4.1 Introduction

Nitrogen (N) is one of the most important nutrients for plant growth. About 1–5 % of total plant dry matter consists of N. It is an essential constituent of proteins, nucleic acids, chlorophyll, co-enzymes, phytohormones and secondary metabolites (Hawkesford et al. 2012). Due to its immense cellular need, N is required in large quantities. Although atmosphere contains 80% of dinitrogen (N2) (Abd-Alla et al. 2014a), it is relatively inert, and most organisms are unable to utilize it (Allito et al. 2015). Generally, the most reduced form of N that is ammonia (NH3) or most oxidized form, nitrate (NO− 3) are required to fulfil needs of plants. The fixed form of N is generated by conversion of N2 to NH3, a process also known as nitrogen fixation. Nitrogen fixation may occur by biological or non-biological means (Fig. 4.1). Non-biological fixation includes geochemical fixation by lightning (10% of the total N2 fixation) (Bezdicek and Kennedy 1998) and industrial fixation by Haber-Bosch process (15% of total N2 fixation) (Bezdicek and Kennedy 1998). However, BNF is carried out by some prokaryotes, including a small but diverse group of bacteria and archea, commonly referred as diazotrophs (Zehr et al. 2003; Kneip et al. 2007) (Fig. 4.2). They encode enzyme complex nitrogenase, that catalyses the conversion of N2 gas to NH3 (Santi et al. 2013). Amongst all of the fixed form of N2, BNF is of immense importance. It is estimated that over half of the fixed N2 is supplied biologically and has a profound agronomic, economic, and ecological impact (Smil 2001). Although BNF is a boon to agro-ecosystems, but N fertilizers are also used for meeting the total N requirement for agricultural production throughout the world. That is why a substantial increase has been noticed in demand for chemical N fertilizers as in comparsion to a century ago (Galloway et al. 2008). The increased use of chemical N fertilizers is problematic because it is susceptible to loss by leaching or denitrification (Luce et al. 2011) and regarded economically and environmentally undesirable. Moreover, manufacturing N fertilizers requires six times more energy than other fertilizers such as that of phosphorus or potassium (Da Silva et al. 1978).

Fig. 4.1
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Nitrogen fixation on earth (Modified from Bezdicek and Kennedy 1998)

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Fig. 4.2
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Biological nitrogen fixation (Modified from Bouizgarne et al. 2015)

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There are various types of associations/interactions occurring between diazotrophs and their host plants (Santi et al. 2013). Amongst them most efficient are diazotrophic bacteria known as rhizobia, involved in the formation of root nodules in legumes (Santi et al. 2013). Currently, more than 98 species belonging to 14 genera of α, β and ϒ proteobacteria have been described as rhizobia (Berrada and Fikri-Benbrahim 2014). Till date 12,000 nodulated legume species are known, and each has its own root nodulating partner(s) (Maróti and Kondorosi 2014). Legumes are second largest group of food and feed crops. These represent the third largest family of angiosperms and cover 12–15% of all available arable land which contributes more than 25% in the world’s primary crop production (247 million tons of grain legumes annually; Ferguson et al. 2010). Rhizobia-legume symbiosis provides approximately 40 million tons of nitrogen into agricultural systems each year (Herridge et al. 2008) and plays a crucial role in increasing productivity and quality of crops specially protein content (Krapp et al. 2011). Besides this, these have been recognized as potential candidates to replace mineral N-fertilizers (Tairo and Ndakidemi 2013). It is estimated that the value of total nitrogen fixed by BNF process is equal to US $ 160–180 billion (Rajwar et al. 2013). Mostly the agricultural legumes have been studied for their symbiotic partner and others including wild and with little economic value are neglected (Ogasawara et al. 2003).

