The Open Agriculture Journal




ISSN: 1874-3315 ― Volume 13, 2019

Enhancing Non-symbiotic N2 Fixation in Agriculture



M. M. Ropera, V. V. S. R. Guptab, *
a CSIRO Agriculture, Private Bag No. 5, Wembley WA 6913 Australia
b CSIRO Agriculture, PMB No. 2, Glen Osmond SA 5064 Australia

Abstract

Much of the demand for nitrogen (N) in cereal cropping systems is met by using N fertilisers, but the cost of production is increasing and there are also environmental concerns. This has led to a growing interest in exploring other sources of N such as biological N2 fixation. Non-symbiotic N2 fixation (by free-living bacteria in soils or associated with the rhizosphere) has the potential to meet some of this need especially in the lower input cropping systems worldwide. There has been considerable research on non-symbiotic N2 fixation, but still there is much argument about the amount of N that can potentially be fixed by this process largely due to shortcomings of indirect measurements, however isotope-based direct methods indicate agronomically significant amounts of N2 fixation both in annual crop and perennial grass systems. New molecular technologies offer opportunities to increase our understanding of N2-fixing microbial communities (many of them non-culturable) and the molecular mechanisms of non-symbiotic N2 fixation. This knowledge should assist the development of new plant-diazotrophic combinations for specific environments and more sustainable exploitation of N2-fixing bacteria as inoculants for agriculture. Whilst the ultimate goal might be to introduce nitrogenase genes into significant non-leguminous crop plants, it may be more realistic in the shorter-term to better synchronise plant-microbe interactions to enhance N2 fixation when the N needs of the plant are greatest. The review explores possibilities to maximise potential N inputs from non-symbiotic N2 fixation through improved management practices, identification of better performing microbial strains and their successful inoculation in the field, and plant based solutions.

Keywords: Agriculture, associative, free-living, non-symbiotic, N2 Fixation.


Article Information


Identifiers and Pagination:

Year: 2016
Volume: 10
First Page: 7
Last Page: 27
Publisher Id: TOASJ-10-7
DOI: 10.2174/1874331501610010007

Article History:

Received Date: 6/11/2015
Revision Received Date: 24/3/2016
Acceptance Date: 2/4/2016
Electronic publication date: 13/05/2016
Collection year: 2016

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open-access license: This is an open access article licensed under the terms of the Creative Commons Attribution-Non-Commercial 4.0 International Public License (CC BY-NC 4.0) (https://creativecommons.org/licenses/by-nc/4.0/legalcode), which permits unrestricted, non-commercial use, distribution and reproduction in any medium, provided the work is properly cited.


* Address correspondence to this author at the CSIRO Agriculture PMB No. 2, Glen Osmond SA 5064 Australia; Tel: +61 8 8303 8579; Fax: +61 8 8303 8590; E-mail: Gupta.Vadakattu@csiro.au




1. INTRODUCTION

Non-symbiotic (NS) N2 fixation includes N2 fixation by free-living soil bacteria (autotrophic and heterotrophic) that are not in a direct symbiosis with plants, and associative N2-fixation (e.g. associated with the rhizospheres of grasses and cereals). Free-living N2 fixation can also be associated with decomposing plant residues, aggregates with decomposable particulate organic matter and in termite habitats. A conceptual diagram of the different NS N2-fixing possibilities and their relationship with soil N cycle is presented in Fig. (1).

Globally, the demand for N fertilisers is expected to exceed 112 million tonnes in 2015 [1FAO. In: FAO Current world fertilizer trends and outlook to 2015, Rome, Food and Agriculture organization of the United Nations . 2015; pp. 1-41.] and much of this is produced by the Haber-Bosch process [2Jenkinson D. The impact of humans on the nitrogen cycle, with focus on temperate arable agriculture. Plant Soil 2001; 228(1): 3-15.
[http://dx.doi.org/10.1023/A:1004870606003]
], a process which uses large amounts of fossil fuel [3Jensen ES, Hauggaard-Nielsen H. How can increased use of biological N2 fixation in agriculture benefit the environment? Plant Soil 2003; 252(1): 177-86.
[http://dx.doi.org/10.1023/A:1024189029226]
]. This, together with the increasing demand for organically grown agricultural and horticultural products, and the need to address economic and environmental concerns, has rekindled interest in promoting biological N2 fixation in non-leguminous crops. Nitrogen is a critical element for sustainable agriculture but inappropriate use of fertiliser results in lower efficiency and has the potential to contribute to (1) greenhouse gas loads such as N2O thereby contributing to climate change, and (2) leaching of N from agricultural lands as NO-3 causing eutrophication of rivers, lakes and oceans, and reducing the quality of water supplies [4Good AG, Beatty PH. Fertilizing nature: a tragedy of excess in the commons. PLoS Biol 2011; 9(8): e1001124.
[http://dx.doi.org/10.1371/journal.pbio.1001124] [PMID: 21857803]
]. Since 1993, the use of N fertilisers in Australia has more than doubled (1.314 million tonnes of N in 2013 [5Australian Agricultural Commodity Statistics 2014. Available from http://www.agriculture.gov.au/abares/publications 2014.]) and more than doubled in China in the last 25 years [4Good AG, Beatty PH. Fertilizing nature: a tragedy of excess in the commons. PLoS Biol 2011; 9(8): e1001124.
[http://dx.doi.org/10.1371/journal.pbio.1001124] [PMID: 21857803]
]. The need to maximize the nutrient (N) inputs from natural processes such as biological N2 fixation is greater today than ever before [6Beatty PH, Good AG. Future prospects for cereals that fix nitrogen. Science 2011; 333(6041): 416-7.
[http://dx.doi.org/10.1126/science.1209467] [PMID: 21778391]
]. Recent increases in fossil fuel costs have resulted in significant increases in N fertiliser costs and the increased variability in rainfall patterns escalates the risk associated with higher input costs in the rainfed farming systems. Cleveland et al. [7Cleveland CC, Townsend AR, Schimel DS, et al. Global patterns of terrestrial biological nitrogen (N2) fixation in natural ecosystems. Global Biogeochem Cycles 1999; 13: 623-45.
[http://dx.doi.org/10.1029/1999GB900014]
] estimated that the potential global biological N2 fixation (symbiotic and NS) in natural ecosystems is between 100 and 290 million tonnes N year-1. In soils under agricultural production, estimates of biologically fixed N range from approximately 33 million tonnes N year-1 [8Smil V. Nitrogen in crop production: an account of global flows. Global Biogeochem Cycles 1999; 13(2): 647-62.
[http://dx.doi.org/10.1029/1999GB900015]
] to 50-70 million tonnes N year-1 [9Herridge DF, Peoples MB, Boddey RM. Global inputs of biological nitrogen fixation in agricultural systems. Plant Soil 2008; 311(1-2): 1-18.
[http://dx.doi.org/10.1007/s11104-008-9668-3]
].

This review summarizes the current knowledge on NS N2 fixation, including measurement techniques, factors that control the function, ecology of N2-fixing bacteria and identifies opportunities to harness this biological process for production and environmental benefits.

Fig. (1)

A conceptual diagram highlighting the diverse set of microsites that can support N2 fixation by NS N2-fixing bacteria and its role in the soil N cycle. (1) A diverse array of bacterial genera have been found in the rhizosphere and endophytic environment of a variety of cereals and other crop plants; the amount of N2 fixation in the field environment is yet to be properly quantified, (2) A significant amount of N2 fixation has been shown to occur in the below- and above-ground plant environments with sugarcane, (3) fresh decomposing residues, especially with wide C:N ratios, provide optimal conditions for N2 fixation, (4) stable aggregates not only provide protective sites for free-living, N2-fixing bacteria but also provide the required low oxygen conditions for nitrogenase activity, and (5) a diverse group of microflora in termite guts and nests possess nifH genes that show potential for N2 fixation in natural environments mainly in semi-arid and arid ecosystems.



2. CROP N DEMAND AND SUPPLY

It takes ~26 kg N to produce 1 t of wheat grain including straw [10Angus J. Nitrogen supply and demand in Australian agriculture. Aust J Exp Agric 2001; 41(3): 277-88.
[http://dx.doi.org/10.1071/EA00141]
]. Of this ~20 kg N is removed in every tonne of grain harvested, and hence, for grain yields between 2-8 t ha-1, this represents an annual removal of 40-160 kg N ha-1 [11Crews TE, Peoples MB. Can the synchrony of nitrogen supply and crop demand be improved in legume and fertilizer-based agroecosystems? A review. Nutr Cycl Agroecosyst 2005; 72(2): 101-20.
[http://dx.doi.org/10.1007/s10705-004-6480-1]
]. At peak demand in a growing crop, N demand exceeds supply from N mineralisation [10Angus J. Nitrogen supply and demand in Australian agriculture. Aust J Exp Agric 2001; 41(3): 277-88.
[http://dx.doi.org/10.1071/EA00141]
] and N fertilisers compensate for much of this shortfall.

In Australia since 2001, wheat yields averaged ~1.7 t/ha [5Australian Agricultural Commodity Statistics 2014. Available from http://www.agriculture.gov.au/abares/publications 2014.] and, based on the calculations above, this represents an average annual N removal of ~ 32 kg N ha-1. N fertiliser use across Australia ranged from 0.7 million tonnes in 2003 to 1.34 million tonnes in 2013 [5Australian Agricultural Commodity Statistics 2014. Available from http://www.agriculture.gov.au/abares/publications 2014.]. If biological systems could be manipulated to increase the inputs of N from NS N2 fixation it should be possible to reduce the requirement for industrially fixed N fertilisers.

Agronomically-significant amounts of N2 fixation (25-50 kg N ha-1 year-1) have been measured in C4 grasses including sugar cane [12Chalk P. The contribution of associative and symbiotic nitrogen fixation to the nitrogen nutrition of non-legumes. Plant Soil 1991; 132(1): 29-39.
[http://dx.doi.org/10.1007/BF00011009]
-16Weier K, Pittaway P, Wildin J. Role of N2-fixation in the sustainability of the ponded grass pasture system. Soil Biol Biochem 1995; 27(4): 441-5.
[http://dx.doi.org/10.1016/0038-0717(95)98616-V]
], but quantification of N2 fixation associated with cereals under natural conditions is limited [12Chalk P. The contribution of associative and symbiotic nitrogen fixation to the nitrogen nutrition of non-legumes. Plant Soil 1991; 132(1): 29-39.
[http://dx.doi.org/10.1007/BF00011009]
]. Kennedy and Islam [17Kennedy I, Islam N. The current and potential contribution of asymbiotic nitrogen fixation to nitrogen requirements on farms: a review. Aust J Exp Agric 2001; 41(3): 447-57.
[http://dx.doi.org/10.1071/EA00081]
] concluded that 10-30 kg N ha-1 crop-1 could be fixed by free-living and rhizosphere N2-fixing bacteria associated with wheat, and similar ranges were indicated by Dart [18Dart P. Nitrogen fixation associated with non-legumes in agriculture. Plant Soil 1986; 90: 303-34.
[http://dx.doi.org/10.1007/BF02277405]
] for cereal production systems in temperate and tropical environments. Field measurement of N2 fixation by free-living bacteria using cereal residues as an energy source indicated 1-12 kg N ha-1 fixed during short periods of 2-4 weeks [19Roper MM. Field measurements of nitrogenase activity in soils amended with wheat straw. Aust J Agric Res 1983; 34(6): 725-39.
[http://dx.doi.org/10.1071/AR9830725]
-21Roper MM, Turpin JE, Thompson JP. Nitrogenase activity (C2H2 reduction) by free-living bacteria in soil in a long-term tillage and stubble management experiment on a vertisol. Soil Biol Biochem 1994; 26(8): 1087-91.
[http://dx.doi.org/10.1016/0038-0717(94)90125-2]
], but where warm, moist conditions coincide with fresh stubbles, annual potentials of up to 38 kg N ha-1 year-1 have been calculated [22Gupta VVSR, Roper MM, Roget DK. Potential for non-symbiotic N2-fixation in different agroecological zones of southern Australia. Aust J Soil Res 2006; 44(4): 343-54.
[http://dx.doi.org/10.1071/SR05122]
]. Giller and Merckx [23Giller KE, Merckx R. Exploring the boundaries of N2-fixation in cereals and grasses: An hypothetical and experimental framework. Symbiosis 2003; 35(1-3): 3-17.], on the other hand, were less optimistic and estimated that inputs from NS N2 fixation are likely to be <5kg N ha-1 year-1. Kennedy and Islam [17Kennedy I, Islam N. The current and potential contribution of asymbiotic nitrogen fixation to nitrogen requirements on farms: a review. Aust J Exp Agric 2001; 41(3): 447-57.
[http://dx.doi.org/10.1071/EA00081]
] calculated that, based on an average yield of straw of 2 t ha-1 with a C content of 43%, a contribution of 50-150 kg N2 ha-1 fixed by free-living bacteria is theoretically possible, provided that metabolism of the straw is substantially directed towards N2 fixation.

Most estimates of NS N2 fixation have been determined by indirect measures such as C2H2 reduction or calculation from N balance. Rates of NS N2 fixation are significantly less than estimated N inputs from symbiotic N2 fixation which range from 2 to 284 kg N ha-1 year-1 in legume pastures [24Peoples M, Baldock JA. Nitrogen dynamics of pastures: nitrogen fixation inputs, the impact of legumes on soil nitrogen fertility, and the contributions of fixed nitrogen to Australian farming systems. Aust J Exp Agric 2001; 41(3): 327-46.
[http://dx.doi.org/10.1071/EA99139]
] and 0-271 kg N ha-1year-1 in grain legumes [25Evans J, McNeill A, Unkovich M, Fettell N, Heenan D. Net nitrogen balances for cool-season grain legume crops and contributions to wheat nitrogen uptake: a review. Aust J Exp Agric 2001; 41(3): 347-59.
[http://dx.doi.org/10.1071/EA00036]
, 26Unkovich M, Pate J, Hamblin J. The nitrogen economy of broadacre lupin in southwest Australia. Aust J Agric Res 1994; 45(1): 149-64.
[http://dx.doi.org/10.1071/AR9940149]
].

