List of endophytic diazotrophic bacteria recently isolated and associated with agricultural crops.
Endophytic bacteria represents a unique class of bacteria that can colonize interior tissues of plant and provide a range of benefits to the plant similar to those provided by the rhizospheric bacteria. Certain endophytic bacteria can provide nitrogen to the plants through biological nitrogen fixation, which is an important source of nitrogen input in agriculture and represents a promising substitute for chemical fertilizers, and are known as endophytic diazotrophic bacteria. Besides fixing nitrogen, endophytic bacteria can produce plant growth hormones like auxin and gibberellin, help in nutrient uptake, and increase the plant’s tolerance to biotic and abiotic stresses. Various direct and indirect methods have been used to quantify the amount of nitrogen fixed by these bacteria, including the acetylene reduction assay, which is a quick but indirect method, and the 15N isotopic dilution assay, which is a robust and accurate method. Research on endophytic diazotrophic bacteria has come a long way, and in this chapter, we have briefly discussed the mechanisms of biological nitrogen fixation and methods to quantify the fixed nitrogen along with reviewing recent studies focused on evaluating the role of endophytic diazotrophic bacteria in promoting plant growth in both native and nonnative crop hosts.
- endophytic bacteria
- biological nitrogen fixation
- plant growth promotion
- agricultural crops
Nitrogen (N) is an essential component of all proteins and enzymes, nucleic acids that make up DNA, and chlorophyll that enables the process of photosynthesis in plants . It is a very common element in nature that is present in abundant amounts in atmosphere, lithosphere, and hydrosphere of the earth . However, much of this N is in the form of dinitrogen (N2), which is inert and cannot be used by plants. In order for plants to use this dinitrogen, it has to be reduced/fixed into forms like nitrate (NO3−) and ammonium (NH4+). N fixation, the process by which dinitrogen is reduced to plant-available forms, is, therefore, a vital process for the sustenance of life on earth. A major industrial process by which dinitrogen is converted into ammonia is known as the Haber-Bosch process. This artificial N-fixation process was established in 1913 and uses a catalyst (iron with a small amount of aluminum added) at high pressure (as much as 5.06 × 107 Pa) and high temperature (600–800 K) consuming large amounts of fossil fuel. Ammonia produced through this highly expensive process is combined with other elements to produce nitrogenous fertilizers like urea and ammonium nitrate. Although the use of these fertilizers is inevitable in meeting rising food demand to sustain the growing global population, their indiscriminate use has set off very negative effects on the environment . Naturally, N is commonly fixed by two processes. The first is atmospheric N fixation by lightning, in which the enormous amount of energy contained in lightning breaks dinitrogen molecules and enables their atoms to combine with oxygen in the air forming N oxides that dissolve in rain. These oxides of N then form nitrates that are carried to the earth in rainfall . The second is biological N fixation (BNF), in which certain prokaryotic microorganisms, known as diazotrophs, fix N by breaking down the triple bond of dinitrogen using a highly specialized enzyme complex called nitrogenase enzyme and convert it to ammonia . This chapter mainly focuses on diazotrophic bacteria that can fix N while living in the internal tissues of plants. In this chapter, only recent developments (from last 5 years) related to this subject have been discussed.
2. Biological nitrogen fixation (BNF)
Farmers since ancient Chinese and Roman civilizations practiced crop rotation with legumes to increase soil fertility and agricultural productivity. However, the science behind such practice was first revealed by Boussingault in 1838, who established that legumes can fix N. But it was not until 1886 when Hellriegel and Wilfarth provided a firm evidence that microbes are responsible for N fixation occurring in leguminous plants .
2.1. Chemistry and genetics of BNF
The overall chemical reaction of BNF catalyzed by the nitrogenase enzyme is represented below:
Nitrogenase is a complex enzyme comprised of two metalloproteins: the Mo-Fe protein, also called dinitrogenase protein, and the Fe protein, also called dinitrogenase reductase protein. The dinitrogenase protein is a heterotetramer composed of two α- and two β-subunits with an overall molecular weight of 240kDa. This protein contains two types of metal centers, the FeMo-cofactor and the P-cluster pair, of which the FeMo cofactor is the active site where dinitrogen binds, whereas the P-cluster mediates electron transfer between the Fe protein and the FeMo cofactor. The dinitrogenase reductase protein is a homodimer of two identical subunits, with an overall molecular mass of ~60 kDa. It contains two ATP/ADP molecules and one Fe4-S4 cluster [6, 7].