In the era of intensive agriculture, rising costs of N fertilizers and their adverse effects on environment have posed a threat to agroecosystems. Using rhizobial inoculants in the form of biofertilizers in place of N fertilzers has been considered as cheap and sustainable alternative (Arora et al. 2001; Kennedy et al. 2004; Mia et al. 2007). Use of biofertilizers is becoming more popular at the global level. The market trends also indicate that this could further increase in near future. The global biofertilizers market was estimated at US $ 535.8 million in 2014 and projected to reach up to US $ 1.88 billion by 2020 (Markets and Markets 2015). Amongst all types of biofertilizers, nitrogen fixers contribute maximum (75%) in agriculture (Grand View Research 2015) (Fig. 4.3).

Fig. 4.3
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Market share of different types of biofertilizers at global level (Grand View Research 2015)

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Rhizobial biofertilizers have been applied to crops since more than a century. However, in last few years, research on field application of rhizobia has addressed the task of identifying the essential conditions required for its survival, usability and quality in the formulation products for enhancing crop production. Generally, the term bioformulation entails application of microorganism(s) as partial or complete substitute for chemical fertilizers/pesticides (Arora et al. 2010) but in a broad sense it is essential to define role of an active ingredient, a carrier material and an additive in preparation of bioformulation (Mishra and Arora 2016). Various types of bioformulations having different rhizobial species as active ingredients are being used, and indeed, have a profound effect on crop N requirement. However, newer techqinues for identification of different carriers, additives and delivery systems have provided robustness to convential bioformulations and show potential to subside the current need of mineral N fertilizers. In the present review, the journey of development and advances in rhizobial inoculants since their inception in the market, are discussed.

4.2 History

Legumes have been used as a food source since ancient times and also known as soil improvers. However, real role of rhizobia was identified much later. German botanist Leonhard Fuchsius, published the first drawings of nodulated legumes in 1542 (Fuchsius 1542). Malpighi (1679) also observed nodules on the bean roots (Phaseolus vulgaris and Vicia faba). BNF by legumes was first time proposed by Boussingault (1838) when he was doing crop rotation experiments with legumes and found increased N content causes superior nutritive quality in legumes and benefits to the soil. Lachmann (1858) during the microscopic study of nodules found that the nodules contain vibrio like particles. Further, these particles were also described as bacteria like and Woronin (1866) validated that root nodules in legumes were formed by a specific group of bacteria. Another milestone in the field of rhizobia-legume symbiosis was the discovery of the ability of root nodules to fix gaseous N which was demonstrated by German scientists Hellriegel and Wilfarth, in 1886 and two years later, they published their observations (Hellriegel and Wilfarth 1888). In the same year Dutch microbiologist Beijerinck, first time isolated a bacterium from root nodules and named it as Bacillus radicicola (Beijerinck 1888) and later Frank renamed it Rhizobium leguminosarum (Frank 1889).

The first commercial N biofertilizer of rhizobia, ‘Nitragin’ was patented by Nobbe and Hiltner (1896). Famous agricultural chemist Guthrie stated about rhizobial inoculants “one of the most valuable contributions ever made by science to practical agriculture” (Guthrie 1896). In the nineteenth century, Löhis and Hansen (1921) classified the rhizobia into two groups according to their growth patterns as slow growers and fast growers. Baldwin and Fred (1929) proposed the cross inoculation between some leguminous host plants and rhizobia. This cross inoculation concept was suggested for taxonomic characterization of rhizobia, based on cross inoculation groups (Eckhardt et al. 1931; Fred et al. 1932).