Many of the measurements of NS N2 fixation were made more than 20 years ago. Since then, across the world, there have been significant changes towards the adoption of conservation farming practices. No-tillage and stubble retention practices have been widely adopted [27Conant RT, Easter M, Paustian K, Swan A, Williams S. Impacts of periodic tillage on soil C stocks: A synthesis. Soil Tillage Res 2007; 95(1): 1-10.
[http://dx.doi.org/10.1016/j.still.2006.12.006]
, 28D'Emden F, Llewellyn R. No-tillage adoption decisions in southern Australian cropping and the role of weed management. Aust J Exp Agric 2006; 46(4): 563-9.
[http://dx.doi.org/10.1071/EA05025]
]. Farming systems worldwide during this time have moved towards intensive cropping systems and in particular intensive cereals in Australia. These changes have resulted in increases in productivity of more than 50% [5Australian Agricultural Commodity Statistics 2014. Available from http://www.agriculture.gov.au/abares/publications 2014.] with associated increases in nutrient and carbon turnover. All of these changes are likely to have significant impacts on NS N2 fixation through the provision of larger C resources for biological activity, and the creation and preservation of ideal soil conditions, e.g. habitable microsites for NS N2 fixation.

3. MOLECULAR ECOLOGY OF N2-FIXING (DIAZOTROPHIC) POPULATIONS

More than 50 different genera of culturable diazotrophic bacteria have been identified [17Kennedy I, Islam N. The current and potential contribution of asymbiotic nitrogen fixation to nitrogen requirements on farms: a review. Aust J Exp Agric 2001; 41(3): 447-57.
[http://dx.doi.org/10.1071/EA00081]
, 29Dalton H. The cultivation of diazotrophic microorganisms. Chichester: John Wiley & Sons 1980., 30Roper MM, Ladha J. Biological N2 fixation by heterotrophic and phototrophic bacteria in association with straw. Plant Soil 1995; 174: 211-24.
[http://dx.doi.org/10.1007/BF00032248]
], but more recently, new technologies have expanded our knowledge of NS microbial communities and their function. Molecular technologies utilising analysis of the nifH gene (a structural gene encoding for the highly conserved nitrogenase reductase) and stable isotope (15N2) probing have identified a suite of previously unrecognised diazotrophic microorganisms and helped to unravel the complexity of N2-fixing communities in a range of natural and agricultural ecosystems (e.g. [31Zehr JP, Jenkins BD, Short SM, Steward GF. Nitrogenase gene diversity and microbial community structure: a cross-system comparison. Environ Microbiol 2003; 5(7): 539-54.
[http://dx.doi.org/10.1046/j.1462-2920.2003.00451.x] [PMID: 12823187]
-35Roesch LF, Camargo FA, Bento FM, Triplett EW. Biodiversity of diazotrophic bacteria within the soil, root and stem of field-grown maize. Plant Soil 2008; 302(1-2): 91-104.
[http://dx.doi.org/10.1007/s11104-007-9458-3]
]). Varying growth requirements of a phylogenetically heterogeneous group of N2-fixing microorganisms have precluded cultivation of a significant proportion of these organisms. Notwithstanding this, there is a significant body of work cited by Buckley et al. [32Buckley DH, Huangyutitham V, Hsu S-F, Nelson TA. Stable isotope probing with 15N2 reveals novel noncultivated diazotrophs in soil. Appl Environ Microbiol 2007; 73(10): 3196-204.
[http://dx.doi.org/10.1128/AEM.02610-06] [PMID: 17369332]
] which suggests that these non-culturable diazotrophs may be the dominant components of N2-fixing communities in soils compared with their culturable cousins.

The great diversity of diazotrophic microorganisms ensures the adaptability of populations of N2-fixing microorganisms to a wide range of conditions. This is reflected in the studies by Bürgmann et al. [36Bürgmann H, Meier S, Bunge M, Widmer F, Zeyer J. Effects of model root exudates on structure and activity of a soil diazotroph community. Environ Microbiol 2005; 7(11): 1711-24.
[http://dx.doi.org/10.1111/j.1462-2920.2005.00818.x] [PMID: 16232286]
] and Zhang et al. [37Zhang L, Hurek T, Reinhold-Hurek B. A nifH-based oligonucleotide microarray for functional diagnostics of nitrogen-fixing microorganisms. Microb Ecol 2007; 53(3): 456-70.
[http://dx.doi.org/10.1007/s00248-006-9126-9] [PMID: 17186154]
] who observed that at any one time, those organisms actively fixing N2 represented only a very small subset of the total diazotrophic community. Recent research combining the use of isotopes with molecular studies is shedding new light on changes in the structure of free-living N2-fixing communities in soils and relating this to function [38Hsu S-F, Buckley DH. Evidence for the functional significance of diazotroph community structure in soil. ISME J 2009; 3(1): 124-36.
[http://dx.doi.org/10.1038/ismej.2008.82] [PMID: 18769458]
]. This type of diversity analysis should help identify which members of the bacterial community are contributing to the soil N cycle in different crops and environments.

The nitrogenase enzyme is mainly found in Bacteria and Archaea and predominantly in chemotrophs, phototrophs and heterotrophs [33Bürgmann H, Widmer F, Von Sigler W, Zeyer J. New molecular screening tools for analysis of free-living diazotrophs in soil. Appl Environ Microbiol 2004; 70(1): 240-7.
[http://dx.doi.org/10.1128/AEM.70.1.240-247.2004] [PMID: 14711647]
] in soils, termite guts, lakes, rivers, estuaries, algal mats and sediments and oligotrophic oceans. This enzyme complex is encoded by nifH, nifD and nifK genes, although nifH gene has been used as the signature gene for molecular diversity studies. By using the nifH sequences available in public databases, five major clusters with homology to nifH have been described [31Zehr JP, Jenkins BD, Short SM, Steward GF. Nitrogenase gene diversity and microbial community structure: a cross-system comparison. Environ Microbiol 2003; 5(7): 539-54.
[http://dx.doi.org/10.1046/j.1462-2920.2003.00451.x] [PMID: 12823187]
, 39Raymond J, Siefert JL, Staples CR, Blankenship RE. The natural history of nitrogen fixation. Mol Biol Evol 2004; 21(3): 541-54.
[http://dx.doi.org/10.1093/molbev/msh047] [PMID: 14694078]
, 40Gaby JC, Buckley DH. A global census of nitrogenase diversity. Environ Microbiol 2011; 13(7): 1790-9.
[http://dx.doi.org/10.1111/j.1462-2920.2011.02488.x] [PMID: 21535343]
]. Gaby and Buckely [40Gaby JC, Buckley DH. A global census of nitrogenase diversity. Environ Microbiol 2011; 13(7): 1790-9.
[http://dx.doi.org/10.1111/j.1462-2920.2011.02488.x] [PMID: 21535343]
] found that the diversity of diazotrophs, based on nifH sequence homology, is not distributed evenly across phylogenetic groups or environments, and that the majority of this diversity is still undiscovered, particularly in soils and in anaerobic environments. Diversity estimates (Chao 1 richness estimates) indicated that soils account for the highest diversity of diazotroph sequences compared to marine environments. Most diazotroph sequences from the α, β and γ Proteobacteria have been recovered from soils [40Gaby JC, Buckley DH. A global census of nitrogenase diversity. Environ Microbiol 2011; 13(7): 1790-9.
[http://dx.doi.org/10.1111/j.1462-2920.2011.02488.x] [PMID: 21535343]
]. Members of the Subcluster IA (e.g. members of (Δ-Proteobacteria) account for a large portion of nifH sequences in some soils and are actively involved in N2 fixation [38Hsu S-F, Buckley DH. Evidence for the functional significance of diazotroph community structure in soil. ISME J 2009; 3(1): 124-36.
[http://dx.doi.org/10.1038/ismej.2008.82] [PMID: 18769458]
]. Gupta et al. [41Gupta VVSR, Kroker S, Hicks M, Davoren CW, Descheemaeker K, Llewellyn RS. Nitrogen cycling in summer active perennial grass systems in South Australia: non-symbiotic nitrogen fixation. Crop Pasture Sci 2014; 65(10): 1044-56.
[http://dx.doi.org/10.1071/CP14109]
] found that members belonging to α-proteobacteria or Cluster Ik/j were the most abundant group in Australian wheat fields. They suggested that on a continental scale, habitat and environment determine the composition of the diazotrophic community, while plant type and management associated factors drive the composition, genetic potential and NS N2 fixation regionally and within fields.

The use of nifH gene analysis, using nifH amplicon sequencing and nifH microarray methods, of isolated organisms and entire N2-fixing microbial communities at the plant interface (rhizosphere, rhizoplane, and phyllosphere) have all shown that the nifH gene is widely distributed in phylogenetically diverse groups of bacteria and archaea [31Zehr JP, Jenkins BD, Short SM, Steward GF. Nitrogenase gene diversity and microbial community structure: a cross-system comparison. Environ Microbiol 2003; 5(7): 539-54.
[http://dx.doi.org/10.1046/j.1462-2920.2003.00451.x] [PMID: 12823187]
, 33Bürgmann H, Widmer F, Von Sigler W, Zeyer J. New molecular screening tools for analysis of free-living diazotrophs in soil. Appl Environ Microbiol 2004; 70(1): 240-7.
[http://dx.doi.org/10.1128/AEM.70.1.240-247.2004] [PMID: 14711647]
, 37Zhang L, Hurek T, Reinhold-Hurek B. A nifH-based oligonucleotide microarray for functional diagnostics of nitrogen-fixing microorganisms. Microb Ecol 2007; 53(3): 456-70.
[http://dx.doi.org/10.1007/s00248-006-9126-9] [PMID: 17186154]
, 40Gaby JC, Buckley DH. A global census of nitrogenase diversity. Environ Microbiol 2011; 13(7): 1790-9.
[http://dx.doi.org/10.1111/j.1462-2920.2011.02488.x] [PMID: 21535343]
] and that a large contingent of these organisms is non-culturable, e.g. in the rhizosphere of graminaceous plants [32Buckley DH, Huangyutitham V, Hsu S-F, Nelson TA. Stable isotope probing with 15N2 reveals novel noncultivated diazotrophs in soil. Appl Environ Microbiol 2007; 73(10): 3196-204.
[http://dx.doi.org/10.1128/AEM.02610-06] [PMID: 17369332]
, 34Hamelin J, Fromin N, Tarnawski S, Teyssier-Cuvelle S, Aragno M. nifH gene diversity in the bacterial community associated with the rhizosphere of Molinia coerulea, an oligonitrophilic perennial grass. Environ Microbiol 2002; 4(8): 477-81.
[http://dx.doi.org/10.1046/j.1462-2920.2002.00319.x] [PMID: 12153588]
, 42Hurek T, Handley LL, Reinhold-Hurek B, Piché Y. Azoarcus grass endophytes contribute fixed nitrogen to the plant in an unculturable state. Mol Plant Microbe Interact 2002; 15(3): 233-42.
[http://dx.doi.org/10.1094/MPMI.2002.15.3.233] [PMID: 11952126]
].

Other associations of N2-fixing bacteria with plants can be endophytic - both obligate and facultative (e.g. [35Roesch LF, Camargo FA, Bento FM, Triplett EW. Biodiversity of diazotrophic bacteria within the soil, root and stem of field-grown maize. Plant Soil 2008; 302(1-2): 91-104.
[http://dx.doi.org/10.1007/s11104-007-9458-3]
, 42Hurek T, Handley LL, Reinhold-Hurek B, Piché Y. Azoarcus grass endophytes contribute fixed nitrogen to the plant in an unculturable state. Mol Plant Microbe Interact 2002; 15(3): 233-42.
[http://dx.doi.org/10.1094/MPMI.2002.15.3.233] [PMID: 11952126]
-45James E. Nitrogen fixation in endophytic and associative symbiosis. Field Crops Res 2000; 65(2): 197-209.
[http://dx.doi.org/10.1016/S0378-4290(99)00087-8]
]), but whether their relationship with the plant is symbiotic or NS is uncertain [45James E. Nitrogen fixation in endophytic and associative symbiosis. Field Crops Res 2000; 65(2): 197-209.
[http://dx.doi.org/10.1016/S0378-4290(99)00087-8]
]. Endophytic bacteria are at an advantage compared with free-living or rhizosphere bacteria because they have ready access to carbon (energy source) nutrients and water from within the plant [46Wilson D. Endophyte: the evolution of a term, and clarification of its use and definition. Oikos 1995; 73: 274-6.
[http://dx.doi.org/10.2307/3545919]
] and are not vulnerable to competition from other microorganisms in the rhizosphere or soil. Therefore, such organisms are more likely to be successful as inoculants. As with non-endophyte diazotrophs, non-culturable N2-fixing microorganisms appear to be dominant. From phylogenetic analyses of nitrogenase sequences, Hurek et al. [42Hurek T, Handley LL, Reinhold-Hurek B, Piché Y. Azoarcus grass endophytes contribute fixed nitrogen to the plant in an unculturable state. Mol Plant Microbe Interact 2002; 15(3): 233-42.
[http://dx.doi.org/10.1094/MPMI.2002.15.3.233] [PMID: 11952126]
] predicted that non-culturable grass endophytes (such as Azoarcus sp.) are ecologically dominant and could play an important role in N2 fixation in natural grass ecosystems.

Termites and termite habitats are important components of soil food webs contributing to soil N cycle in rangelands and low rainfall regions in Australia, Africa and India. Molecular analysis using nifH PCR primers has extended our understanding of the diversity of N2-fixing communities in guts of termites that feed on cellulose substrates [31Zehr JP, Jenkins BD, Short SM, Steward GF. Nitrogenase gene diversity and microbial community structure: a cross-system comparison. Environ Microbiol 2003; 5(7): 539-54.
[http://dx.doi.org/10.1046/j.1462-2920.2003.00451.x] [PMID: 12823187]
, 47Okuma M. Molecular ecological studies on bacterial symbiosis in termite. Seibutsu-kogaku Kaishi 2007; 85: 215-7., 48Yamada A, Inoue T, Noda S, Hongoh Y, Ohkuma M. Evolutionary trend of phylogenetic diversity of nitrogen fixation genes in the gut community of wood-feeding termites. Mol Ecol 2007; 16(18): 3768-77.
[http://dx.doi.org/10.1111/j.1365-294X.2007.03326.x] [PMID: 17850544]
].

4. MEASUREMENT/QUANTIFICATION

Current methods of quantifying NS N2 fixation are far from perfect and measurement may be flawed if inappropriate techniques or inadequate controls are used. However, used appropriately, these techniques can provide some valuable insight into the role and importance of NS N2 fixation. New molecular technologies promise further advances and this will be addressed later in this review. Advantages and disadvantages of current techniques and their appropriate use are described here briefly.

