1. Introduction
Nitrogen is one of the most important elements in biological systems, comprising the main building blocks of nucleic acids, enzymes and proteins among its multiple functions. In nature, it exists primarily in the gaseous form and constitutes approximately 78% of the atmosphere. Despite its abundance, nitrogen (N) is one of the most growth-limiting nutrients in terrestrial and aquatic ecosystems (Dalton & Krammer, 2006) because its gaseous form is inert and unusable by most living organisms except for nitrogen fixing microorgansisms. For it to become biologically available, atmospheric nitrogen must be transformed or “fixed” from its inert gaseous form (N2) to ammonia (NH3), which can then be assimilated into a variety of important biochemicals. This transformation, which requires a large amount of energy to break apart the triple-bonded N atoms that comprise gaseous N2, is called ‘nitrogen fixation’ (NF). Nitrogen is fixed naturally through energy-releasing abiotic processes such as lightening, forest fires and volcanic activity. These processes produce oxides of N in the atmosphere that subsequently dissolve in rain and descend to the ground as NH3 molecules. Approximately 12% of annual global NF is fixed in this way (Bezdicek & Kennedy, 1998). Fertilizer production using high temperatures and pressures in the Haber-Bosch process occurs widely and accounts for approximately 20% of annual global NF (Bezdicek & Kennedy, 1998). However, the process is fossil-fuel intensive and consumes 3-5% of the world’s natural gas annually (Myrold & Bottomley, 2007). Alternatively, NF occurs through the normal metabolic activity of many prokaryotic microorganisms, known as diazotrophs, through a process commonly referred to as biological nitrogen fixation (BNF). This essentially “free” process is responsible for the addition of almost all biologically available N that enters terrestrial ecosystems, some 140 million metric tons per year (Bezdicek & Kennedy, 1998; Galloway et al., 2008). Biological nitrogen fixation is an ATP-demanding process that is catalyzed by the enzyme complex known as nitrogenase, which is found in many members of the Bacteria and Archaea (Galloway et al., 2008). Terrestrial BNF occurs primarily in the soil, by either free-living diazotrophs or those associated to varying degrees with plants (see below).
2. Free-living bacteria
Free-living diazotrophic bacteria are those that do not associate with plants (
3. Rhizosphere bacteria
The rhizosphere is defined as soil that surrounds plant roots and is under their direct metabolic influence (Curl & Truelove, 1987). Proximity to plant roots is important for soil organisms as actively growing plant roots deposit approximately 20% of annual photosynthate in the rhizosphere (Nguyen, 2003), but depending on the type of plant and its growth stage, more than 50% of newly-fixed carbon may be deposited in the rhizosphere at any given time. The soluble carbon compounds that plants deposit through their root systems are known as root exudates. These comprise a wide range of carbon compounds (amino acids, peptides, proteins, enzymes, “growth factors”, vitamins and phytohormones) (Grayston et al., 1997; Jones et al., 2004; Shi et al., 2012) and are released continuously during the growing season through a process known as root exudation (Jones et al., 2004). This process has been shown to significantly stimulate growth and population sizes of most soil microorganisms, but effects are particularly noticeable in soil bacteria and fungi. The degree of stimulation is significant: in comparison with bulk soil not under the influence of plant roots, the rhizosphere typically supports 5-100 x larger bacterial and fungal populations than non-rhizosphere or “bulk” soil (Warembourg, 1997; Dobelaere et al., 2003). Due to their ability to fix N2, diazotrophs can have a competitive advantage over non-N2 fixing bacteria in the rhizosphere and prevail in it particularly when soil N is limited (Döbereiner & Pedrosa, 1987). In addition to stimulating their own growth, rhizosphere diazotrophs representing several genera (
4. Phyllosphere
The leaf surface, or phyllosphere is another microsite known to be colonized by a wide range of microorganisms, including diazotrophic bacteria (Lindow & Brandl, 2003). While comparatively little work has been done on phyllosphere-colonizing diazotrophs, it is likely that their contribution to plant N nutrition is modest, owing to the energy constraints and problems associated with oxygen toxicity of nitrogenase such N2 fixing microorganisms would experience. Nevertheless, phyllosphere-colonizing diazotrophs should be evaluated further for possible contributions to plant nutrition.