Before the discovery of rhizobia, the inoculation of seed or soil was done in the crop by “soil transfer method”, in which soil from legume grown field to field or field to seed were applied before planting (Fred et al. 1932). However, artificial inoculation techniques by using pure cultures on agar slants and broths also began (Nobbe and Hiltner 1896; Fred et al. 1932). Inoculant production techniques started to change from those of the early 1900s, and use of solid carrier based formulations were started for legume inoculants, which were developed to enhance the shelf life and field efficacy of inoculants. In this context, soil and peat formulations were used (Fred et al. 1932). Peat was the most important carrier for long term storage of inoculants because of some beneficial properties, e.g., high water holding capacity, chemical and physical evenness, non-toxic and environment friendly nature (Ferreira and Castro 2005). Although peat was being used as the most common type of carrier for rhizobia based inoculants (Bezdicek et al. 1978; Khavazi et al. 2007; Albareda et al. 2008) but some constraints were also reported, such as it may contain inhibitory factors for the microbes (Brockwell 1985) and lack of availability in many parts of the world. Hence, the interest in using other carrier materials was also on. The carrier materials such as lignite (Kandasamy and Prasad 1971), filter mud (Philpotts 1976), coal-bentonite mixture (Deschodt and Strijdom 1976), cellulose (Pugashetti et al. 1971), coal (Crawford and Berryhill 1983), bagasse, wheat straw, compost of coir dust and soil, charcoal, manure, compost, powdered coconut shells, ground teak leaves also started to find their way in rhizobial inoculants (Tilak and Subba Rao 1978). Solid carrier based inoculants were also developed as granular inoculants for direct application in soil (Brockwell et al. 1980), and key benefits of this technique were easy storage, handling, and application (Smith 1992).

It is reported that liquid based formulations for rhizobia were developed as alternatives and were effective (Van Schreven et al. 1953; Singleton et al. 2002). Smith et al. (1981) reported that nodule numbers increased when applied with liquid rhizobial inoculants. The freeze-dried inoculants based on lyophilization techniques were first time identified as commercially beneficial in 1958 (Brockwell 1982). Bonish (1979) applied diluted soil samples to inoculate clover seedlings growing in lab conditions. At that time gel based microbial inoculants were also developed as alternatives to powdered carrier-based inoculants by entrapping rhizobia in polymer gels such as polyacrylamide-entrapped Rhizobium (PER) (Dommergues et al. 1979), alginate-entrapped Rhizobium (AER), xanthan-entrapped Rhizobium (XER); which gave satisfactory results in wet conditions (Jung et al. 1982). Kremer and Peterson (1982) reported that freeze-dried rhizobia suspended in dried oil were resistant against high soil temperatures. Brockwell et al. (1988) found whole soil inoculation technique (WSIT) suitable for clover production. In this method, rhizobial populations of soil were used to inoculate plants for assessing the N2 fixing capability of that soil. Thies et al. (1991) developed simple functions to predict the need for inoculation based on numbers of rhizobia in the soil and soil nitrate levels.

Use of flavonoids (genistein/daidzein) for enhancing soybean yield was patented by Smith and Zhang (1999) which was commercialized as SoyaSignal™ (Leibovitch et al. 2001). Ballard and Charman (2000) used the Brockwell technique (WSIT) to evaluate the symbiotic N2-fixing potential of soil samples. Other formulation types also appeared in the market, such as the vermiculite-based Gold Coat TM Rhizobium inoculant (Paau et al. 1991), liquid seed-applied soybean inoculant, Cell-Tech® (Smith 1995), liquid in-furrow inoculant LIFT (Smith 1995) and air-dried clay powder for alfalfa, Nitragin® Gold (Smith 1995). It can be said that rhizobial species have been successfully marketed globally and inoculants are produced and used in many countries in all continents of the world (Nelson 2004).

Hussain et al. (1995) introduced precursor based inoculum technology for growth enhancement of lentil crop in which indole acetic acid (IAA) precursor (tryptophan) was added with inoculum. The commercialisation of a genetically engineered strain of Sinorhizobium meliloti was approved in 1997 (EPA 1997). After few years, modifications in liquid inoculants were proposed; Singleton et al. (2002) developed additives and cell protectants based liquid formulations for improved growth performance. The additives promote cell survival in storage and after application to seed or soil (Singleton et al. 2002). Commonly used additives for rhizobial inoculants were polyvinyl pyrrolidone (PVP), carboxymethyl cellulose (CMC), gum arabic, sodium alginate and glycerol. PVP is a synthetic vinyl polymer that improves survival of rhizobia by protecting it from desiccation and also from harmful seed coat exudates (Singleton et al. 2002). The CMC has important rheological property and increases the gel viscosity of carriers to make it more suitable for viability of rhizobial cells (Rohr 2007). Gum arabic is a complex carbohydrate extracted from Acacia and commonly used as adhesive to protect the rhizobia against desiccation (Wani et al. 2007). Sodium alginate is non-toxic compound and used to enhance the survival of inoculant because it has limited heat transfer property and high water activity (Jung et al. 1982). Glycerol is used as additive because it protects rhizobial cells from desiccation by slowing the drying rate (Manikandan et al. 2010).