4.1. C2H2 Reduction Assay

The C2H2 reduction assay (based on the reduction C2H2 to C2H4 by nitrogenase) is a rapid, sensitive, simple and low cost method which if used with appropriate controls and calibrations can be useful for evaluating nitrogenase activity in time and space [49Witty J. Acetylene reduction assay can overestimate nitrogen-fixation in soil. Soil Biol Biochem 1979; 11(2): 209-10.
[http://dx.doi.org/10.1016/0038-0717(79)90103-2]
]. Under controlled conditions it can be extremely useful for comparative purposes where absolute values of N2 fixation are not critical. Hardy et al. [50Hardy RW, Holsten RD, Jackson EK, Burns RC. The acetylene-ethylene assay for N2 fixation: laboratory and field evaluation. Plant Physiol 1968; 43(8): 1185-207.
[http://dx.doi.org/10.1104/pp.43.8.1185] [PMID: 16656902]
] found a direct correlation between N2 fixation (N2→2NH3) and C2H2→C2H4 in pure cultures of diazotrophs and in legumes, and calculated that the theoretical relationship of C2H2 reduced to N2 fixed was 3. However, measured values of conversion factors in different environments can vary widely; these are summarized in Table 1.

Such variations in ratios of C2H4 produced to N2 fixed have been the basis of most criticism of the acetylene reduction assay, although research has shown that for all systems including pure cultures, legumes, non-legumes and soils that this ratio averaged between 2.6 and 6.9 [51Hardy R, Burns R, Holsten R. Applications of the acetylene-ethylene assay for measurement of nitrogen fixation. Soil Biol Biochem 1973; 5(1): 47-81.
[http://dx.doi.org/10.1016/0038-0717(73)90093-X]
]. The only exception to this was anaerobic soil which had conversion factors of up to 25. Under other conditions a reasonable estimate is possible, although any experimental procedure should always include a calibration of the assay using 15N2 gas exposure [52Steyn PL, Delwiche CC. Nitrogen fixation by nonsymbiotic microorganisms in some California soils. Environ Sci Technol 1970; 4(12): 1122-8.
[http://dx.doi.org/10.1021/es60047a007]
-54Giller K. Use and abuse of the acetylene reduction assay for measurement of “associative” nitrogen fixation. Soil Biol Biochem 1987; 19(6): 783-4.
[http://dx.doi.org/10.1016/0038-0717(87)90066-6]
].

4.2. Use of 15N2 Gas as a Direct Measure of N2 Fixation

This method can be sensitive, accurate and provide absolute proof of N2 fixation and has been used to demonstrate N2 fixation associated with cereals and grasses [41Gupta VVSR, Kroker S, Hicks M, Davoren CW, Descheemaeker K, Llewellyn RS. Nitrogen cycling in summer active perennial grass systems in South Australia: non-symbiotic nitrogen fixation. Crop Pasture Sci 2014; 65(10): 1044-56.
[http://dx.doi.org/10.1071/CP14109]
, 55Giller K, Wani S, Day J, Dart P. Short-term measurements of uptake of nitrogen fixed in the rhizospheres of sorghum (Sorghum bicolor) and millet (Pennisetum americanum). Biol Fertil Soils 1988; 7(1): 11-5.
[http://dx.doi.org/10.1007/BF00260725]
, 56Witty J, Day J. Use of 15N2 in evaluating asymbiotic N2 fixation. 1978. In: IAEA; 21-25 November; Vienna, Austria. 1977.] and in soils [19Roper MM. Field measurements of nitrogenase activity in soils amended with wheat straw. Aust J Agric Res 1983; 34(6): 725-39.
[http://dx.doi.org/10.1071/AR9830725]
, 56Witty J, Day J. Use of 15N2 in evaluating asymbiotic N2 fixation. 1978. In: IAEA; 21-25 November; Vienna, Austria. 1977., 57Azam F, Mulvaney RL, Stevenson FJ. Quantification and potential availability of non-symbiotically fixed 15N in soil. Biol Fertil Soils 1988; 7(1): 32-8.
[http://dx.doi.org/10.1007/BF00260729]
]. It is a most useful method for calibrating other measures of N2 fixation. Although difficulties in controlling environmental conditions can be encountered when using the method to measure N2 fixation associated with plants, it has been successfully used to estimate N2 fixation with cereals and sorghum [55Giller K, Wani S, Day J, Dart P. Short-term measurements of uptake of nitrogen fixed in the rhizospheres of sorghum (Sorghum bicolor) and millet (Pennisetum americanum). Biol Fertil Soils 1988; 7(1): 11-5.
[http://dx.doi.org/10.1007/BF00260725]
, 56Witty J, Day J. Use of 15N2 in evaluating asymbiotic N2 fixation. 1978. In: IAEA; 21-25 November; Vienna, Austria. 1977.].

Table 1

Different values used for conversion factor from acetylene reduced to N2 fixed




Demonstrating the incorporation of 15N2 into free-living microbial populations in the soil is much more difficult. While N2 fixation in soils was confirmed both in the laboratory and in the field by incorporation of 15N2 [19Roper MM. Field measurements of nitrogenase activity in soils amended with wheat straw. Aust J Agric Res 1983; 34(6): 725-39.
[http://dx.doi.org/10.1071/AR9830725]
, 41Gupta VVSR, Kroker S, Hicks M, Davoren CW, Descheemaeker K, Llewellyn RS. Nitrogen cycling in summer active perennial grass systems in South Australia: non-symbiotic nitrogen fixation. Crop Pasture Sci 2014; 65(10): 1044-56.
[http://dx.doi.org/10.1071/CP14109]
], absolute measures of N2 fixation in the field using 15N2 gas are costly and are influenced by seasonal factors (M. M. Roper, G. L. Turner, F. J. Bergersen, unpublished data; [58Bei Q, Liu G, Tang H, et al. Heterotrophic and phototrophic 15N2 fixation and distribution of fixed 15 N in a flooded rice–soil system. Soil Biol Biochem 2013; 59: 25-31.
[http://dx.doi.org/10.1016/j.soilbio.2013.01.008]
]). Many free-living, diazotrophic bacteria require reduced oxygen concentrations to fix N2 and are located within microsites of low oxygen tension. Sites that restrict oxygen availability may also limit access by 15N2 and this could result in an underestimation of N2 fixation.

4.3. 15N Isotope Dilution and Natural Abundance (δ15N) to Measure Associative N2 Fixation

Both 15N isotope dilution and natural abundance methods depend upon differences in isotopic composition of the sources of N used for plant growth, i.e. atmospheric N, soil N and fertiliser N. Both methods require a non-N2-fixing reference plant and therefore it is essential that the reference plant and the test plant with associative N2 fixation have a similar root architecture and can extract N from the soil at the same rate in space and time [59Boddey RM, Chalk PM, Victoria R, Matsui E. The 15N-isotope dilution technique applied to the estimation of biological nitrogen fixation associated with Paspalum notatum cv. Batatais in the field. Soil Biol Biochem 1983; 15(1): 25-32.
[http://dx.doi.org/10.1016/0038-0717(83)90114-1]
]. Neither 15N method is suitable for quantification of the total amount of N2 fixation by free-living bacteria because of the difficulty of separating N2-fixing microorganisms from the soil for 15N analysis.

The 15N isotope dilution technique involves supplying a 15N enriched (or depleted) source of N to the soil so that it is significantly different from the natural abundance of the atmospheric N2 [59Boddey RM, Chalk PM, Victoria R, Matsui E. The 15N-isotope dilution technique applied to the estimation of biological nitrogen fixation associated with Paspalum notatum cv. Batatais in the field. Soil Biol Biochem 1983; 15(1): 25-32.
[http://dx.doi.org/10.1016/0038-0717(83)90114-1]
, 60Chalk PM. Estimation of N2 fixation by isotope dilution: An appraisal of techniques involving 15N enrichment and their application. Soil Biol Biochem 1985; 17(4): 389-410.
[http://dx.doi.org/10.1016/0038-0717(85)90001-X]
]. For accurate measurement, the spatial and temporal availability of the isotope should be uniform [60Chalk PM. Estimation of N2 fixation by isotope dilution: An appraisal of techniques involving 15N enrichment and their application. Soil Biol Biochem 1985; 17(4): 389-410.
[http://dx.doi.org/10.1016/0038-0717(85)90001-X]
]. The 15N isotope dilution method has been used to estimate N2 fixation associated with sugar cane, forage grasses, cereals and actinorhizal plants grown in soil, mostly in tropical systems.

The natural abundance (δ15N) method is exactly analogous to the isotope dilution method except that endogenous 15N in the soil is used [61Boddey RM, Polidoro JC, Resende AS, Alves BJ, Urquiaga S. Use of the 15N natural abundance technique for the quantification of the contribution of N2 fixation to sugar cane and other grasses. Aust J Plant Physiol 2001; 28(9): 889-95., 62Shearer G, Kohl D. Natural 15N abundance as a method of estimating the contribution of biologically fixed nitrogen to N2-fixing systems: Potential for non-legumes. Plant Soil 1988; 110(2): 317-27.
[http://dx.doi.org/10.1007/BF02226812]
]. The natural abundance method has an advantage over isotope enrichment methods in natural ecosystems because disturbance of the system is unnecessary [62Shearer G, Kohl D. Natural 15N abundance as a method of estimating the contribution of biologically fixed nitrogen to N2-fixing systems: Potential for non-legumes. Plant Soil 1988; 110(2): 317-27.
[http://dx.doi.org/10.1007/BF02226812]
], but other factors can affect δ15N in plants, such as N from precipitation (NOx, NH3), the depths in the soil from which N is taken up and the form of soil N that is used (organic N, NH4+ or NO3-) [63Högberg P. 15N natural abundance in soil–plant systems. New Phytol 1997; 137: 179-203.
[http://dx.doi.org/10.1046/j.1469-8137.1997.00808.x]
]. The ability of the natural abundance method to measure associative N2 fixation depends on N2 fixed by associative microorganisms being predominantly taken up by the plant rather than going into the soil N pool [62Shearer G, Kohl D. Natural 15N abundance as a method of estimating the contribution of biologically fixed nitrogen to N2-fixing systems: Potential for non-legumes. Plant Soil 1988; 110(2): 317-27.
[http://dx.doi.org/10.1007/BF02226812]
]. Application of the 15N natural abundance technique in oil palms in the field in Brazil identified diazotrophs with a high potential for N2 fixation, but estimates of N2 fixation could not be calculated because of the absence of a suitable reference plant [64de Carvalho AL, Alves BJ, Baldani VL, Reis VM. Application of 15N natural abundance technique for evaluating biological nitrogen fixation in oil palm ecotypes at nursery stage in pot experiments and at mature plantation sites. Plant Soil 2008; 302(1-2): 71-8.
[http://dx.doi.org/10.1007/s11104-007-9456-5]
] highlighting a significant limitation of this method.

4.4. N Budget (N2 Fixed by Difference)

Giller and Merckx [23Giller KE, Merckx R. Exploring the boundaries of N2-fixation in cereals and grasses: An hypothetical and experimental framework. Symbiosis 2003; 35(1-3): 3-17.] suggested that the ultimate test of the contribution of N from fixation is to measure net inputs of N over long periods (>10 years) in the field, i.e. an N budget. However, this may be difficult as it requires measuring all inputs and outputs of N over this period, including inputs from fertilisers, wet N deposition, dry N deposition, run-on and uptake from lateral flow, outputs from crop/animal removal, gaseous losses, N leaching and soil erosion. A number of studies using long-term field experimental data have shown considerable N gains which were attributed to inputs from NS N2 fixation (for example [18Dart P. Nitrogen fixation associated with non-legumes in agriculture. Plant Soil 1986; 90: 303-34.
[http://dx.doi.org/10.1007/BF02277405]
, 22Gupta VVSR, Roper MM, Roget DK. Potential for non-symbiotic N2-fixation in different agroecological zones of southern Australia. Aust J Soil Res 2006; 44(4): 343-54.
[http://dx.doi.org/10.1071/SR05122]
, 65Boddey RM, De Oliveira O, Urquiaga S, et al. Biological nitrogen fixation associated with sugar cane and rice: contributions and prospects for improvement. Plant Soil 1995; 174(1-2): 195-209.
[http://dx.doi.org/10.1007/BF00032247]
-67Shabaev V. The effect of cropping and fertiliser nitrogen rates on nitrogen balance in soil. Plant Soil 1986; 91(2): 249-56.
[http://dx.doi.org/10.1007/BF02181792]
]).

In the Rothamsted Broadbalk experiment, during the period from 1852-1967, Jenkinson [68Jenkinson D. Rothamsted Rep 1976 Part 2. Dorking, UK. 1976; pp. 103-09.] calculated that inputs from N2 fixation were between 18-28 kg N ha-1 year-1 in a plot that received no fertiliser, and 23-35 kg N ha-1 year-1 in a plot that received inorganic fertiliser without N. These estimates were obtained after adjusting for wet deposition from rainfall (5 kg N ha-1 year-1), dry N deposition (10 kg N ha-1 year-1) and for inputs in the seed (3 kg N ha-1 year-1) all of which were measured at some time during the course of the experiment [68Jenkinson D. Rothamsted Rep 1976 Part 2. Dorking, UK. 1976; pp. 103-09.]. Estimates indicate N deposition in rainfall and dust may range between 3-5 kg N ha-1 year-1 over much of Africa and Australia, and 10-50 kg N ha-1 year-1 in more densely populated areas such as in Europe [23Giller KE, Merckx R. Exploring the boundaries of N2-fixation in cereals and grasses: An hypothetical and experimental framework. Symbiosis 2003; 35(1-3): 3-17., 69McNeill A, Unkovich M. Nutrient cycling in terrestrial ecosystems. Berlin: Springer-Verlag 2007; pp. 37-64.
[http://dx.doi.org/10.1007/978-3-540-68027-7_2]
].

To achieve a reliable N balance it is necessary to have a very high repeatability and accuracy of N measurements through strict sampling protocols and extremely high sample numbers to enable the mean soil N to be precise enough to determine statistically significant changes in soil N [70Chalk P. Dynamics of biologically fixed N in legume-cereal rotations: a review. Aust J Agric Res 1998; 49: 303-16.
[http://dx.doi.org/10.1071/A97013]
, 71Vallis I. Sampling for soil nitrogen changes in large areas of grazed pastures. Commun Soil Sci Plant Anal 1973; 4(2): 163-70.
[http://dx.doi.org/10.1080/00103627309366430]
]. From a scientific viewpoint, N balance studies only give an indirect measure of N gains due to NS N2 fixation which can be useful for supporting other more direct measures. However, from a grower’s perspective, statistically significant measures of N accumulation provide valuable information for planning N fertiliser inputs for a crop.