5. Cyanobacterial associations
Cyanobacteria are prokaryotes belonging to the domain
Many diazotrophic cyanobacterial species enter into symbioses with eukaryotes including phytoplankton, fungi and terrestrial plants. Cyanobacterial symbionts (cyanobionts) in these associations may contribute a significant portion of N required for growth of both organisms through BNF in N-limited aquatic and terrestrial environments (Schell & Alexander,1973; Hobara et al., 2006). In the ocean, they are frequently found in association with diatoms (Ferrario et al., 1995) and brown algae (Carpenter, 1972), while in fresh water, the cyanobiont
6. Legume x Rhizobium symbiosis
The legume x
The legume x
7. Actinorhizal symbiosis
The actinorhizal symbiosis refers to a root nodule-forming, nitrogen-fixing symbiotic relationship that is functionally analogous to the legume x
In contrast to the easily cultured, gram negative rod shaped cells that typify
8. Bacterial endophytes
The presence of microorganisms in plant tissues might reasonably be considered an indication that a disease state is imminent, however this is not necessarily the case. Several decades ago, Trevet and Hollis (1948) reported the occurrence of bacteria within tissues of healthy potato plants and several studies have since demonstrated that internal tissues of healthy plants are colonized by bacteria. The term ‘bacterial endophytes’ has been used to describe bacteria that reside within living plant tissues without causing disease (Wilson, 1995; van der Lelie et al., 2009), however it does not differentiate whether such bacteria are (i) truly harmless, (ii) latent pathogens (Sinclair & Cerkauskas, 1996) or (iii) able to elicit production of symbiotic structures such as root nodules on the host. We use the term ‘endophyte’ in this paper to describe bacteria that ‘can be detected at a particular moment within the tissue of apparently healthy plant hosts’ (Schultz & Boyle, 2005) without inducing disease or organogenesis (Iniguez et al., 2005). In contrast to free-living, rhizosphere or phyllosphere microorganisms, bacterial endophytes are better protected from abiotic stresses such as extreme variations in temperature, pH, nutrient and water availability as well as biotic stresses such as competition (Loper et al., 1985; Cocking, 2003; Rosenblueth & Martinez-Romero, 2006). In addition, bacterial endophytes colonize niches that are more conducive to forming mutualistic relationships with plants through NF, for example, as suggested in sugarcane and other crops (see below) (Richardson et al., 2009).
8.1. Bacterial endophytes of sugarcane and other agricultural crops
In the 1980’s, Brazilian researchers were perplexed by the consistently high yields of field-grown sugarcane, an N-demanding crop, without exogenous N fertilizer application and looked for a microbiological explanation for this apparently anomalous observation. After it was determined that rhizospheric NF did not occur at sufficient rates to facilitate high sugarcane yields, Cavalcante & Döbereiner (1988) looked for microorganisms within sugarcane tissues that might be involved and isolated a diazotrophic bacterium,
Despite sugarcane’s apparent potential to derive much of its N from BNF, it has not been proven that
If endophytic diazotrophs are ultimately proven to be the primary cause of BNF and growth promotion of their host plants, such a plant x microbe association would represent another type of mutualistic symbiosis where the plant provides photosynthate and a competition-free, microaerobic environment for effective N2 fixation (Hallman et al., 1997; Reinhold-Hurek & Hurek, 1998a,b; Santi et al., 2013) for microorganisms in return for plant growth-promoting amounts of N from BNF. In contrast to the legume x
8.2. Nitrogen fixation in gymnosperms: Effects on seedling growth and N content
Lodgepole pine (
Based on these results, we hypothesized that soil N depletion would eventually restrict the growth rate of control seedlings to a point where they would be outperformed by N2-fixing seedlings, and set up longer term growth experiments to evaluate this possibility. After a 13- month growth period, pine seedlings treated with P2b-2R were observed to derive most of their foliar N (79%) from BNF (Anand et al., 2013), which was confirmation of the ability of lodgepole pine to fix N after colonization by
8.3. Nitrogen fixation in gymnosperms: Colonization of seedlings
The ability of
We also observed intact cells from different pine stem sections that were colonized internally by GFP-labeled bacteria (Anand & Chanway, 2013a). In some cases, GFP-labeled bacteria were observed in pine cells in close proximity to chloroplasts (Fig. 1a), which raises the possibility that bacteria colonized microsites near these energy-generating organelles. GFP labelling were also observed tightly packed within other pine cells (Fig. 1b). Whether bacteria in either of these pine cells (Fig. 1) were fixing N cannot be determined, but these unique endophytic colonization patterns warrant further study. Endophytic bacteria have also been observed inside cells of grape (Compant et al., 2005), grasses (Hurek et al., 1994), sugarcane (James & Olivares, 1998) and poplar (
Results with lodgepole pine strongly suggest that seedlings can fix N2 when colonized by
9. Conclusions and future research
Biological nitrogen fixation is a free and environmentally benign process through which biologically useful N can be generated for plant growth. Because nitrogen fixation is most effective when bacteria associate with plants to some degree, future research should focus on the various known plant associative and symbiotic nitrogen fixing systems. However, owing to the complexity of root-nodule based symbioses, colonization of plants by endophytic diazotrophs seems to hold the greatest potential for expansion of BNF to plant species that do not normally fix N2. Initial steps might include mass screening of known endophytic diazotrophs with commercially important plant species while continuing studies of effective nitrogen fixing associations involving plants that are colonized by endophytic diazotrophs. Such studies will help to elucidate the molecular, physiological and ecological details of this potentially useful plant x microbe interaction.
Acknowledgments
Funding for this study was provided by an NSERC Discovery Grant to CPC.
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