Besides above mentioned carriers for rhizobial inoculants, waste water sludge was also used as a carrier and it was firstly reported by Ben Rebah et al. (2002a). There are various other types of formulations with different carriers developed for rhizobial inoculants and some of them have been patented, e.g., the patent no. 521.850 (Belgian) for Rhizobium which uses diatomaceous earth and colloidal silica; the British patent no. 1.777.077 for the use of bentonite for Rhizobium. For more advancement in formulation techniques, genetic modification of rhizobia is also being done to increase the efficiency of N2 fixation such as genes that regulate the Hup system (recycles the hydrogen released during nitrogen fixation) have been identified and transferred (Brito et al. 2002).

From the beginning of twentieth century, extensive research has been carried out for development of state of the art rhizobial bioformulations and advents of newer techniques have provided inputs in this direction. There are various modifications related to rhizobial inoculants that have been done since their inception and the major events in the history are compiled in Fig. 4.4. The rhizobial inoculants are being used from long time in diverse types of formulations and detail of some formulations are given in Table 4.1.

Fig. 4.4
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Timeline of major discoveries in the development of rhizobial bioformulations

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Table 4.1 Overview of some of the rhizobial bioformulations around the globe
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4.3 Present Scenario

The application of rhizobial bioformulations is one of the cheapest and eco-friendly approaches for improving production of leguminous plants and fixation of atmospheric nitrogen (Thakare and Rasal 2000). It has been estimated that 2000 tons of rhizobial inoculants of worth US$ 50 million are produced worldwide every year (Ben Rebah et al. 2007) and this quantity is sufficient to inoculate 20 million hectares of legumes (Herridge et al. 2002). Currently, there are various types of rhizobial bioformulations available in the market for agricultural purposes and all are categorized mainly into two major groups - solid formulations and liquid formulations (Burges and Jones 1998). Peat is still most common carrier material in rhizobial inoculant production (Kaljeet et al. 2011) and other carrier materials such as coal, bagasse, coir dust, perlite are also used (Albareda et al. 2008). In a recent study, Ruíz-Valdiviezo et al. (2015) worked on the granular peat based and perlite based bioformulations. The additive based solid inoculants are also used such as sawdust based formulations amended with CMC (Aeron et al. 2012).

Liquid formulations are used for legume inoculation as more suitable technique for mechanical sowing in large areas (Fernandes-Júnior et al. 2009). Liquid formulations typically contain aqueous, oil or polymer based products and these formulations may have the desired strain and its nutrients, which are more tolerant to adverse conditions (Brahmaprakash and Sahu 2012). One of the methods for liquid formulations is water-in-oil emulsions (Vandergheynst et al. 2007), which is beneficial for desiccation sensitive organisms as it slows down water evaporation. Currently, additive based liquid formulations are in greater use and demand (Rivera et al. 2014; Ruíz-Valdiviezo et al. 2015).