4.5. Use of Multiple Techniques and New Methods

Used in conjunction with other measures such as the C2H2 reduction assay, N budgets may increase the certainty of estimates. For example, Shearman et al. [72Shearman RC, Pedersen WL, Klucas RV, Kinbacher EJ. Nitrogen fixation associated with ‘Park’ Kentucky bluegrass (Poa pratensis L.). Can J Microbiol 1979; 25(10): 1197-200.
[http://dx.doi.org/10.1139/m79-186] [PMID: 394819]
] found that in grass pastures inoculated with a range of known N2-fixing bacteria, rates of C2H2 reduction were strongly correlated (r=0.92) with N accumulation measured by the Kjeldahl method. 15N aided N balance studies have been used to strengthen evidence for associative biological N2 fixation in sugar cane [65Boddey RM, De Oliveira O, Urquiaga S, et al. Biological nitrogen fixation associated with sugar cane and rice: contributions and prospects for improvement. Plant Soil 1995; 174(1-2): 195-209.
[http://dx.doi.org/10.1007/BF00032247]
, 73Lima E, Boddey RM, Döbereiner J. Quantification of biological nitrogen fixation associated with sugar cane using a 15N aided nitrogen balance. Soil Biol Biochem 1987; 19(2): 165-70.
[http://dx.doi.org/10.1016/0038-0717(87)90077-0]
, 74Urquiaga S, Cruz KH, Boddey RM. Contribution of nitrogen fixation to sugar cane: nitrogen-15 and nitrogen-balance estimates. Soil Sci Soc Am J 1992; 56(1): 105-14.
[http://dx.doi.org/10.2136/sssaj1992.03615995005600010017x]
], where there was good agreement between estimates of biological N2 fixation from N balance and isotope dilution. However, the authors were careful not to assume the same rates of fixation occurred in the field because the conditions of the experiment differed from those in the field [73Lima E, Boddey RM, Döbereiner J. Quantification of biological nitrogen fixation associated with sugar cane using a 15N aided nitrogen balance. Soil Biol Biochem 1987; 19(2): 165-70.
[http://dx.doi.org/10.1016/0038-0717(87)90077-0]
].

Much of the information on estimates of N2 fixation using techniques that are currently available apply to one instant in space and time or over a short period of assay [58Bei Q, Liu G, Tang H, et al. Heterotrophic and phototrophic 15N2 fixation and distribution of fixed 15 N in a flooded rice–soil system. Soil Biol Biochem 2013; 59: 25-31.
[http://dx.doi.org/10.1016/j.soilbio.2013.01.008]
]. However, knowledge of the conditions that favour N2 fixation and the rates at which fixation responds to changes in environmental conditions can be used to obtain estimates for a wider region if environmental conditions in those regions are known (e.g. meteorological records; cropping statistics and soil maps). Gupta et al. [22Gupta VVSR, Roper MM, Roget DK. Potential for non-symbiotic N2-fixation in different agroecological zones of southern Australia. Aust J Soil Res 2006; 44(4): 343-54.
[http://dx.doi.org/10.1071/SR05122]
] used this principle to derive estimates for parts of the southern agroecological zones of Australia. Using information from other studies on the effects of different soil moistures, temperatures and carbon sources, potential N2 fixation in different zones was determined using a spatial analytical tool (ArcviewGIS Spatial Analyst, v3.1). Use of this principle with a range of measurement strategies may provide useful information about regions that are most likely to benefit from NS N2 fixation and where new advances can be made.

New techniques using microarray technologies have the potential to simultaneously measure the dynamics and/or activities of most microbial populations in the complex soil environment [75Gentry TJ, Wickham GS, Schadt CW, He Z, Zhou J. Microarray applications in microbial ecology research. Microb Ecol 2006; 52(2): 159-75.
[http://dx.doi.org/10.1007/s00248-006-9072-6] [PMID: 16897303]
, 76He Z, Gentry TJ, Schadt CW, et al. GeoChip: a comprehensive microarray for investigating biogeochemical, ecological and environmental processes. ISME J 2007; 1(1): 67-77.
[http://dx.doi.org/10.1038/ismej.2007.2] [PMID: 18043615]
]. Zhang et al. [37Zhang L, Hurek T, Reinhold-Hurek B. A nifH-based oligonucleotide microarray for functional diagnostics of nitrogen-fixing microorganisms. Microb Ecol 2007; 53(3): 456-70.
[http://dx.doi.org/10.1007/s00248-006-9126-9] [PMID: 17186154]
], used a nifH-based short oligonucleotide microarray and showed that this technique allows quantification and mapping of the abundance, diversity and activities of N2-fixing populations. This approach is likely to assist in the identification of regions and managements that favour inputs of N from NS N2 fixation. With the availability of complete genomes for several diazotrophic rhizobacteria and our increased ability to conduct in-depth genomic and functional analysis of candidate genes, we can now interrogate the specific features of diazotrophic endophytes. Such information on the molecular mechanisms of NS N2 fixation should enable the development of agronomic options to improve N2 fixation in non-leguminous crops [77Santi C, Bogusz D, Franche C. Biological nitrogen fixation in non-legume plants. Ann Bot (Lond) 2013; 111(5): 743-67.
[http://dx.doi.org/10.1093/aob/mct048] [PMID: 23478942]
].

5. FACTORS AFFECTING NS N2 FIXATION

5.1. Soil and Environmental Factors

Edaphic, environmental and management factors have a significant impact on the composition of diazotrophic communities and potentially their function [41Gupta VVSR, Kroker S, Hicks M, Davoren CW, Descheemaeker K, Llewellyn RS. Nitrogen cycling in summer active perennial grass systems in South Australia: non-symbiotic nitrogen fixation. Crop Pasture Sci 2014; 65(10): 1044-56.
[http://dx.doi.org/10.1071/CP14109]
,78Reed SC, Cleveland CC, Townsend AR. Functional ecology of free-living nitrogen fixation: a contemporary perspective. Annu Rev Ecol Evol Syst 2011; 42: 489-512.
[http://dx.doi.org/10.1146/annurev-ecolsys-102710-145034]
, 79Wakelin S, Gupta VVSR, Forrester S. Regional and local factors affecting diversity, abundance and activity of free-living, N2-fixing bacteria in Australian agricultural soils. Pedobiologia (Jena) 2010; 53(6): 391-9.
[http://dx.doi.org/10.1016/j.pedobi.2010.08.001]
]. Varietal-based differences in the diversity of diazotrophic communities have been reported for wheat, barley, rice, sorghum [80Tan Z, Hurek T, Reinhold-Hurek B. Effect of N-fertilization, plant genotype and environmental conditions on nifH gene pools in roots of rice. Environ Microbiol 2003; 5(10): 1009-15.
[http://dx.doi.org/10.1046/j.1462-2920.2003.00491.x] [PMID: 14510855]
-82Gupta VV, Hicks M. Diversity and activity of free-living bacteria in south Australian soils 2011. Rhizosphere 3 International conference held during 25-30 September 2011; Perth, Australia. Available from: http://rhizosphere3.com/conference-program/rhizoabstract00331.] and perennial grasses [41Gupta VVSR, Kroker S, Hicks M, Davoren CW, Descheemaeker K, Llewellyn RS. Nitrogen cycling in summer active perennial grass systems in South Australia: non-symbiotic nitrogen fixation. Crop Pasture Sci 2014; 65(10): 1044-56.
[http://dx.doi.org/10.1071/CP14109]
]. Differences in soil environments at the micro-scale can also influence the composition of diazotrophic communities [83Widmer F, Shaffer BT, Porteous LA, Seidler RJ. Analysis of nifH gene pool complexity in soil and litter at a Douglas fir forest site in the Oregon cascade mountain range. Appl Environ Microbiol 1999; 65(2): 374-80.
[PMID: 9925556]
].

Nitrogenase proteins are extremely sensitive to O2 and on exposure to air they are rapidly and irreversibly inactivated [84Eady R. Methods for evaluating biological nitrogen fixation. Chichester: John Wiley & Sons 1980; pp. 213-64.]. Therefore, NS N2-fixing bacteria need mechanisms to exclude O2 for N2 fixation (nitrogenase activity) to occur. Some bacteria, e.g. Azotobacter, Azomonas, Beijerinckia and Derxia exclude O2 through rapid respiration or the formation of extracellular polysaccharide [29Dalton H. The cultivation of diazotrophic microorganisms. Chichester: John Wiley & Sons 1980., 85Postgate J. Biochemical and physiological studies with free-living, nitrogen-fixing bacteria. Plant Soil 1971; 35(1): 551-9.
[http://dx.doi.org/10.1007/BF02661878]
]. However, most culturable diazotrophic bacteria will only fix N2 under microaerophilic or anaerobic conditions [29Dalton H. The cultivation of diazotrophic microorganisms. Chichester: John Wiley & Sons 1980.]. Anaerobic conditions can be created by saturating soil moistures and substantial amounts of N2 fixation have been measured under these conditions [86Rice WA, Paul EA. The organisms and biological processes involved in asymbiotic nitrogen fixation in waterlogged soil amended with straw. Can J Microbiol 1972; 18(6): 715-23.
[http://dx.doi.org/10.1139/m72-114] [PMID: 4556097]
]. In aerated soils, aggregate formation in the soil allows microaerophilic and anaerobic conditions to coexist simultaneously under aerobic conditions. Furthermore, substrates such as dissolved organic C can be allocated into both aerobic and anaerobic fractions and processes [87Li C, Aber J, Stange F, Butterbach-Bahl K, Papen H. A process-oriented model of N2O and NO emissions from forest soils: 1. Model development. J Geophys Res 2000; 105(D4): 4369-84.
[http://dx.doi.org/10.1029/1999JD900949]
] and so, it is possible for soluble products from organic matter decomposed under aerobic conditions, to supply C energy to microaerophilic and anaerobic N2-fixing bacteria within aggregates.

The second major condition required for NS N2 fixation is the availability of C as an energy source. Free-living N2-fixing bacteria generally rely on decomposing plant material above and below ground from crops and pastures. Associative N2-fixing bacteria utilise root exudates within a rhizosphere association with plants and other organisms. In both environments, other microbial groups compete for limited energy resources. Endophytic N2-fixing bacteria, on the other hand, have ready access to C and nutrients from within the plant [46Wilson D. Endophyte: the evolution of a term, and clarification of its use and definition. Oikos 1995; 73: 274-6.
[http://dx.doi.org/10.2307/3545919]
].

Crop residues contain cellulose and hemicellulose which comprise 50-70% of its dry weight [88Harper SH, Lynch JM. The chemical components and decomposition of wheat straw leaves, internodes and nodes. J Sci Food Agric 1981; 32(11): 1057-62.
[http://dx.doi.org/10.1002/jsfa.2740321103]
]. A few species of N2-fixing bacteria (Azospirillum spp.) are able to use straw directly for fixation [89Halsall DM, Turner GL, Gibson AH. Straw and xylan utilization by pure cultures of nitrogen-fixing Azospirillum spp. Appl Environ Microbiol 1985; 49(2): 423-8.
[PMID: 16346730]
], but most N2-fixing bacteria rely on decomposition to smaller components by other organisms [90Adl SM. The Ecology of Soil Decomposition. Wallingford: CABI Publishing 2003; p. 335.
[http://dx.doi.org/10.1079/9780851996615.0000]
]. Almost all N2-fixing heterotrophic bacteria are able to utilise the products of cellulose decomposition including carbohydrates and some organic acids and alcohols [91Rao VR. Effect of carbon sources on asymbiotic nitrogen fixation in a paddy soil. Soil Biol Biochem 1978; 10(4): 319-21.
[http://dx.doi.org/10.1016/0038-0717(78)90029-9]
, 92Roper MM, Halsall DM. Use of products of straw decomposition by N2-fixing (C2H2-reducing) populations of bacteria in three soils from wheat-growing areas. Aust J Agric Res 1986; 37(1): 1-9.
[http://dx.doi.org/10.1071/AR9860001]
]. Rates of N2 fixation are proportional to the amount of crop residue available and to rates of decomposition [19Roper MM. Field measurements of nitrogenase activity in soils amended with wheat straw. Aust J Agric Res 1983; 34(6): 725-39.
[http://dx.doi.org/10.1071/AR9830725]
]. The retention of crop residues can alter the composition of diazotrophic community structure, increase nifH gene abundance and N2 fixation [82Gupta VV, Hicks M. Diversity and activity of free-living bacteria in south Australian soils 2011. Rhizosphere 3 International conference held during 25-30 September 2011; Perth, Australia. Available from: http://rhizosphere3.com/conference-program/rhizoabstract00331., 93Wakelin SA, Colloff MJ, Harvey PR, Marschner P, Gregg AL, Rogers SL. The effects of stubble retention and nitrogen application on soil microbial community structure and functional gene abundance under irrigated maize. FEMS Microbiol Ecol 2007; 59(3): 661-70.
[http://dx.doi.org/10.1111/j.1574-6941.2006.00235.x] [PMID: 17116166]
, 94Nelson DR, Mele PM. The impact of crop residue amendments and lime on microbial community structure and nitrogen-fixing bacteria in the wheat rhizosphere. Aust J Soil Res 2006; 44(4): 319-29.
[http://dx.doi.org/10.1071/SR06022]
]. Root exudates, e.g. carbon containing compounds and quorum-sensing compounds, have been shown to influence the composition and function of nifH communities in the rhizosphere, and N2 fixation rates of 4-20 kg N ha-1 year-1 were predicted by Jones et al. [95Jones DL, Farrar J, Giller KE. Associative nitrogen fixation and root exudation-What is theoretically possible in the rhizosphere? Symbiosis 2003; 35(1): 19-38.]. Gupta et al. [41Gupta VVSR, Kroker S, Hicks M, Davoren CW, Descheemaeker K, Llewellyn RS. Nitrogen cycling in summer active perennial grass systems in South Australia: non-symbiotic nitrogen fixation. Crop Pasture Sci 2014; 65(10): 1044-56.
[http://dx.doi.org/10.1071/CP14109]
] observed a plant-based selection of nifH communities in the root environments of different summer-active perennial grass species. They found that diversity of diazotrophic bacteria was significantly higher in the rhizosphere than in the roots and that both the rhizosphere and roots supported higher N2 fixation than in cropping soils during summer.