The overall functioning of nitrogenase can be summarized as a key biochemical cycle that involves five steps [6, 7]: (i) the reduction of Fe protein by electron carriers such as flavodoxin or ferredoxin; (ii) association of the reduced Fe protein (including two MgATP complexes) with the Mo-Fe protein in preparation for electron transfer; (iii) hydrolysis of MgATP, which enables transfer of one electron to the Mo-Fe protein (via Fe4S4 and the P-cluster); (iv) electron transfer to dinitrogen and thus its reduction, while it is bound to the active site within the Mo-Fe protein; and (v) dissociation of the two protein molecules, exchange of ATP back into the Fe protein, and rereduction of the Fe protein.
The structure and function of nitrogenase enzyme are encoded by ~20 genes, known as N-fixation genes (
2.2. Quantification of biologically fixed N
BNF can be measured using various methods, the most common being: N balance method, xylem solute analysis, acetylene reduction assay, and stable isotope (15N) method . In the N balance method, the amount of N fixed is estimated by calculating the difference between total N content of plants inoculated by diazotrophs and those that are not inoculated. In this method, it is assumed that both inoculated and noninoculated plants absorb equal amounts of N from the soil, which is hard to justify as there are differences in root morphology and physiological attributes . In the xylem solute analysis, the composition of N compounds flowing through the xylem sap to the shoot of the plant is determined. The N absorbed by plants from the soil is predominantly nitrate, whereas the fixed N is primarily in the form of amides and ureides . This difference in composition of N compounds is used to make quantitative measurements of N fixation . However, its major disadvantage is that only a very small proportion of N-fixing plants export fixed N in the form of ureides . The acetylene reduction assay is a popular technique used to indirectly measure BNF by estimating the nitrogenase enzyme activity. It is based on the ability of nitrogenase to reduce acetylene (H─C≡C─H) to ethylene by breaking the triple bond between carbon atoms. Samples are incubated in a gas-tight chamber and a portion of the head space is injected with acetylene. After incubation, gas samples are collected from the chamber and analyzed for ethylene production using gas chromatography . It is a simple, low cost, and sensitive assay that can measure BNF in bacterial cultures, detached nodules, plant parts, or even whole plants. The major disadvantage is the short-term nature of the assay and the autoinhibition of acetylene conversion to ethylene . The stable isotope method using 15N is a widely used and accepted method. This method is based on the principle that soil has a noticeably different 15N to 14N ratio as compared to the atmosphere, which has a constant ratio (0.3663%). Therefore, plants absorbing fixed N from the atmosphere will have a different 15N to 14N ratio as compared to the ones absorbing N only from the soil. When plants inoculated with diazotrophs are grown in air labeled with 15N, they are expected to have an enhanced ratio as compared to the noninoculated ones (15N incorporation method). When available soil N is labeled with 15N, a reduction in the ratio is expected since the inoculated plants tend to incorporate fixed N from the air as compared to the noninoculated plants, which take up labeled N from the soil (15N isotope dilution method) .
2.3. N-fixing organisms
The ability to fix N, in other words, the presence of nitrogenase enzyme, is only limited to certain bacteria and archaea . Within these groups, it is quite widely distributed revealing considerable phylogenetic diversity among diazotrophs. A comprehensive list of N-fixing bacteria and archaea, under 12 broad phylogenetic groups based on 16S rDNA phylogeny was prepared by Young . Diazotrophs are also widely distributed ecologically. They can be found living in soils and water freely, in the rhizosphere and phyllosphere and inside the plant tissues, in symbiotic association with legumes and actinorhizal association with woody plants, and in cyanobacterial symbiosis with phytoplankton, fungi, and terrestrial plants . Free-living diazotrophs are those that do not associate with plants and are found in soils that are free from the direct influence of plant roots. These microorganisms are ubiquitous in terrestrial and aquatic environments and are physiologically very diverse . Many diazotrophs can be found dwelling in the rhizosphere of a plant. Due to their ability to fix N, diazotrophs can have a competitive advantage over other microbes in the rhizosphere. They prevail in the rhizosphere particularly when soil N is limited . The phyllosphere (leaf surface) is another microsite known to be colonized by diazotrophs . The symbiotic association between legume and
The presence of diazotrophs in nonleguminous plants was first detected by Brazilian researchers in the rhizosphere and rhizoplane of sugarcane (
3. Endophytic bacteria
The term ‘endophyte’ was first coined more than 150 years ago by de Bary  for pathogenic fungi entering the internal tissues of leaves. Since then, many authors have redefined this term, but each has its own restrictions. Taken literally, the word endophyte means ‘in the plant’ (endon = within; phyton = plant) . Since our main focus in this chapter is on ‘endophytic bacteria,’ we would like to reiterate the definition notated by Chanway et al. : “bacteria that can be detected at a particular moment within the tissue of apparently healthy plant hosts without inducing disease or organogenesis are known as endophytic bacteria.” The occurrence of endophytic bacteria in internal tissues was first reported inside a healthy potato plant . Since then, many scientific studies have been focused on isolating the endophytic bacteria from a variety of plant species and evaluating their benefits for agricultural plants [44, 45, 46, 47]. In contrast to free-living, rhizosphere or phyllosphere microorganisms, endophytic bacteria 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 [48, 49, 50]. In addition, endophytic bacteria colonize niches that are more conducive to forming mutualistic relationships with plants , for example, providing fixed N to the plant and getting photosynthate in return [52, 53, 54]. Following the rhizospheric colonization, endophytic bacteria can colonize various plant organs such as roots, stem, leaves, flowers, fruits, and seeds [55, 56, 57, 58, 59, 60, 61], indicating different capacities of endophytic bacteria to colonize various plant compartments. They can even colonize legume nodules  and tubercles of mycorrhizal fungi . The endophytic bacterial population is extremely variable in different plant organs and tissues and have been shown to vary from as low as hundreds to as high as 109 cfu per gram plant tissue [64, 65, 66, 67].