Polymer gel based formulation techniques and synthetic polymer based techniques are also in focus (Fernandes-Júnior et al. 2009). Synthetic formulations based on a mixture of polymers have been continuously investigated (Bashan 1998; John et al. 2011). Alvarez et al. (2010) used silica gel as efficient formulation technique for rhizobial inoculants. Denardin and Freire (2000) reported that blends of natural or synthetic polymers are able to maintain viability of rhizobial cells for over 6 months. For agricultural and environmental uses, these polymers include alginate, agar, λ and κ carrageenan, pectin, chitosan, bean gum, and proprietary polymers (Bashan et al. 2014). Granular vermicompost, produced from essential oil bearing crop, scented geranium (Pelargonium graveolens), is used as efficient carrier for rhizobia (Ben Rebah et al. 2007). Some natural compounds such as cow urine and calliterpenone added to rhizobial bioformulations increased the numbers of rhizobial cells by tenfold when supplemented at 12.5–25.0 μl/ml (Kalra et al. 2010).

In case of rhizobial inoculants, mono-inoculation, co-inoculation or multistrain inoculation are also being used (Arora et al. 2014; Malusá and Vassilev 2014; Verma et al. 2014). The application of rhizobia in combination with other plant growth promoting rhizobacteria (PGPR) are used as suitable alternatives to promote plant growth both under normal and stress conditions, for example, plant growth promoting bacteria (PGPB) and rhizobia enhance nodulation, nitrogen fixation symbiotically (Rodrigues et al. 2013; Arora et al. 2014; Chibeba et al. 2015) and increase the grain yield by involving diverse mechanisms (Hungria et al. 2015). It is reported that Rubiya (2006) developed the “Multigeneric diazotrophic co-flocs” (Azospirillum, Azotobacter and Rhizobium) and reported good improvement in rice yield. The biofilm based inoculants containing a fungal-rhizobia consortium were also applied significantly for increased N2 fixation in legumes (Jayasinghearachchi and Seneviratne 2004). The soil-made inoculants are also used by mixing clay soil inoculant with powdered elemental sulphur and inoculation of sulphur oxidizing bacteria (Thiobacillus sp.) with rhizobia synergistically promoted the yield and oil content of groundnut in sulphur-deficient soils (Anandham et al. 2007). Khare and Arora (2011) reported that on applying pyocyanin-producing pseudomonads together with rhizobia, an enhancement in nodulation ability is observed, which causes better growth and productivity of groundnut even in the presence of fungal phytopathogens. The consortium based liquid bioformulation technique is now being used as an important way for sustainable agriculture (Pindi and Satyanarayana 2013). Some of the recent trends include arbuscular mycorrhizal fungi (AMF) and rhizobia co-inoculation, which enhance the growth and yield of crops due to higher nutrient uptake (Abd-Alla et al. 2014b). AMF causes the enhancement in nitrogen uptake process by rhizobia and their association with legumes (Tajini et al. 2011). Meng et al. (2015) stated that AMF and rhizobia simplify the nitrogen uptake process and transfer in soybean/maize inter-cropping system. This AMF and rhizobia co-inoculation has great potential for stressed soils. Zhu et al. (2016) reported that AMF and nitrogen fixing bacteria enhance alfalfa yield under saline conditions.

There is also an emerging formulation technique in which the addition of microbial/plant associated secondary metabolites to bioformulations increases agricultural productivity by improving the inoculants efficiency (Morel et al. 2015). In the current market, metabolite based formulations for rhizobial inoculants are highly focused and the additions of flavonoids and phytohormones are being used for rhizobial inoculants. Plant inoculation with rhizobial cells, previously induced with flavonoids during growth, significantly alleviates the effects of adverse conditions (Nápoles et al. 2009; Muñoz et al. 2014). The addition of flavonoids to inoculated crops enhances the nitrogen fixation (Dashti et al. 2000), improves the rhizobial competitiveness and nodulation (Pan and Smith 2000). Although flavonoids are expensive, these act at very low concentrations and produced industrially for sustainable agriculture (Mishra and Arora 2016; Morel et al. 2016).