It is well known that inorganic mineral N in soil can inhibit N2 fixation by NS microorganisms [96Knowles R. Methods for evaluating biological nitrogen fixation. Chichester: John Wiley & Sons 1980; pp. 557-82.]. However, the dynamics of N2-fixing microbial populations are linked to available C:N ratios. For example, when C is abundant, excess ammonium N can be assimilated by other microbial populations allowing N2 fixation to occur, but with low C, excess ammonium N concentrations inhibit N2-fixing populations [97Kavadia A, Vayenas D, Pavlou S, Aggelis G. Dynamics of free-living nitrogen-fixing bacterial populations in antagonistic conditions. Ecol Modell 2007; 200(1): 243-53.
[http://dx.doi.org/10.1016/j.ecolmodel.2006.07.037]
]. In the presence of large amounts of crop residue with wide C:N ratios, decomposition can be slow. But the addition of N increases the rate of decomposition (making C available for use by N2-fixing bacteria [30Roper MM, Ladha J. Biological N2 fixation by heterotrophic and phototrophic bacteria in association with straw. Plant Soil 1995; 174: 211-24.
[http://dx.doi.org/10.1007/BF00032248]
, 90Adl SM. The Ecology of Soil Decomposition. Wallingford: CABI Publishing 2003; p. 335.
[http://dx.doi.org/10.1079/9780851996615.0000]
]. Other mineral nutrients may influence NS N2 fixation. Mo and Fe are components of the nitrogenase enzyme, but they are rarely limiting in natural environments [98Jensen V. Nitrogen fixation In: Volume I, Ecology. Oxford, UK: Clarendon Press 1981; pp. 30-56.]. On the other hand, applications of P can significantly increase NS N2 fixation in crops [99Reed SC, Cleveland CC, Townsend AR. Controls over leaf litter and soil nitrogen fixation in two lowland tropical rain forests. Biotropica 2007; 39(5): 585-92.
[http://dx.doi.org/10.1111/j.1744-7429.2007.00310.x]
, 100Smith VH. Effects of nitrogen: phosphorus supply ratios on nitrogen fixation in agricultural and pastoral ecosystems. Biogeochemistry 1992; 18(1): 19-35.
[http://dx.doi.org/10.1007/BF00000424]
] and in grasslands [101Eisele K, Schimel D, Kapustka L, Parton W. Effects of available P and N:P ratios on NS dinitrogen fixation in tallgrass prairie soils. Oecologia 1989; 79: 471-4.
[http://dx.doi.org/10.1007/BF00378663]
, 102Reed SC, Seastedt TR, Mann CM, Suding KN, Townsend AR, Cherwin KL. Phosphorus fertilization stimulates nitrogen fixation and increases inorganic nitrogen concentrations in a restored prairie. Appl Soil Ecol 2007; 36(2): 238-42.
[http://dx.doi.org/10.1016/j.apsoil.2007.02.002]
] particularly in nutrient poor soils. Reed et al. [99Reed SC, Cleveland CC, Townsend AR. Controls over leaf litter and soil nitrogen fixation in two lowland tropical rain forests. Biotropica 2007; 39(5): 585-92.
[http://dx.doi.org/10.1111/j.1744-7429.2007.00310.x]
] and Smith [100Smith VH. Effects of nitrogen: phosphorus supply ratios on nitrogen fixation in agricultural and pastoral ecosystems. Biogeochemistry 1992; 18(1): 19-35.
[http://dx.doi.org/10.1007/BF00000424]
] concluded that the strong inverse relationship between N2 fixation and mineral N content in the soil is mitigated by the availability of P.

High soil water contents have been used to promote N2 fixation in soils by reducing O2 at the sites of fixation [86Rice WA, Paul EA. The organisms and biological processes involved in asymbiotic nitrogen fixation in waterlogged soil amended with straw. Can J Microbiol 1972; 18(6): 715-23.
[http://dx.doi.org/10.1139/m72-114] [PMID: 4556097]
]. However, in unsaturated soils, it is important to maintain aggregate structure and O2 gradients. In disturbed soils in the laboratory, a minimum of 50% water holding capacity was required for nitrogenase activity [103Roper MM. Straw decomposition and nitrogenase activity (C2H2 reduction): effects of soil moisture and temperature. Soil Biol Biochem 1985; 17(1): 65-71.
[http://dx.doi.org/10.1016/0038-0717(85)90091-4]
], whereas in in situ assays in undisturbed soils in the field, nitrogenase activity occurred at soil water contents below 30% water holding capacity [19Roper MM. Field measurements of nitrogenase activity in soils amended with wheat straw. Aust J Agric Res 1983; 34(6): 725-39.
[http://dx.doi.org/10.1071/AR9830725]
]. In some environments, N2-fixing bacteria have adapted to harsh semi-arid environments e.g. lichens (containing cyanobacteria or other free-living bacteria in association with a fungus) [104Russow R, Veste M, Böhme F. A natural 15N approach to determine the biological fixation of atmospheric nitrogen by biological soil crusts of the Negev Desert. Rapid Commun Mass Spectrom 2005; 19(23): 3451-6.
[http://dx.doi.org/10.1002/rcm.2214] [PMID: 16261635]
, 105Rychert R, Skujiņš J, Sorensen D, Porcella D. Nitrogen fixation by lichens and free-living microorganisms in deserts 1978; 9: 20-30. USB/IBP Synth Ser.] and rhizosheaths around the roots of perennial grass species, and can contribute significant amounts of biologically fixed N [106Othman AA, Amer WM, Fayez M, Hegazi NA. Rhizosheath of Sinai desert plants is a potential repository for associative diazotrophs. Microbiol Res 2004; 159(3): 285-93.
[http://dx.doi.org/10.1016/j.micres.2004.05.004] [PMID: 15462528]
, 107Shane M, McCully M, Huang C, Cawthray G, Pate J, Lambers H. Sand-binding roots down-under: formation and functioning; Rhizosphere 2 International Conference 26-31 August; Montpellier, France. 2007.]. Rhizosheaths support enriched organic materials, greater water contents and a higher density of microorganisms including associative diazotrophs [106Othman AA, Amer WM, Fayez M, Hegazi NA. Rhizosheath of Sinai desert plants is a potential repository for associative diazotrophs. Microbiol Res 2004; 159(3): 285-93.
[http://dx.doi.org/10.1016/j.micres.2004.05.004] [PMID: 15462528]
] and up to 9 kg N ha-1 year-1 has been measured [108Tjepkema J, Burris R. Nitrogenase activity associated with some Wisconsin prairie grasses. Plant Soil 1976; 45(1): 81-94.
[http://dx.doi.org/10.1007/BF00011131]
].

N2 fixation has been shown to occur in situ in temperature extremes from near 0oC in Antarctica [109Fogg G, Stewart W. British Antartic Survey Bulletins. 1968; pp. 39-46., 110Horne A. The ecology of nitrogen fixation on Signy Island, South Orkney Islands. British Antartic Survey Bulletin 1972; 27: 1-18.] and in the Arctic [111Liengen T. Conversion factor between acetylene reduction and nitrogen fixation in free-living cyanobacteria from high arctic habitats. Can J Microbiol 1999; 45(3): 223-9.
[http://dx.doi.org/10.1139/w98-219]
] to desert environments where N2-fixing bacteria utilise morning dew or summer rains [105Rychert R, Skujiņš J, Sorensen D, Porcella D. Nitrogen fixation by lichens and free-living microorganisms in deserts 1978; 9: 20-30. USB/IBP Synth Ser.] but must survive during intervening hot dry conditions up to 60oC [98Jensen V. Nitrogen fixation In: Volume I, Ecology. Oxford, UK: Clarendon Press 1981; pp. 30-56.]. Laboratory experiments indicated that the most favourable temperatures for N2 fixation were between 30 and 35oC, with a range from 4-45oC [103Roper MM. Straw decomposition and nitrogenase activity (C2H2 reduction): effects of soil moisture and temperature. Soil Biol Biochem 1985; 17(1): 65-71.
[http://dx.doi.org/10.1016/0038-0717(85)90091-4]
]. The variation for the best temperature range for activity may depend upon the organisms present and the climatic conditions at each environment [98Jensen V. Nitrogen fixation In: Volume I, Ecology. Oxford, UK: Clarendon Press 1981; pp. 30-56., 103Roper MM. Straw decomposition and nitrogenase activity (C2H2 reduction): effects of soil moisture and temperature. Soil Biol Biochem 1985; 17(1): 65-71.
[http://dx.doi.org/10.1016/0038-0717(85)90091-4]
].

Soil characteristics can significantly alter the potential for NS N2 fixation. For example, nitrogenase activity by free-living bacteria extracted from soil is best at pH 7-7.5 regardless of the pH of the original soil [112Roper MM, Smith NA. Straw decomposition and nitrogenase activity (C2H2 reduction) by free-living microorganisms from soil: effects of pH and clay content. Soil Biol Biochem 1991; 23(3): 275-83.
[http://dx.doi.org/10.1016/0038-0717(91)90064-Q]
], and liming can increase the abundance of nifH-containing rhizobacteria in acidic soils [94Nelson DR, Mele PM. The impact of crop residue amendments and lime on microbial community structure and nitrogen-fixing bacteria in the wheat rhizosphere. Aust J Soil Res 2006; 44(4): 319-29.
[http://dx.doi.org/10.1071/SR06022]
]. Clays are highly reactive colloidal particles that interact strongly with microorganisms [113Marshall K. Clay mineralogy in relation to survival of soil bacteria. Annu Rev Phytopathol 1975; 13(1): 357-73.
[http://dx.doi.org/10.1146/annurev.py.13.090175.002041]
]. Roper and Smith [112Roper MM, Smith NA. Straw decomposition and nitrogenase activity (C2H2 reduction) by free-living microorganisms from soil: effects of pH and clay content. Soil Biol Biochem 1991; 23(3): 275-83.
[http://dx.doi.org/10.1016/0038-0717(91)90064-Q]
] observed that the presence of montmorillonite clay increased N2 fixation by free-living bacteria. Clays increase macroaggregate formation [114Denef K, Six J, Merckx R, Paustian K. Short-term effects of biological and physical forces on aggregate formation in soils with different clay mineralogy. Plant Soil 2002; 246(2): 185-200.
[http://dx.doi.org/10.1023/A:1020668013524]
] which creates sites of low O2 concentration [115Angert A, Luz B, Yakir D. Fractionation of oxygen isotopes by respiration and diffusion in soils and its implications for the isotopic composition of atmospheric O2. Global Biogeochem Cycles 2001; 15(4): 871-80.
[http://dx.doi.org/10.1029/2000GB001371]
] thus favouring N2 fixation. Microsites within intra-aggregate pore spaces and interior parts of aggregates not only provide suitable environments for nitrogenase activity but also protect bacteria from environmental extremes [116Gupta VVSR, Roper MM. Protection of free-living nitrogen-fixing bacteria within the soil matrix. Soil Tillage Res 2010; 109(1): 50-4.
[http://dx.doi.org/10.1016/j.still.2010.04.002]
]. Although halophilic bacteria (e.g. Halomonas maura) isolated from saline soils have been shown to contain nifH genes and fix N2 [117Argandoña M, Fernández-Carazo R, Llamas I, et al. The moderately halophilic bacterium Halomonas maura is a free-living diazotroph. FEMS Microbiol Lett 2005; 244(1): 69-74.
[http://dx.doi.org/10.1016/j.femsle.2005.01.019] [PMID: 15727823]
], there is little other information on the impact of salt on NS N2-fixing bacteria in terms of growth and N2 fixation [118Hassouna M, Madkour M, Helmi S, Yacout S. Salt tolerance of some free-living nitrogen fixers. Alex J Agric Res 1995; 40: 389-413.] particularly in agricultural soils.

Heavy metal (Zn, Cu, Ni, Cd, Cr, Pb, Hg, As) contamination can reduce abundance of NS N2-fixing bacteria [119Athar R, Ahmad M. Heavy metal toxicity: Effect on plant growth and metal uptake by wheat, and on free living Azotobacter. Water Air Soil Pollut 2002; 138(1-4): 165-80.
[http://dx.doi.org/10.1023/A:1015594815016]
, 120Oliveira A, Pampulha ME. Effects of long-term heavy metal contamination on soil microbial characteristics. J Biosci Bioeng 2006; 102(3): 157-61.
[http://dx.doi.org/10.1263/jbb.102.157] [PMID: 17046527]
] and N2-fixing activity [121Lorenz SE, Mcgrath SP, Giller KE. Assessment of free-living nitrogen fixation activity as a biological indicator of heavy metal toxicity in soil. Soil Biol Biochem 1992; 24(6): 601-6.
[http://dx.doi.org/10.1016/0038-0717(92)90086-D]
]. However, some strains of Azospirillum brasilense showed adaptation to heavy metals (Co, Cu and Zn [122Kamnev AA, Tugarova AV, Antonyuk LP, Tarantilis PA, Polissiou MG, Gardiner PH. Effects of heavy metals on plant-associated rhizobacteria: comparison of endophytic and non-endophytic strains of Azospirillum brasilense. J Trace Elem Med Biol 2005; 19(1): 91-5.
[http://dx.doi.org/10.1016/j.jtemb.2005.03.002] [PMID: 16240678]
]). The response of associative N2 fixation to heavy metals seems dependent on the tolerance of the plants themselves to the contaminant [123Christiansen-Weniger C, Groneman A, Van Veen J. Associative N2 fixation and root exudation of organic acids from wheat cultivars of different aluminium tolerance. Plant Soil 1992; 139(2): 167-74.
[http://dx.doi.org/10.1007/BF00009307]
]. For example, roots of aluminium-tolerant plants exude significantly higher amounts of low molecular weight dicarboxylic acids which not only chelate Al3+ protecting the plant, but are also C sources for Azospirillum spp. and other N2-fixing bacteria [36Bürgmann H, Meier S, Bunge M, Widmer F, Zeyer J. Effects of model root exudates on structure and activity of a soil diazotroph community. Environ Microbiol 2005; 7(11): 1711-24.
[http://dx.doi.org/10.1111/j.1462-2920.2005.00818.x] [PMID: 16232286]
, 91Rao VR. Effect of carbon sources on asymbiotic nitrogen fixation in a paddy soil. Soil Biol Biochem 1978; 10(4): 319-21.
[http://dx.doi.org/10.1016/0038-0717(78)90029-9]
].

