fi xed nitrogen with a very high risk for leaking reactive nitrogen to the environment

26 Subbarao et al.

capacity (Subbarao et al., 2007a), this would have major implications for N2O emissions and the leakage of reactive N into the environment. Approximately 11 Mha of grassland in the Cerrados region of Brazil has already been converted to soybean and maize cultivation (Zimmer et al., 2004), and an additional 35 to 40 Mha could suffer such conversion in the near future. Such land-use changes could have major consequences for N2O emissions from this region, in addition to the leaching of nitrate. A major research objective for the use of low fertility acid soils in Latin America is to improve the economic and ecological sustainability of the crop-livestock systems (Guimaraes et al., 2004; Rao et al., 2004; Amezquita et al., 2007; Ayarza et al., 2007). Acid soil-adapted forage grass with the BNI capacity could suppress soil nitrifi cation and reduce N2O emissions to improve N-use effi ciency of crop-livestock systems.

4. FUTURE OUTLOOKThe availability of large amounts of fi xed nitrogen (i.e. N fertilizer) from industrial-scale production using the Haber-Bosch process, has been a major driver of the green revolution and is largely responsible for the doubling of food grain production during the last-half century. Currently, fi xed N inputs into agricultural systems (about 150 Tg.yr–1) exceeds the total amount of N fi xed by all the natural systems of our planet (about 100 Tg.yr–1) (Vitousek et al., 1997; Tilman et al., 2001). Fertilization is responsible for the massive amounts of reactive N (reduced forms of N, i.e. nitrogen fertilizer) (Liu et al., 2010) that is moving through the agricultural ecosystems that make up only 11% of the earth’s surface (Newbould, 1989) (Figures 1, 17). A major portion (>90%) of this reactive N (i.e. N-fertilizer) goes through nitrifi cation under the current agricultural production systems (Northup et al., 1995; Nasholm et al., 1998). This is largely due to the failure of production systems to limit nitrifi cation, resulting in high-nitrifying systems with a high potential for reactive N leakage into the environment. This has a cascading effect on the ecosystem functioning, services and human health (Galloway et al., 2008; Schlesinger, 2009).

Fertilizer-N consumption is expected to reach 300 Tg-y–1 by 2050 from the present 150 Tg.y–1 (IFA, 2005; Schlesinger, 2009; Charles et al., 2010). Environmental damage will result, given the pervasive ineffi ciencies of crops in N use (<40% of applied N is recovered by most fi eld crops) (Hauck, 1990; Smil, 1999; Schlesinger, 2009; Ju et al., 2009), due largely to nitrifi cation and its associated processes (Subbarao et al., 2006a; Galloway et al., 2008). The economic implications of this ‘wasted N” can be enormous; it is expected to reach close to 81 billion US$ at the current fertilizer prices (estimated at 450 US$ per Mg of urea-N) from the lost fertilizer alone, leaving aside the potential environmental pollution and the ecological destruction caused. Nitrous oxide emissions from agricultural systems are expected to reach close to 38.6 Tg of N per year by 2050, contributing signifi cantly to global warming (Kroeze, 1994; Smith et al., 1997; IPCC, 2007; Schlesinger, 2009).

Given our current environmental concerns, it is highly desirable to develop new technologies and approaches for combating the rampant and rapid nitrifi cation in agricultural systems, to reduce N pollution and improve NUE (Harrison et al., 2000; Galloway et al., 2008; Fedoroff et al., 2010; Nordhaus, 2010; Tester and


Langridge, 2010). Exploiting the naturally occurring BNI function in plants to suppress nitrifi cation, would be a step towards the development of N-use effi cient food and feed production systems. The evidence presented in this bulletin on BNI function points toward the potential for genetically exploiting the BNI capacity in crops and pasture grasses. Development of improved forage grasses for low-nitrifying pasture-based production systems is possible given the signifi cant genetic variability found for the BNI function within Brachiaria spp. (Subbarao et al., 2007a). Also, introducing high BNI capacity from wild wheat (Leymus racemosus) into cultivated wheat could be an option in the foreseeable future (Subbarao et al., 2007b; Zahn, 2007).

From a broader ecological perspective, BNI-process should be integrated at the ecosystem level to close the N-cycle. A major question remains from an evolutionary viewpoint as to under what environmental conditions/perturbations can BNI-plants outcompete non-inhibiting plants? It is hypothesized that the ability to depress nitrifi cation should give a crucial competitive advantage to plant populations for N acquisition. This competitive advantage could explain the invasive dynamics of some African tropical pasture grasses in South America or Australia. Another example is the potential importance of the BNI in understanding the plant/bacteria competition, co-evolution and succession. Experiments on competition among grasses have shown that plants with high BNI-capacity often out-compete those without BNI-capacity (Lata, 1999) (Figure 13).