The phytohormones produced by microbes or plants show positive effects on plant growth (Hedin and McCarty 1994; Dazzo et al. 2000; Chandra et al. 2007; Tank and Saraf 2010; Kudoyarova et al. 2015). The inoculations of plants with phytohormones producing rhizobia have positive effects on plant development and seed priming with phytohormones (IAA, gibberellins, abscisic acid and ethylene) increase the germination rate and finally crop yields (Roberto et al. 2012; Bianco et al. 2014; Kudoyarova et al. 2014; Qiu et al. 2014). Addition of phytohormones to bioformulations increases plant development and yield in comparison to bioformulations alone (Morel et al. 2016). Although various types of rhizobial bioformulations are commercially available in the current market having diverse types of additives, adjuvants and metabolites, it needs more exploration for future use.

4.4 Limitations: With the Current System

The demand of bioinoculants, mainly rhizobial inoculants, is high and these are produced commercially at global level (Brockwell and Bottomley 1995). Although rhizobial bioformulations have very significant value in agriculture, these also have some limitations. It is obvious that on application of inoculants in field, the introduced microbes face a very hostile environment and sometimes their population decreases, which leads to failure (Bashan and Levanony 1988; Arora et al. 2001). In some cases, applied rhizobial inoculants are unable to increase sufficient crop yield because of competitions faced by indigenous rhizosphere microflora of plants (Merwe et al. 1974; Olufajo and Adu 1993; Mazid and Khan 2014). Generally, predatory organisms, protozoans and bacteriophages are already present in the soil (Somasegaran and Hoben 1994). The environmental conditions also affect the inoculant efficacy and adverse abiotic stresses (hot, dry and saline conditions) can cause rapid decrease in rhizobial populations (Mazid et al. 2011; Deshwal et al. 2013). Other factors such as bacterial survival on the seed are mainly affected by three factors: desiccation, the toxic nature of seed coat exudates and high temperatures (Deaker et al. 2004). Bacterial survival on seed directly affects the total legume/crop yield (Brockwell and Bottomley 1995). Sterilization techniques of inoculant carrier (Strijdom and van Rensburg 1981) and compatibility towards crop also affect the applicability of inoculants (Lupwayi et al. 2006). Shelf life of inoculants is a very major factor for their efficacy which mainly depends on several factors (production technology, carrier and packaging material used, transport activity) to sustain the quality of inoculants (Arora et al. 2010). Often import and proper storage of inoculants are also problematic because in absence of proper care, viability of inoculants decreases with loss in their beneficial properties (Kaljeet et al. 2011). The storage of bioformulations needs special facilities and skills, which most producers, shopkeepers, and farmers do not possess (Arora et al. 2010). The use of genetically improved rhizobia as inoculants has some legislative constraints because it requires permission from environmental protection agencies to release into the environment and the second problem is less understanding of microbial ecology (Geetha and Joshi 2013).

All inoculant producers claim that their products promote crop productivity but actually most of the products are available in the market without robust scientific data to favour their efficacy (Herrmann et al. 2015). In this regard, Brockwell et al. (1995) stated that most of the inoculants (90%) have no practical impact on the yield of target crop. Similarly, Olsen et al. (1996) reported that commercially available rhizobial inoculants lack proper population of rhizobial cells. Recently, Herrmann et al. (2015) analysed various inoculants and reported that more than 50% of the inoculants have high levels of contamination. It is also reported that contaminants have detrimental effects on the quality of rhizobial inoculants (Sparrow and Ham 1983; Rodriguez-Navarro et al. 1991) and 25% contaminants of the commercial inoculants can be opportunistic human pathogens (Olsen et al. 1996; Gomez et al. 1997).

The quality of inoculants is judged by their efficacy on application in field and if their quality and field performance is poor, then the product become unsuccessful in the market. Hence a lot of inoculants produced globally are of poor quality and the reason behind this issue is the lack of efficient quality control programmes (Somasegaran 1991; Brockwell and Bottomley 1995; Catroux et al. 2001). This drawback can lead to a negative impact on the future of the inoculant industry. In this context, various researchers suggested the need of an appropriate regulatory quality control program at international level for successful production and use of inoculants by end users (farmers) (Olsen et al. 1994; Herrmann et al. 2015; Arora et al. 2016). Thus, there is a requirement of strict regulations for rhizobial bioformulations to overcome above mentioned problems related with its worldwide productions and applications. All these factors have to be taken into accord to develop future rhizobial inoculants.