5.2. Management Practices

Minimum tillage systems support the stability of the aggregates especially macroaggregates that are critical for the development and maintenance of microsites of reduced O2 tension and for protection against biocidal exposure [116Gupta VVSR, Roper MM. Protection of free-living nitrogen-fixing bacteria within the soil matrix. Soil Tillage Res 2010; 109(1): 50-4.
[http://dx.doi.org/10.1016/j.still.2010.04.002]
]. Any increase in soil disturbance reduces aggregation, reduces soil C and disrupts the soil pore network by which soil organisms interact [124Six J, Elliott E, Paustian K. Aggregate and soil organic matter dynamics under conventional and no-tillage systems. Soil Sci Soc Am J 1999; 63(5): 1350-8.
[http://dx.doi.org/10.2136/sssaj1999.6351350x]
-126Young I, Ritz K. Tillage, habitat space and function of soil microbes. Soil Tillage Res 2000; 53(3): 201-13.
[http://dx.doi.org/10.1016/S0167-1987(99)00106-3]
]. As a result of all these factors, NS N2 fixation under no-till is characteristically higher than in cultivated soils [126Young I, Ritz K. Tillage, habitat space and function of soil microbes. Soil Tillage Res 2000; 53(3): 201-13.
[http://dx.doi.org/10.1016/S0167-1987(99)00106-3]
]. However, biological changes in NS N2 fixation in response to adopting reduced/no- tillage practices can be slow sometimes taking several years to develop [20Roper M, Marschke G, Smith N. Nitrogenase activity (C2H2 reduction) in soils following wheat straw retention: effects of straw management. Aust J Agric Res 1989; 40(2): 241-53.
[http://dx.doi.org/10.1071/AR9890241]
, 116Gupta VVSR, Roper MM. Protection of free-living nitrogen-fixing bacteria within the soil matrix. Soil Tillage Res 2010; 109(1): 50-4.
[http://dx.doi.org/10.1016/j.still.2010.04.002]
, 127Lamb J, Doran J, Peterson G. Nonsymbiotic dinitrogen fixation in no-till and conventional wheat-fallow systems. Soil Sci Soc Am J 1987; 51(2): 356-61.
[http://dx.doi.org/10.2136/sssaj1987.03615995005100020018x]
].

In no-till systems, populations of soil macrofauna such as ants (and termites) and earthworms are generally more abundant [126Young I, Ritz K. Tillage, habitat space and function of soil microbes. Soil Tillage Res 2000; 53(3): 201-13.
[http://dx.doi.org/10.1016/S0167-1987(99)00106-3]
]. Significant amounts of N2 fixation can occur in the guts of earthworms [128Striganova B, Pantoshderimova T, Tiunov A. Comparative estimation of nitrogen-fixation activity in the intestine of various species of earthworms. Izv Akad Nauk Biol 1993; 2: 257-63.], termites [129Graber JR, Leadbetter JR, Breznak JA. Description of Treponema azotonutricium sp. nov. and Treponema primitia sp. nov., the first spirochetes isolated from termite guts. Appl Environ Microbiol 2004; 70(3): 1315-20.
[http://dx.doi.org/10.1128/AEM.70.3.1315-1320.2004] [PMID: 15006748]
, 130Lilburn TG, Kim KS, Ostrom NE, Byzek KR, Leadbetter JR, Breznak JA. Nitrogen fixation by symbiotic and free-living spirochetes. Science 2001; 292(5526): 2495-8.
[http://dx.doi.org/10.1126/science.1060281] [PMID: 11431569]
] and arthropods [131Nardi JB, Mackie RI, Dawson JO. Could microbial symbionts of arthropod guts contribute significantly to nitrogen fixation in terrestrial ecosystems? J Insect Physiol 2002; 48(8): 751-63.
[http://dx.doi.org/10.1016/S0022-1910(02)00105-1] [PMID: 12770053]
], e.g. 4-10 kg N ha-1 year-1 [131Nardi JB, Mackie RI, Dawson JO. Could microbial symbionts of arthropod guts contribute significantly to nitrogen fixation in terrestrial ecosystems? J Insect Physiol 2002; 48(8): 751-63.
[http://dx.doi.org/10.1016/S0022-1910(02)00105-1] [PMID: 12770053]
, 132Yamada A, Inoue T, Wiwatwitaya D, et al. Nitrogen fixation by termites in tropical forests, Thailand. Ecosystems (NY) 2006; 9(1): 75-83.
[http://dx.doi.org/10.1007/s10021-005-0024-7]
]. Crop rotations can profoundly modify the soil environment by influencing the removal of nutrients from the soil, return of crop residues (including quality and quantity), development and distribution of bio-pores, and dynamics of microbial communities [133Ball B, Bingham I, Rees R, Watson C, Litterick A. The role of crop rotations in determining soil structure and crop growth conditions. Can J Soil Sci 2005; 85(5): 557-77.
[http://dx.doi.org/10.4141/S04-078]
] and therefore, are likely to affect the potential for N2 fixation. Information on the impact of pesticides on NS N2 fixation is relatively sparse and the effects are mixed. Among the pesticides, herbicides appear to have least significant effects on soil organisms, whereas insecticides and especially copper fungicides can be quite toxic [134Bünemann E, Schwenke G, Van Zwieten L. Impact of agricultural inputs on soil organisms-a review. Aust J Soil Res 2006; 44(4): 379-406.
[http://dx.doi.org/10.1071/SR05125]
]. Fungicides (methyl N-(1H-benzimidazo-2yl) carbamate and tetramethylthiuram disulfide) [135Gaind S, Rathi MS, Kaushik BD, Nain L, Verma OP. Survival of bio-inoculants on fungicides-treated seeds of wheat, pea and chickpea and subsequent effect on chickpea yield. J Environ Sci Health B 2007; 42(6): 663-8.
[http://dx.doi.org/10.1080/03601230701465759] [PMID: 17701702]
] and the herbicide glyphosate [136Santos A, Flores M. Effects of glyphosate on nitrogen fixation of free-living heterotrophic bacteria. Lett Appl Microbiol 1995; 20(6): 349-52.
[http://dx.doi.org/10.1111/j.1472-765X.1995.tb01318.x]
] have been shown to have negative impacts on the abundance of diazotrophs, whereas other studies indicated stimulation of populations and activities of N2-fixing bacteria, e.g. with the insecticide (hexachlorcyclohexane) [137Das A, Mukherjee D. Insecticidal effects on soil microorganisms and their biochemical processes related to soil fertility. World J Microb Biot 1998; 14(6): 903-9.
[http://dx.doi.org/10.1023/A:1008820908917]
] and a range of herbicides [138Das AC, Debnath A. Effect of systemic herbicides on N2-fixing and phosphate solubilizing microorganisms in relation to availability of nitrogen and phosphorus in paddy soils of West Bengal. Chemosphere 2006; 65(6): 1082-6.
[http://dx.doi.org/10.1016/j.chemosphere.2006.02.063] [PMID: 16630642]
].

Associative N2 fixation has been suggested to be under the genetic control of the host plant [12Chalk P. The contribution of associative and symbiotic nitrogen fixation to the nitrogen nutrition of non-legumes. Plant Soil 1991; 132(1): 29-39.
[http://dx.doi.org/10.1007/BF00011009]
, 139Belimov A, Kunakova A, Vasilyeva N, et al. Relationship between survival rates of associative nitrogen-fixers on roots and yield response of plants to inoculation. FEMS Microbiol Ecol 1995; 17(3): 187-96.
[http://dx.doi.org/10.1111/j.1574-6941.1995.tb00142.x]
]. Differences in associative N2 fixation have been observed between different lines of rice [140Knauth S, Hurek T, Brar D, Reinhold-Hurek B. Influence of different Oryza cultivars on expression of nifH gene pools in roots of rice. Environ Microbiol 2005; 7(11): 1725-33.
[http://dx.doi.org/10.1111/j.1462-2920.2005.00841.x] [PMID: 16232287]
], wheat [82Gupta VV, Hicks M. Diversity and activity of free-living bacteria in south Australian soils 2011. Rhizosphere 3 International conference held during 25-30 September 2011; Perth, Australia. Available from: http://rhizosphere3.com/conference-program/rhizoabstract00331., 141Neal J, Larson RI. Acetylene reduction by bacteria isolated from the rhizosphere of wheat. Soil Biol Biochem 1976; 8(2): 151-5.
[http://dx.doi.org/10.1016/0038-0717(76)90081-X]
], maize and sorghum [142Krotzky A, Berggold R, Werner D. Analysis of factors limiting associative N2-Fixation (C2H2 reduction) with two cultivars of Sorghum nutans. Soil Biol Biochem 1986; 18(2): 201-7.
[http://dx.doi.org/10.1016/0038-0717(86)90028-3]
, 143Werner D, Berggold R, Jaeger D, et al. Plant, microbial and soil factors, determining nitrogen fixation in the rhizosphere. Z Pflanz Bodenkunde 1989; 152(2): 231-6.
[http://dx.doi.org/10.1002/jpln.19891520215]
], millet [144Bouton J, Albrecht S, Zuberer D. Screening and selection of pearl millet for root associated bacterial nitrogen fixation. Field Crops Res 1985; 11: 131-40.
[http://dx.doi.org/10.1016/0378-4290(85)90097-8]
], and among various species of grasses [41Gupta VVSR, Kroker S, Hicks M, Davoren CW, Descheemaeker K, Llewellyn RS. Nitrogen cycling in summer active perennial grass systems in South Australia: non-symbiotic nitrogen fixation. Crop Pasture Sci 2014; 65(10): 1044-56.
[http://dx.doi.org/10.1071/CP14109]
, 145Rönkkö R, Smolander A, Nurmiaho-Lassila E-L, Haahtela K. Frankia in the rhizosphere of nonhost plants: A comparison with root-associated N2-fixing Enterobacter, Klebsiella and Pseudomonas. Plant Soil 1993; 153(1): 85-95.
[http://dx.doi.org/10.1007/BF00010547]
] and weeds [146Conklin A Jr, Biswas P. A survey of asymbiotic nitrogen fixation in the rhizosphere of weeds. Weed Sci 1978; 26: 148-50.]. Characteristics which contribute to high N2-fixing genotypes include a reduced transpiration rate, lower numbers of stomata and increased root exudates with a high concentration of dicarboxylic acids [143Werner D, Berggold R, Jaeger D, et al. Plant, microbial and soil factors, determining nitrogen fixation in the rhizosphere. Z Pflanz Bodenkunde 1989; 152(2): 231-6.
[http://dx.doi.org/10.1002/jpln.19891520215]
, 147Krotzky A, Berggold R, Werner D. Plant characteristics limiting associative N2-fixation (C2H2-reduction) with two cultivars of Sorghum nutans. Soil Biol Biochem 1988; 20(2): 157-62.
[http://dx.doi.org/10.1016/0038-0717(88)90032-6]
]. Wood et al. [148Wood CC, Islam N, Ritchie RJ, Kennedy IR. A simplified model for assessing critical parameters during associative 15N2 fixation between Azospirillum and wheat. Aust J Plant Physiol 2001; 28(9): 969-74.] suggested that plants with an increased release of photosynthate to the rhizosphere should be a priority for the future development of broad-acre agricultural systems that are more self-sufficient for N nutrition.

6. TRANSFER OF FIXED N FROM DIAZOTROPHS TO PLANTS AND OTHER ORGANISMS

The transfer of N fixed to plants is likely to depend on the location at which N2 fixation occurs. Endophytic diazotrophs can supply biologically fixed N directly to the host [149Sturz A, Christie B, Nowak J. Bacterial endophytes: potential role in developing sustainable systems of crop production. Crit Rev Plant Sci 2000; 19(1): 1-30.
[http://dx.doi.org/10.1016/S0735-2689(01)80001-0]
], e.g. N2 fixation by endophytic bacteria associated with sugarcane can directly contribute more than half the crop’s N requirement [65Boddey RM, De Oliveira O, Urquiaga S, et al. Biological nitrogen fixation associated with sugar cane and rice: contributions and prospects for improvement. Plant Soil 1995; 174(1-2): 195-209.
[http://dx.doi.org/10.1007/BF00032247]
, 150Baldani JI, Reis VM, Baldani VL, Döbereiner J. Review: A brief story of nitrogen fixation in sugarcane-reasons for success in Brazil. Funct Plant Biol 2002; 29(4): 417-23.
[http://dx.doi.org/10.1071/PP01083]
]. Because endophytic diazotrophs have only been observed in intercellular spaces, vascular tissue, aerenchyma and dead cells and not within living host cells, James [45James E. Nitrogen fixation in endophytic and associative symbiosis. Field Crops Res 2000; 65(2): 197-209.
[http://dx.doi.org/10.1016/S0378-4290(99)00087-8]
] suggested that N transfer from these organisms is likely to be dependent on their death and release of fixed N. Transfer to plants of N fixed by diazotrophs or N contained in non-fixing microbial biomass in the soil or rhizosphere is also likely to be dependent on the death of these bacteria and release of ammonium or amino acids [151Lethbridge G, Davidson M. Root-associated nitrogen-fixing bacteria and their role in the nitrogen nutrition of wheat estimated by 15N isotope dilution. Soil Biol Biochem 1983; 15(3): 365-74.
[http://dx.doi.org/10.1016/0038-0717(83)90085-8]
, 152Lethbridge G, Davidson M. Microbial biomass as a source of nitrogen for cereals. Soil Biol Biochem 1983; 15(3): 375-6.
[http://dx.doi.org/10.1016/0038-0717(83)90086-X]
], although excretion of nitrogenous substances during bacterial growth can also occur (e.g. Beijerinckia derxii [153Miyasaka NR, Thuler DS, Floh EI, et al. During stationary phase, Beijerinckia derxii shows nitrogenase activity concomitant with the release and accumulation of nitrogenated substances. Microbiol Res 2003; 158(4): 309-15.
[http://dx.doi.org/10.1078/0944-5013-00209] [PMID: 14717451]
]; cyanobacteria [154Balachandar D, Kumar K, Kannaiyan S, Arulmozhiselvan K. Evaluating nitrogen transfer efficiency of immobilized cyanobacteria to rice seedlings by 15N technique. Int Rice Res Notes 2004; 29: 53-4.]).