A fundamental shift toward NH4+-dominated crop N nutrition can be achieved

by using crops and pasture grasses that have high BNI-capacity, which can benefi t both agriculture and the environment. We hope that this bulletin will bring awareness about the ineffi cient utilization of N in the current agricultural production systems that use large amounts of reactive N to drive food production. The fundamental weakness of the present production systems is the dominance of nitrifi cation in the soil N cycle. This needs to be effectively curbed to reduce/minimize the leakage of reactive N to the environment, a critical requirement for improving NUE of agricultural systems. The BNI-function/trait should be exploited in major fi eld crops and pastures to bring a balance in soil N forms (i.e., NH4

+ vs NO3–)

that could increase NUE while reducing N pollution from agricultural and agro-pastoral production systems.

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Biological Nitrifi cation Inhibition A

ING Bulletins on Regional Assessment of Reactive Nitrogen (2010)

1. Nitrogen Management in Rice-Wheat Cropping Systems of Asia and its Environmental and Health Implications

Rajendra Prasad, Himanshu Pathak and J.K. Ladha Editor: Bijay Singh2. Nitrogen Losses, N-Use Efficiency and N-Management in Rice and Rice-based Cropping System T.K. Adhya, Arvind K. Shukla and D. Panda Editor: Bijay Singh3. Reactive Nitrogen Emissions from Crop and Livestock Farming in India Viney P. Aneja, William H. Schlesinger, Jan Willem Erisman, Mukesh Sharma,

Sailesh N. Behera and William Battye Editor: Himanshu Pathak4. Atmospheric Deposition of Reactive Nitrogen over Continental Sites and Oceanic

Regions of India: A Review R. Rengarajan, B. Srinivas and M.M. Sarin Editor: Himanshu Pathak5. Nitrogen and Sustainabilty Issues of Waste Management in Urban Ecosystems H.N. Chanakya and H.C. Sharatchandra Editor: N Raghuram 6. Role of Soil Nitrogen in Efficient Management of Fertilizer Nitrogen in India D.K. Benbi and Bijay Singh Editor: Himanshu Pathak7. Management of Solid Wastes in Agricultural Systems

for Nitrogen Conservation in India Reena Singh, Alok Adholeya and Deepak Pant Editor: N. Raghuram 8. Harnessing the Nitrogen Fixing Potential of Cyanobacteria in Integrated Nutrient Management

Strategies for Sustainable Agriculture Dolly Wattal Dhar, Radha Prasanna and K. Swarnalakshmi Editor: N. Raghuram 9. Nitrogen Use in Agriculture in Arid and Semiarid Regions–Indian Perspective K.L. Sharma, M. Maheswari, B. Venkateswarlu and Y.P. Abrol Editor: Bijay Singh10. Nitrogen in the North Indian Ocean M. Dileep Kumar Editor: N. Raghuram 11. Slow-Release and Controlled-Release Nitrogen Fertilizers Chandrika Varadachari and Harvey M. Goertz Editor: Bijay Singh12. Nutrient Dynamics under Shifting Agricultural (Jhum) Landscape in North-East India

and Linked Sustainability Issues P.S. Ramakrishnan Editor: Bijay Singh13. Nitrification: Is it a Strategic Point of Intervention to Limit Nitrogen Losses from Agricultural Systems? G.V. Subbarao, K.L. Sahrawat, K. Nakahara, M. Kishii, I.M. Rao, C.T. Hash, T.S. George,

W. Berry, J.C. Lata and O. Ito Editor: N. Raghuram 14. Green Manure Approaches to Crop Production and Sustainable Agriculture Yadvinder Singh, Bijay Singh and H.S. Thind Editor: N. Raghuram 15. Contribution of Energy Sector to Nitrogen Emissions S.K. Goyal and C.V. Chalapati Rao Editor: Himanshu Pathak16. N-Dynamics in the Coastal Regions of India T.K. Adhya, S. Adhikari, M. Muralidhar and S. Ayyappan Editor: Himanshu Pathak17. Hydrogen from Biomass Parag A. Deshpande, M.S. Hegde and Giridhar Madras Editor: Bijay Singh18. Physiological Approaches for Improving Nitrogen use Efficiency Altaf Ahmad, Lata, Vanita Jain and YP Abrol Editor: N. Raghuram 19. Greenhouse Gas Emission and Mitigation in Indian Agriculture – A Review H. Pathak, A. Bhatia, N. Jain and P.K. Aggarwal Editor: Bijay Singh20. Pathophysiology of Nitrate Toxicity in Human and its Mitigation Measures Sunil Gupta, R.C. Gupta, A.B. Gupta, E.V.S. Prakasa Rao, K. Puttanna and

Aditi Singhvi Editor: N. Raghuram

COMPREHENSIVE STATUS REPORT (2010)REACTIVE NITROGEN: GOOD, BAD AND UGLY – V. Balasubramanian

Other Publications from the Society for Conservation of Nature1. Policy Options for Efficient Nitrogen Use

Eds. Y.P. Abrol. and B.N. Johri2. Agricultural Nitrogen Use and Its Environmental Implications

Eds. Y.P. Abrol, N. Raghuram and M.S. Sachdev3. Reactive Nitrogen in Agriculture, Industry and Environment in India

Bijay Singh, M.K. Tiwari and Y.P. Abrol4. Reactive Nitrogen in Agriculture, Industry and Environment in India

Y.P. Abrol, N. Raghuram and H.N. Chanakya

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