4.5 Future Prospects

The future of rhizobial formulations is directed to overcome or improve the lacunae associated with the present systems. As there are various types of rhizobial formulations available in the market, but each product has some limitations especially regarding their use, efficacy, survival or market availability. Hence, it is challenging task to develop a state of art formulation technology that fulfils all the required traits and make it available to farmers with global acceptancy. In this context, novel scientific approaches and information has also led to some good results and promise. For example, genotypic diversity of rhizobia can be assessed accurately by polymerase chain reaction (PCR) fingerprinting techniques such as enterobacterial repetitive intergenic consensus PCR (ERIC- PCR; Pongslip 2012), repetitive element palindromic PCR (rep-PCR; Menna et al. 2009) and enterobacterial repetitive sequences (BOX- PCR; Granada et al. 2014). These techniques help in the characterization of rhizobial strain which controls quality of inoculants (Pongslip 2012). Similarly, two primers random amplification polymorphic DNA (TP-RAPD) and amplified fragment length polymorphism (AFLP) are highly discriminating fingerprinting techniques and differentiate at species or below species level (Gzyl et al. 2005). However, emphases should also be given on techniques for increasing population density and survival of rhizobial strains in inoculants. Damasceno et al. (2013) devised electrospinning technique of rhizobia immobilization in nanofibers. They showed that encapsulation of rhizobial cell with polyvinyl alcohol (PVA) nanofibers enhances protection from dehydration and also minimized the effects of toxic chemicals. Nanoparticles made of inorganic or organic materials may enhance the quality of carrier-based microbial inoculants (Malusa et al. 2012). Sivasakthivelan and Saranraj (2013) also stated that survival of cells is mandatory for better commercialization of rhizobial inoculants in the global market. Suman et al. (2016) used the hydrogel based inoculants and experimentally proved that this method has low-cost and long shelf-life and also increased plant development in drought-prone environments. There has been a conflict in choosing a carrier for rhizobia inoculant development, as the carrier choice differs greatly in different inoculants. It is clear that same basic carriers are still in use. Various workers addressed that to increase the inoculant quality and efficiency, and to reduce costs and environmental impacts, alternative carrier materials have to be explored (Ben Rebah et al. 2007; Albareda et al. 2008). Polymeric inoculants (Bashan et al. 2014) and alginate beads (Sivakumar et al. 2014) have already been tested and need more exploration for their future use in inoculants.

Recently, Arora and Mishra (2016) provided their view on using metabolites and additives in bioformulations. Metabolites such as EPS can be used as carriers and also can play role in protection of cells and help in the nodulation process. The rhizobia nodulation genes nod ABC are involved in the synthesis of lipo-chitin oligosaccharides (LCOs) metabolites which are synthesized in the response of root exudates or flavonoids (Abdel-Lateif et al. 2012). LCOs increase plant growth development on applying alone or in combination with rhizobia and have given better results in field conditions (Miransari and Smith 2009; Marks et al. 2013). LCOs also act as growth regulators in a wide variety of plants, including non-legumes (Zhang and Smith 2002; Prithiviraj et al. 2003). Novozyme, first started to manufacture bioformulations containing LCOs and this technology is known as LCO promoter technology. These days, single formulations containing LCOs (Ratchet® and Torque®) or formulations with bacteria (OptimizeII®, Signum® and DynaStartMax®) are being marketed (Bioag.novozymes.com 2015).