In rhizosphere associations, N fixed can either be directly taken up by the plant or remain in the surrounding soil N pool (Fig. 1). There is little information about the proportions of N transfer to each of these pools. However, transfer of fixed N to plants from associative N2-fixing bacteria has been demonstrated using 15N2 by Giller et al. [55Giller K, Wani S, Day J, Dart P. Short-term measurements of uptake of nitrogen fixed in the rhizospheres of sorghum (Sorghum bicolor) and millet (Pennisetum americanum). Biol Fertil Soils 1988; 7(1): 11-5.
[http://dx.doi.org/10.1007/BF00260725]
, 155Giller K, Day J, Dart P, Wani S. A method for measuring the transfer of fixed nitrogen from free-living bacteria to higher plants using 15N 2. J Microbiol Methods 1984; 2(6): 307-16.
[http://dx.doi.org/10.1016/0167-7012(84)90049-6]
] and others reviewed by Boddey [156Boddey RM. Methods for quantification of nitrogen fixation associated with gramineae. Crit Rev Plant Sci 1987; 6(3): 209-66.
[http://dx.doi.org/10.1080/07352688709382251]
] and James [45James E. Nitrogen fixation in endophytic and associative symbiosis. Field Crops Res 2000; 65(2): 197-209.
[http://dx.doi.org/10.1016/S0378-4290(99)00087-8]
]. Release of N following the death of diazotrophic bacteria in the rhizosphere can be rapid due to wetting and drying cycles and microbial predation.

7. ENHANCING THE VALUE OF NS N2 FIXATION – A WAY FORWARD

Kennedy and Islam [17Kennedy I, Islam N. The current and potential contribution of asymbiotic nitrogen fixation to nitrogen requirements on farms: a review. Aust J Exp Agric 2001; 41(3): 447-57.
[http://dx.doi.org/10.1071/EA00081]
] expressed an optimism that up to half the N requirements of some cereal crops might be met from NS N2 fixation in the future through the use of genetic tools and inoculant biofertilisers. In addition, Beatty and Good [6Beatty PH, Good AG. Future prospects for cereals that fix nitrogen. Science 2011; 333(6041): 416-7.
[http://dx.doi.org/10.1126/science.1209467] [PMID: 21778391]
] proposed two other strategies (1) developing root nodule symbioses in important cereal crops such as wheat, rice and maize and (2) introducing nitrogenase genes into a plant organelle.

7.1. Inoculation

There have been many studies on inoculation with N2-fixing bacteria of non-legumes (predominantly cereals and grasses), with reported above- and below-ground increases in total plant growth and N content [157Bashan Y, Levanony H. Current status of Azospirillum inoculation technology: Azospirillum as a challenge for agriculture. Can J Microbiol 1990; 36(9): 591-608.
[http://dx.doi.org/10.1139/m90-105]
]. The most successful inoculation responses have been in pot trials under controlled conditions (e.g. [158Alam MS, Cui Z, Yamagishi T, Ishii R. Grain yield and related physiological characteristics of rice plants (oryza sativa L.) inoculated with free-living rhizobacteria. Plant Prod Sci 2001; 4: 125-30.
[http://dx.doi.org/10.1626/pps.4.126]
-161Yanni Y, El-Fattah F. Towards integrated biofertilization management with free living and associative dinitrogen fixers for enhancing rice performance in the Nile delta. Symbiosis 1999; 27(3-4): 319-31.], but inoculation experiments in the field have been less consistent [162Baldani V, Baldani J, Döbereiner J. Inoculation of field-grown wheat (Triticum aestivum) with Azospirillum spp. in Brazil. Biol Fertil Soils 1987; 4(1-2): 37-40.]. Andrews et al. [163Andrews M, James E, Cummings S, et al. Use of nitrogen fixing bacteria inoculants as a substitute for nitrogen fertiliser for dryland graminaceous crops: progress made, mechanisms of action and future potential. Symbiosis 2003; 35(1): 209-29.] concluded that currently no NS N2-fixing bacterial inoculant is available that can match the consistency of N fertilisers for reducing soil N deficiencies.

One of the difficulties of inoculating soils with bacteria is that the inoculants generally decline rapidly due to competition with the native microflora [164Rao VR, Jena P, Adhya T. Inoculation of rice with nitrogen-fixing bacteria-Problems and perspectives. Biol Fertil Soils 1987; 4(1-2): 21-6., 165Schank S, Smith R. Status and evaluation of associative grass-bacteria N-fixing systems in Florida. In: USA: Proceedings-Soil and Crop Science Society of Florida. 1984; 43: pp. 120-23.]. Inoculants compete with other microflora for available nutrients or become food for indigenous micro- and macro-fauna [166Gupta VVSR, Roper MM. Protozoan diversity in soil and its influence on microbial functions that determine plant growth. In: 17th World Congress of Soil Science Abstracts 2002; Vol 1: 275.]. Hence the ultimate test for even the most effective beneficial organism is the ability to survive and colonise plant roots in the presence of much larger populations of indigenous microorganisms [157Bashan Y, Levanony H. Current status of Azospirillum inoculation technology: Azospirillum as a challenge for agriculture. Can J Microbiol 1990; 36(9): 591-608.
[http://dx.doi.org/10.1139/m90-105]
]. Inoculum formulation and application technology, e.g. along with organic matter (compost or peat) or micro-granulated inoculum, are likely to be crucial for inoculant survival and success [167Fages J. An industrial view of Azospirillum inoculants: formulation and application technology. Symbiosis 1992; 13: 15-26.].

7.1.1. Endophytes and GMOs

Endophytes are more likely to be successful inoculants because they can escape competition from indigenous microflora and can directly access the required energy source from the plant. Increased success with endophytic N2-fixing inoculants may be possible through genetic manipulation. For example, An et al. [168An Q, Dong Y, Wang W, Li Y, Li J. Constitutive expression of the nifA gene activates associative nitrogen fixation of Enterobacter gergoviae 57-7, an opportunistic endophytic diazotroph. J Appl Microbiol 2007; 103(3): 613-20.
[http://dx.doi.org/10.1111/j.1365-2672.2007.03289.x] [PMID: 17714394]
] suggested that manipulation of the promoter of the nifA gene in a N2-fixing bacterium that has a high colonisation competence may achieve stable associative N2 fixation in cereals. A similar approach has been put forward by Bloemberg [169Bloemberg GV. Microscopic analysis of plant-bacterium interactions using auto fluorescent proteins. Eur J Plant Pathol 2007; 119: 301-9.
[http://dx.doi.org/10.1007/s10658-007-9171-3]
]. Other advances using molecular strategies may be possible, e.g. the creation of ammonium excreting mutant diazotrophs, in which the mechanisms by which ammonium inhibits N2 fixation, are disarmed [170Colnaghi R, Green A, He L, Rudnick P, Kennedy C. Strategies for increased ammonium production in free-living or plant associated nitrogen fixing bacteria. Plant Soil 1997; 194(1-2): 145-54.
[http://dx.doi.org/10.1023/A:1004268526162]
] or the increased production of nitrogenase reductase such as in an Azospirillum brasilense mutant [171de Campos SB, Roesch LF, Zanettini MH, Passaglia LM. Relationship between in vitro enhanced nitrogenase activity of an Azospirillum brasilense Sp7 mutant and its growth-promoting activities in situ. Curr Microbiol 2006; 53(1): 43-7.
[http://dx.doi.org/10.1007/s00284-005-0191-y] [PMID: 16775786]
]. However, the survival of such mutants in the field is uncertain [45James E. Nitrogen fixation in endophytic and associative symbiosis. Field Crops Res 2000; 65(2): 197-209.
[http://dx.doi.org/10.1016/S0378-4290(99)00087-8]
].

7.1.2. Inoculants With Dual Benefits

Greater benefits may be possible where inoculants have a dual benefit through increased N nutrition via N2 fixation coupled with the production of plant growth hormones. There are several groups of organisms that are known to fix N2, produce phytohormones and/or provide protection against fungal and bacterial pathogens [171de Campos SB, Roesch LF, Zanettini MH, Passaglia LM. Relationship between in vitro enhanced nitrogenase activity of an Azospirillum brasilense Sp7 mutant and its growth-promoting activities in situ. Curr Microbiol 2006; 53(1): 43-7.
[http://dx.doi.org/10.1007/s00284-005-0191-y] [PMID: 16775786]
-173Vessey JK. Plant growth promoting rhizobacteria as biofertilizers. Plant Soil 2003; 255(2): 571-86.
[http://dx.doi.org/10.1023/A:1026037216893]
]. Hafeez et al. [174Hafeez FY, Yasmin S, Ariani D, Zafar Y, Malik KA. Plant growth-promoting bacteria as biofertilizer. Agron Sustain Dev 2006; 26(2): 143-50.
[http://dx.doi.org/10.1051/agro:2006007]
] showed that amongst 17 rhizobacteria isolated from different ecological regions, 15 fixed N2 and all produced various concentrations of indole-3-acetic acid, and at least one of the isolates produced siderophores. Some actinobacteria (Microbacterium sp., Micromonospora sp. and Arthrobacter sp.) contain nifH genes and fix N2 [82Gupta VV, Hicks M. Diversity and activity of free-living bacteria in south Australian soils 2011. Rhizosphere 3 International conference held during 25-30 September 2011; Perth, Australia. Available from: http://rhizosphere3.com/conference-program/rhizoabstract00331., 175Gtari M, Ghodhbane-Gtari F, Nouioui I, Beauchemin N, Tisa LS. Phylogenetic perspectives of nitrogen-fixing actinobacteria. Arch Microbiol 2012; 194(1): 3-11.
[http://dx.doi.org/10.1007/s00203-011-0733-6] [PMID: 21779790]
], but can also colonise cereals as endophytes where they promote plant growth via phytohormone production, and suppress multiple root pathogens [176Govindasamy V, Franco CM, Gupta VVSR. Gange, AC: Eds. Advances in endophytic research. Springer: India 2014; pp. 27-59.].

7.1.3. Co-Cultures

Combinations of cellulolytic microorganisms and N2-fixing bacteria have been studied, mostly in controlled environments, to understand the synergy between each group of organisms. For example, Veal and Lynch [177Veal D, Lynch J. Associative cellulolysis and dinitrogen fixation by co-cultures of Trichoderma harzianum and Clostridium butyricum. Nature 1984; 63: 245-53., 178Veal D, Lynch J. Associative cellulolysis and N2 fixation by co-cultures of Trichoderma harzianum and Clostridium butyricum: the effects of ammonium-N on these processes. J Appl Bacteriol 1987; 63(3): 245-53.
[http://dx.doi.org/10.1111/j.1365-2672.1987.tb04943.x]
] found that mixed cultures of the cellulolytic fungus Trichoderma harzianum and the N2-fixing bacterium Clostridium butyricum co-operatively degraded cellulose and used the degradation products to fix N2 equivalent to 7.87 mg N fixed / g C lost. A similar rate (12 - 14.6 mg N fixed / g cellulose consumed) was measured with cellulose containing co-cultures of Cellulomonas gelida and Azospirillum lipoferum or A. brasilense or Bacillus macerans [179Halsall DM, Gibson AH. Cellulose decomposition and associated nitrogen fixation by mixed cultures of Cellulomonas gelida and Azospirillum species or Bacillus macerans. Appl Environ Microbiol 1985; 50(4): 1021-6.
[PMID: 16346898]
]. With wheat straw and the same organisms, these authors measured 17-19 mg N / g straw consumed, a value not dissimilar to that found by Lynch and Harper [180Lynch J, Harper S. Straw as a substrate for cooperative nitrogen fixation. J Gen Microbiol 1983; 129(1): 251-3.] (11.5 mg N / g straw lost) for a Penicillium corylophilumClostridium butyricum association. However, in co-cultures of a mutant strain of Cellulomonas sp. (strain CS1-17) and Azospirillum spp. with cereal straw, Halsall and Gibson [181Halsall DM, Gibson AH. Comparison of two Cellulomonas strains and their interaction with Azospirillum brasilense in degradation of wheat straw and associated nitrogen fixation. Appl Environ Microbiol 1986; 51(4): 855-61.
[PMID: 16347043]
] measured much larger rates of fixation (72 and 63 mg N / g straw utilised) which concurs with the theoretical upper limit of 75 mg / g straw calculated by Kennedy and Islam [17Kennedy I, Islam N. The current and potential contribution of asymbiotic nitrogen fixation to nitrogen requirements on farms: a review. Aust J Exp Agric 2001; 41(3): 447-57.
[http://dx.doi.org/10.1071/EA00081]
]. Halsall and Gibson [181Halsall DM, Gibson AH. Comparison of two Cellulomonas strains and their interaction with Azospirillum brasilense in degradation of wheat straw and associated nitrogen fixation. Appl Environ Microbiol 1986; 51(4): 855-61.
[PMID: 16347043]
] attributed these vastly increased rates to the efficiency of the mutant strain of Cellulomonas sp. (strain CS1-17) and to low background N levels in the experiment.

Co-culture inoculants of cellulolytic organisms and diazotrophs are unlikely to confer great benefits in the field because of the high diversity of cellulolytic organisms that occur naturally in the soil (e.g. [182Chatterjee S, Nandi B. Biodegradation of wheat stubbles by soil micro-organisms and role of the products on soil fertility. Plant Soil 1981; 59(3): 381-90.
[http://dx.doi.org/10.1007/BF02184542]
-184Zeikus JG. Advances in Microbial Ecology. US: Springer 1981; pp. 211-43.
[http://dx.doi.org/10.1007/978-1-4615-8306-6_5]
]). The exception to this might be soils that have not had a history of significant carbon inputs and cellulolytic populations are not well developed. Combining enhanced cellulolytic capability with nitrogenase activity in the same organism is likely to increase the efficiency of transfer of energy to N2 fixation, but so far efforts to achieve this have not been demonstrated.