Localization of endophytic bacteria within plant tissues requires techniques that facilitate observation on a tiny spatial scale. Various methods have been used to locate bacteria
3.1. Endophytic diazotrophic bacteria
A few years after the discovery of diazotrophs by Cavalcante and Döbereiner  in the stem and root tissues of sugarcane plant, Döbereiner  coined the term “endophytic diazotrophic bacteria” to designate all diazotrophs able to colonize primarily the root interior of graminaceous plants, survive very poorly in soil and fix N in association with these plants . Since the discovery of endophytic diazotrophic bacteria in sugarcane, other agronomically important crop species like rice [87, 88, 89], corn [90, 91, 92, 93], wheat , canola (
|Endophytic diazotrophic bacteria||Isolated from||Colonized into||Method used to confirm N-fixing ability||References|
|Pearl millet (||Wheat (||Amplification of |||
|Rice (||Rice (||Acetylene reduction assay|||
|Rice (||Rice (||Amplification of |||
|Banana tree cultivar ‘Prata Anã’ (||—||Amplification of |||
|Corn (||Corn (||Amplification of |||
|Sugarcane (||Sugarcane (||Kjeldahl method; natural abundance of 15N in leaf samples; isotopic 15N dilution|||
|Rice (||Rice (||Acetylene reduction assay|||
|Lodgepole pine (||Corn (||Amplification of ||[109, 113, 117, 119]|
4. Recent studies highlighting the role of endophytic diazotrophic bacteria in agricultural crops
Rice is a major staple crop in many countries around the world. It is a highly N-demanding crop; thus, it becomes extremely important to find alternatives to reduce the use of chemical N fertilizers applied to rice without decreasing the productivity. Endophytic diazotrophic strains were isolated from root, culm, and leaf tissues of traditional rice varieties (Zebu Branco and Manteiga) cultivated traditionally by the local farmers in the Maranhão state, Brazil . Ten strains showing consistent acetylene reduction activity and capable of producing indole-3-acetic acid (IAA) were identified as belonging to the genera
Corn is an agriculturally important crop that is extensively grown and consumed by a large population around the world. Szilagyi-Zecchin et al.  isolated and identified six endophytic strains from roots of corn growing in the southern Brazilian region of Campo Largo, PR. Out of these six endophytic isolates, four were able to grow on N-free media, consistently reducing acetylene, and were found positive for the presence of
Pearl millet (
Our lab group has been working with endophytic diazotrophic bacteria from many years and has published several reports regarding the role of these bacteria in fixing N and promoting plant growth in both agricultural and forest ecosystems . In 2012, our lab discovered an endophytic diazotrophic bacterium,
|Host plant||Harvest (days)||%Ndfaa||% growth promotion||References|
|Foliar nitrogen concentrationb||Seedling lengthc||Seedling biomassd|
Since their discovery in sugarcane tissues decades ago, endophytic diazotrophic bacteria have been characterized for their role in performing BNF. Studies have suggested that these bacteria can act as N biofertilizer for highly N-demanding crops like sugarcane, corn, and rice. Most recent studies have also focused their attention on testing the PGP characteristics of isolated endophytic diazotrophic strains other than N fixation, which indicates the growing concern of agricultural scientists to develop bacterial inoculants that can enhance plant growth through a variety of mechanisms, so as to decrease the dependence on chemical fertilizers. Endophytic diazotrophic strains like
Authors would like to dedicate this work to Late Mr. Darshan K. Puri (1956–2014). You were, are and always will be an inspirational figure for us.