Precursor in inoculant technology is also emerging as promising technique for efficient rhizobial bioformulations development (Naveed et al. 2015). Qureshi et al. (2012) found that the co-inoculation of Rhizobium and Bacillus sp. in the presence of a phytohormone precursor L-tryptophan (L-TRP) improved the pod and straw yield. Similarly, Qureshi et al. (2013) showed that interaction of L-TRP and rhizobial species increased the fresh fodder and dry matter yield in comparison to their separate application. Micronutrients have important role in nitrogen fixation and in this regard, Arora et al. (2009) recommended that the supplementation of Mo and Fe (up to certain concentrations) in soils along with the rhizobial formulations enhance the symbiotic nitrogen fixation process. Mmbaga et al. (2014) reported that the rhizobial inoculants supplemented with exogenous nutrients (phosphorus and potassium) improved photosynthesis, nutrient uptake, nodulation, growth and yield of the crop. According to Marks et al. (2013) and Morel et al. (2015), the bioformulations based on a mixture of various compounds, e.g., phytohormones, LCOs, flavonoids may be very useful for higher plant growth, and further research is required in this field.

The discovery of novel stress tolerating rhizobial species is also thought to be imperative in developing bioformulations that will survive in stress conditions (high temperature, drought, salinity) (Arora et al. 2012; Laranjo et al. 2014; Rao 2014). Currently, various types of rhizobial species have been discovered but only few of them are used as inoculants (Table 4.2). Stress tolerant bacteria have lots of scope for bioformulations production and also have important role in reclamation of waste lands (Arora et al. 2000, 2006). In a study, Karuppasamy et al. (2011) showed that the growth of tree legumes Samanea saman could be improved by application of stress tolerant rhizobia. Ahmad et al. (2013) also stated that halo-tolerant, auxin producing Rhizobium strains improve osmotic stress tolerance in mung bean. Recently, benefits of using exopolysaccharides (EPS) in bioformulation is documented (Tewari and Arora 2014; Thenmozhi and Dinakar 2014). EPS protects inoculated rhizobial cells from stress factors such as salinity, dessication and pH (Ophir and Gutnick 1994; Qurashi and Sabri 2012). Use of EPS as efficient alternative carriers for inoculant production is also reported (Rodrigues et al. 2015). Maheshwari et al. (2012) suggested that the combined application of microbial inoculants and fertilizer worked as better choice for farmers to reduce the risk and expenses of chemical fertilizers.

Table 4.2 Classification of rhizobia and their inoculants used in global market
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Use of omics-based approaches (genomics and proteomics) can also be very useful in enhancing our understanding of rhizobia-legume symbiosis (Ramalingam et al. 2015). Omics based techniques including genomics, proteomics and metabolomics can go a long way in designing state of the art bioformulation for a particular soil and crop. Discovery of very similar signalling pathway in cereals as used by legumes to fix nitrogen has opened the door of non-legume fixation (de Bruijn 2016) and according to research by Rogers and Oldroyd (2014), in near future cereal crops capable of fixing nitrogen will also be available by the application of synthetic biological approaches.

4.6 Conclusion

Nitrogen is one of the key and limiting nutrients for agricultural ecosystems. The most important process to bring the atmospheric nitrogen in use for living creatures is BNF. Amongst microbes possessing the capacity to carry out BNF, rhizobia are the most important. Rhizobial inoculants are being used since long time around the globe, but still there is room for improvement so as to enhance the efficacy, productivity and credibility amongst the farmers. Liquid formulations are now picking up the pace in comparison to solid carrier based ones. Different types of additives and metabolites are also now being incorporated into the formulations to enhance their shelf life, performance and efficacy. Gel based encapsulated inoculants are also being used and with emergence of biotechnology, genetically improved inoculants and co-inoculants of rhizobia with other PGPRs/AMFs are also being worked upon in present time. Use of biotechnological tools and improvement in regulations can go a long way in designing a rhizobial bioformulation which will be more reliable and effective. To design a tailor made state of the art rhizobial formulation, it is very important to further our knowledge on plant-microbe interactions by using the latest tools and techniques. Also it would be better if the government and non-government organizations help in spreading the knowledge of usefulness of such bioformulations amongst the end-users. This is particularly required in developing nations. In future, we must see an even greater role and share of rhizobial inoculants in the market for sustainable supply of nitrogen to the future generations.