7.1.4. Non-culturable Microorganisms

The finding that non-culturable bacteria, including members of Betaproteobacteria and Actinobacteria, may be dominant N2-fixing microorganisms [32Buckley DH, Huangyutitham V, Hsu S-F, Nelson TA. Stable isotope probing with 15N2 reveals novel noncultivated diazotrophs in soil. Appl Environ Microbiol 2007; 73(10): 3196-204.
[http://dx.doi.org/10.1128/AEM.02610-06] [PMID: 17369332]
, 34Hamelin J, Fromin N, Tarnawski S, Teyssier-Cuvelle S, Aragno M. nifH gene diversity in the bacterial community associated with the rhizosphere of Molinia coerulea, an oligonitrophilic perennial grass. Environ Microbiol 2002; 4(8): 477-81.
[http://dx.doi.org/10.1046/j.1462-2920.2002.00319.x] [PMID: 12153588]
, 42Hurek T, Handley LL, Reinhold-Hurek B, Piché Y. Azoarcus grass endophytes contribute fixed nitrogen to the plant in an unculturable state. Mol Plant Microbe Interact 2002; 15(3): 233-42.
[http://dx.doi.org/10.1094/MPMI.2002.15.3.233] [PMID: 11952126]
, 140Knauth S, Hurek T, Brar D, Reinhold-Hurek B. Influence of different Oryza cultivars on expression of nifH gene pools in roots of rice. Environ Microbiol 2005; 7(11): 1725-33.
[http://dx.doi.org/10.1111/j.1462-2920.2005.00841.x] [PMID: 16232287]
] requires the development of tools to culture them in order to develop effective inoculants. Advances in culturing technology (e.g. Janssen [185Janssen PH. Identifying the dominant soil bacterial taxa in libraries of 16S rRNA and 16S rRNA genes. Appl Environ Microbiol 2006; 72(3): 1719-28.
[http://dx.doi.org/10.1128/AEM.72.3.1719-1728.2006] [PMID: 16517615]
]) including sequence-directed isolation of novel bacteria offer new hope to identify functionally important bacteria suitable for specific crops and environments.

7.2. Management Combinations

Development of combinations of management practices should maximize NS N2 fixation. Cropping systems which combine high C inputs and good soil structure, e.g. conservation farming practices or perennial grass systems, are likely to be ideal. No-tillage practices combined with crop residue retention have increased rapidly world-wide in response to a range of pressures, and by 2003 for example, it was estimated that in Western Australia, 86% of farmers had adopted no-tillage [28D'Emden F, Llewellyn R. No-tillage adoption decisions in southern Australian cropping and the role of weed management. Aust J Exp Agric 2006; 46(4): 563-9.
[http://dx.doi.org/10.1071/EA05025]
]. Many of the field studies on NS N2 fixation in the past were done with soils where stubble retention had recently been adopted (e.g. [19Roper MM. Field measurements of nitrogenase activity in soils amended with wheat straw. Aust J Agric Res 1983; 34(6): 725-39.
[http://dx.doi.org/10.1071/AR9830725]
]). Soil microbial composition and functions respond slowly to changed managements [116Gupta VVSR, Roper MM. Protection of free-living nitrogen-fixing bacteria within the soil matrix. Soil Tillage Res 2010; 109(1): 50-4.
[http://dx.doi.org/10.1016/j.still.2010.04.002]
] and therefore it is likely that if similar experiments were conducted today on sites with long-term no-tillage and crop residue retention, different rates of N2 fixation might be found, particularly in areas under continuous cropping.

A system which supports 100% ground cover 100% of the time is likely to provide continuous inputs of C either by rhizodeposition or by inputs from above-ground residues. In ‘pasture-cropping systems’ where native summer-active perennial grasses are coupled with winter cereals it is proposed that biological N inputs are sufficient to supply the needs of the cereal crop. Using δ15N techniques, Mordelet et al. [186Mordelet P, Cook G, Abbadie L, Grably M, Mariotti A. Natural 15N abundance of vegetation and soil in the Kapalga savanna, Australia. Aust J Ecol 1996; 21(3): 336-40.
[http://dx.doi.org/10.1111/j.1442-9993.1996.tb00617.x]
] and Abbadie et al. [187Abbadie L, Mariotti A, Menaut JC. Independence of savanna grasses from soil organic-matter for their nitrogen supply. Ecology 1992; 73(2): 608-13.
[http://dx.doi.org/10.2307/1940766]
] were able to identify contributions from NS N2 fixation of up to 17 % of the annual savannah requirement for N. In pasture-cropping systems, fixed N is likely to be protected from leaching losses due to uptake by the grasses in autumn and later release by mineralisation from stubble and decomposing roots to a cereal crop during winter when the grass is not active [188Syme H, Acuña TB, Abrecht D, Wade L. Nitrogen contributions in a windmill grass (Chloris truncata)–wheat (Triticum aestivum L.) system in south-western Australia. Aust J Agric Res 2007; 45(8): 635-42.]. Further research is needed to understand the N dynamics of pasture-cropping systems and to evaluate their potential in agriculture.

Little is known about the potential for N inputs via N2 fixation with other plants including weeds and other non-legume components of pastures except for a small study by Conklin and Biswas [146Conklin A Jr, Biswas P. A survey of asymbiotic nitrogen fixation in the rhizosphere of weeds. Weed Sci 1978; 26: 148-50.] who observed NS N2-fixing bacteria and nitrogenase activity (C2H2 reduction) associated with 20 weed species.

7.3. Plant Based Solutions

Only in Brazil are there varieties of sugar cane that have been shown to fix over 60% of their nitrogen (>150 kg N ha-1 year-1 [65Boddey RM, De Oliveira O, Urquiaga S, et al. Biological nitrogen fixation associated with sugar cane and rice: contributions and prospects for improvement. Plant Soil 1995; 174(1-2): 195-209.
[http://dx.doi.org/10.1007/BF00032247]
]). Elsewhere in the world, measurements of contributions to N supply in sugar cane via biological N2 fixation have been small [189Biggs IM, Wilson JR, Keating BA, Critchley C. Does biological N2-fixation contribute to nitrogen requirements in Australian sugarcane? Proc Aust Soc Sugar Cane Technol 2000; 22: 133-8.] although specific associations between diazotrophic bacteria and sugar cane have been observed [190Li R, MacRae I. Specific association of diazotrophic acetobacters with sugarcane. Soil Biol Biochem 1991; 23(10): 999-1002.
[http://dx.doi.org/10.1016/0038-0717(91)90181-I]
]. It has been argued that this may be due to sugar crops in Brazil being systematically bred for high yields with low fertiliser inputs [65Boddey RM, De Oliveira O, Urquiaga S, et al. Biological nitrogen fixation associated with sugar cane and rice: contributions and prospects for improvement. Plant Soil 1995; 174(1-2): 195-209.
[http://dx.doi.org/10.1007/BF00032247]
, 150Baldani JI, Reis VM, Baldani VL, Döbereiner J. Review: A brief story of nitrogen fixation in sugarcane-reasons for success in Brazil. Funct Plant Biol 2002; 29(4): 417-23.
[http://dx.doi.org/10.1071/PP01083]
]. Baldani et al. [150Baldani JI, Reis VM, Baldani VL, Döbereiner J. Review: A brief story of nitrogen fixation in sugarcane-reasons for success in Brazil. Funct Plant Biol 2002; 29(4): 417-23.
[http://dx.doi.org/10.1071/PP01083]
] suggested that such a breeding process with low fertiliser inputs has led to the development of (or preserved) an effective association between N2-fixing bacteria and the plant. Almost all of our modern crop varieties have been developed in conjunction with the use of nitrogen fertilisers suggesting that the capacity for significant associative N2 fixation may have been lost during breeding processes. Therefore, examination of the capacity for associative and endophytic N2 fixation in the wild relatives of wheat and other cereals, and the possibility of transferring this capability into modern varieties may have merit. Support for this notion can be seen with rice, for example Knauth et al. [140Knauth S, Hurek T, Brar D, Reinhold-Hurek B. Influence of different Oryza cultivars on expression of nifH gene pools in roots of rice. Environ Microbiol 2005; 7(11): 1725-33.
[http://dx.doi.org/10.1111/j.1462-2920.2005.00841.x] [PMID: 16232287]
] examined the composition of diazotrophic communities associated with related rice cultivars (Oryza sativa) and wild species (Oryza brachyantha) and found that when grown under identical conditions in the same soil without N fertiliser there were remarkable differences in root associated nifH-gene expressing communities between the two cultivars. Furthermore, NifH fragments expressed in the wild species of rice roots indicated that the active diazotrophs were not related to cultured strains. In a separate study, Engelhard et al. [191Engelhard M, Hurek T, Reinhold-Hurek B. Preferential occurrence of diazotrophic endophytes, Azoarcus spp., in wild rice species and land races of Oryza sativa in comparison with modern races. Environ Microbiol 2000; 2(2): 131-41.
[http://dx.doi.org/10.1046/j.1462-2920.2000.00078.x] [PMID: 11220300]
] found that endophytic populations of diazotrophs differed with rice genotype and that the natural host range of the non-culturable Azoarcus spp. included rice, with wild and old rice varieties being preferred over modern cultivars. On the other hand, culturable species such as Azospirillum spp., Klebsiella sp., Sphingomonas paucimobilis, Burkholderia spp. were associated with more modern cultivars of Oryza sativa.

Evidence that ecosystems with low N promote NS N2 fixation occurs in a perennial grass (Molinia coerulea) which grows in oligotrophic environments. In another example, Gupta et al. [41Gupta VVSR, Kroker S, Hicks M, Davoren CW, Descheemaeker K, Llewellyn RS. Nitrogen cycling in summer active perennial grass systems in South Australia: non-symbiotic nitrogen fixation. Crop Pasture Sci 2014; 65(10): 1044-56.
[http://dx.doi.org/10.1071/CP14109]
] reported diazotrophic N2 fixation of 0.92 to 2.35 mg N / kg root / day with summer active perennial grasses such as Panicum species and Rhodes grass (Chloris gayana) in low organic matter soils of southern Australia. Hamelin et al. [34Hamelin J, Fromin N, Tarnawski S, Teyssier-Cuvelle S, Aragno M. nifH gene diversity in the bacterial community associated with the rhizosphere of Molinia coerulea, an oligonitrophilic perennial grass. Environ Microbiol 2002; 4(8): 477-81.
[http://dx.doi.org/10.1046/j.1462-2920.2002.00319.x] [PMID: 12153588]
] observed that the rhizosphere of the perennial grass Molinia coerulea supported a diversity of N2-fixing bacteria, 56% of which contained NifH sequences that did not match any cultivated diazotrophs, but were dominant in the roots and surrounding soil. Further examination of such oligotrophic systems may yield diazotrophic communities that could be adapted to agricultural systems where they might increase the contribution from associative N2 fixation in agricultural crops.

SUMMARY

There is a range of estimates for NS N2 fixation in different cropping systems. A number of reviews suggest that significant amounts of N2 fixation (>30-40 kg N ha-1 year-1) are possible with C4 grasses including sugar cane in tropical regions (e.g. [12Chalk P. The contribution of associative and symbiotic nitrogen fixation to the nitrogen nutrition of non-legumes. Plant Soil 1991; 132(1): 29-39.
[http://dx.doi.org/10.1007/BF00011009]
]), and where sugar cane has been bred with low N fertiliser inputs >150 kg N ha-1 year-1 has been measured [65Boddey RM, De Oliveira O, Urquiaga S, et al. Biological nitrogen fixation associated with sugar cane and rice: contributions and prospects for improvement. Plant Soil 1995; 174(1-2): 195-209.
[http://dx.doi.org/10.1007/BF00032247]
]. Estimates in temperate and Mediterranean regions are less certain and range from 10-30 kg N ha-1 crop-1 [17Kennedy I, Islam N. The current and potential contribution of asymbiotic nitrogen fixation to nitrogen requirements on farms: a review. Aust J Exp Agric 2001; 41(3): 447-57.
[http://dx.doi.org/10.1071/EA00081]
, 18Dart P. Nitrogen fixation associated with non-legumes in agriculture. Plant Soil 1986; 90: 303-34.
[http://dx.doi.org/10.1007/BF02277405]
] to less than 5 kg N ha-1 year-1 [23Giller KE, Merckx R. Exploring the boundaries of N2-fixation in cereals and grasses: An hypothetical and experimental framework. Symbiosis 2003; 35(1-3): 3-17.], but it is likely that differences in methodology including application of individual methods have contributed to some of the reported variability. Environmental and management factors play an enormous role in the contribution of N from this beneficial microbial function. New technologies using molecular approaches, particularly when combined with isotope methods, are broadening our understanding of NS N2 fixation, and the molecular mechanisms of plant-diazotroph interactions. Microarray, pyrosequencing and Stable Isotope Probing (SIP) technologies offer an opportunity to investigate simultaneously both the diversity and function of diazotrophic microbial communities, and this may lead to the discovery of currently non-culturable bacteria that are functionally significant.

Generally there is a good understanding of the environmental factors controlling NS N2 fixation and this can be helpful in designing farming systems that promote N inputs from fixation, but most estimates of N2 fixation, particularly in the field were determined more than 20 years ago. Since then, farming practices have evolved towards intensive cropping (particularly with cereals), no-tillage and stubble retention, and further evaluation in terms of quantity of N fixed and identity of significant members of the N2-fixing community is needed. Similarly, alternative systems such as ‘pasture-cropping’ that benefit from N2 fixation associated with perennial grasses could be explored.

Further gains may be possible through inoculation with highly efficient N2-fixing bacteria particularly if they have the additional capacity to promote plant growth. However, the ultimate test for even the most beneficial inoculant is to be able to survive in soil and colonise plant roots. Inoculation with bacteria that can form an endophytic relationship within the plant (either in below-ground and/or above-ground parts) may increase the potential for success. However, many effective diazotrophic bacteria remain non-culturable and this may limit our ability to exploit them as inoculants unless new culturing techniques can be developed. New research using molecular techniques will reveal the true diversity of diazotrophic bacteria in agricultural and natural ecosystems and their potential to be used as inoculants in agricultural systems. Additionally, co-occurrence network analysis using nifH sequence data indicated the presence of complex co-occurrence patterns in the free-living diazotrophs than that known in symbiotic diazotrophs [202Tu Q, Zhou X, He Z, et al. The diversity and co-occurrence patterns of N2-fixing communities in a CO2-enriched Grassland Ecosystem. Microb Ecol 2016; 71(3): 604-15.
[http://dx.doi.org/10.1007/s00248-015-0659-7] [PMID: 26280746]
]. Such novel insights in to the ecology of diazotrophs may lead to development of inoculant mixtures that promote overall N2 fixation. Re-introduction into modern varieties of traits that promote the colonisation of highly efficient diazotrophic populations should further contribute biologically fixed N to agricultural systems, particularly in non-leguminous crops. Finally, NS N2 fixation provides an attractive option as an environmentally responsible alternative fertiliser source for sustainable food production, especially in lower organic matter and low fertility soils worldwide.

CONFLICT OF INTEREST

The authors confirm that this article content has no conflict of interest.

ACKNOWLEDGEMENTS

Financial support for the preparation of this review was provided by the Australian Grains Research and Development Corporation (GRDC) and from CSIRO. The authors thank Ramona Jongepier for assistance in preparation of the manuscript.

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