India is the world’s largest producer and consumer of pulses. In India during 2019-20, pulses were cultivated on an area of 27.9 million hectares with a production of 23.0 million tones and productivity of 823 kg/ha (
INDIASTAT, 2021). India plays a key role in the area (42.6%) and production (28.34%) of pulses globally. Among major pulses grown and produced globally, Indian share is maximum for pigeonpea in the area (73%) and production (67%) followed by chickpea (68% area and 66% production), dry beans (37% area and 18% production), lentil (29% area and 18% production) and dry peas (12% area and 6% production) in total pulses production (
FAOSTAT, 2021). The major pulse crops grown and produced in India are chickpea (35% area and 44% production), pigeonpea (15% area and 17% production), urdbean (18% area and 14% production), mungbean (14% area and 8% production), lentil (5% area and 6% production) and fieldpea (1% area and 4% production) in different agro-ecological regions. The major pulse crops grown and produced globally are dry beans (57% area and 48% production), chickpea (17% area and 17% production), pigeonpea (10% area and 10% production), dry peas (9% area and 17% production) and lentil (7% area and 8% production) (
Anonymous, 2018). The major pulse producing states in the country are Rajasthan, Madhya Pradesh, Maharashtra, Uttar Pradesh, Karnataka and Andhra Pradesh, which together contribute to about 75% of the total pulses production in the country.
Nowadays, agrochemicals are extensively applied to obtain higher yields. The intensive application of agrochemicals leads to several agricultural problems. Farmers use more chemical fertilizers than the recommended levels for many crops. Excessive use of chemical nitrogen fertilizer leads to soil acidification, contaminating groundwater and the atmosphere. Successful crop husbandry to a great extent depends upon how successfully one could manage these factors to favour crop production
(Kaur et al., 2015).
In the present scenario of the degradation of natural resources, the beneficial effect of pulse crops in improving soil health and sustaining productivity is very important. Pulses have the ability to fix atmospheric nitrogen thereby improving soil fertility and sustaining crop productivity. Moreover, the use of biofertilizers in pulses may help in increasing the productivity of pulses and improving soil health. Biofertilizers are a cost-effective, eco-friendly and renewable source of plant nutrients to supplement chemical fertilizers in the sustainable agricultural system. Biofertilizers are also ideal input for reducing the cost of cultivation which is a helpful parameter in increasing the farm income.
Biofertilizer refers to a preparation containing live microbes which help in enhancing the soil fertility either by fixing atmospheric nitrogen, solubilization of phosphorus or decomposing organic wastes or by augmenting plant growth by producing growth hormones with their biological activities. Biofertilizers play a very significant role in fixing atmospheric nitrogen, both, in association with plant roots and without roots
(Liu et al., 2011), solubilizing insoluble soil phosphates
(Alori et al., 2017) and producing plant growth substances
(Masciarelli et al., 2014; Oteino et al., 2015) in the soil. Annually, pulses fix about 2.95 Tg nitrogen through biological nitrogen fixation
(Herridge et al., 2008). Biofertilizers play many important roles in pulses (
Singh and Singh, 2018a;
Singh and Singh, 2018b). In order to reduce the use of chemical fertilizers, biofertilizers can play a crucial role by increasing the availability of soil nutrients and also sustain crop production.
Types of biofertilizers
There are different types of biofertilizers (Table 1). Some of them fix nitrogen, some solubilize fixed phosphorus by producing organic acids and enzymes and make them available to the crops. Apart from fixing nitrogen, they are also known to release growth promoting substances such as IAA, gibberellins, pantothenic acid, thiamine and niacin which promote root proliferation, plant growth and yield.
Role of biofertilizers in pulse crops
Biological nitrogen fixation
Biological nitrogen fixation (BNF) can act as a renewable and environmentally sustainable source of nitrogen in pulses. Nitrogen is provided through symbiotic fixation of atmospheric N
2 by nitrogenase in rhizobial bacteroids
(Hayat et al., 2010). The availability and uptake of nitrogen is very important for plant growth and development. Its use can mitigate the need for fertilizer nitrogen. Nitrogen obtained through the process of BNF is used directly by the plant and so is less susceptible to volatilization, denitrification and leaching losses. BNF is a kind of beneficial plant-microbe interaction that provides plants with nitrogen. Significant amount of nitrogen is fixed through the legume-
Rhizobium symbiosis.
Inoculation with
Rhizobium increased nitrogen fixation in mungbean crop
(Hussain et al., 2012). Similarly, co-inoculation of
Mesorhizobium sp. and PGPR enhanced the nodulation and N
2-fixation in cowpea
(Verma et al., 2013). Rhizobium inoculation of dry bean (
Phaseolus vulgaris) had a significant influence on the amount of fixed nitrogen. Inoculation significantly increased the N fixed in different tissues of the plant such as roots, shoots, pods and whole plants of
Phaseolus vulgaris grown both in the greenhouse and in the field
(Bambara et al., 2010). Seed inoculation with biofertilizer containing
Bradyrhizobium strains (
B. japonicum SAY3-7 and
B. elkanii BLY3-8) and
Streptomyces griseoflavusP4 promoted nodulation and thereby increased nitrogen fixation in soybean and mungbean
(Htwe et al., 2019).
Thus, inoculation with biofertilizers improved the N nutrition and resulted in more plant growth and crop grain yield.
Phosphate solubilization and production of growth promoting substances
Application of biofertilizers produces growth promoting substances such as indole acetic acid, gibberellins, auxins
etc.
Bacillus spp. strains B-20 and B-24 produced higher IAA equivalents as compared to
Pseudomonas spp. (
Saini and Khanna, 2012).
Mesorhizobium sp. strain BHURC02,
Pseudomonas fluorescens BHUPSB06,
Acetobacter chroococcum and
Bacillus megaterium BHUPSB14 were found to be positive for NH
3 and IAA production and
Pseudomonas fluorescens BHUPSB06 was found to be positive for HCN, siderophore production and inhibition of growth of soil-borne phytopathogens (
Fusarium oxysporum and
Rhizoctonia solani)
(Verma et al., 2012).
Kaur and Sharma (2013) reported that significant amount of IAA was produced by PGPR-3 (70.05 µg/ml) and PGPR-2 (66.79 µg/ml) as compared to PGPR LK 884 (61.58 µg/ml). Furthermore, PGPR-3 (13.45 mg/100 ml) and PGPR-2 (13.15 mg/100 ml) solubilized phosphorus also. Similarly high level of IAA production was recorded in
Pseudomonas sp.,
Bacillus sp.,
Rhizobium and
Mesorhizobium sp. by other workers
(Verma et al., 2012; Verma et al., 2013). Pseudomonas sp.,
Bacillus sp.,
Azotobacter sp. and
Mesorhizobium ciceri were reported as good phosphate solubilizers
(Verma et al., 2013). Isolates of
Pseudomonas sp. PGPR-2 and PGPR-3 also produced siderophores, HCN, NH
3 and improved seed germination in
kabuli and
desi chickpea (
Kaur and Sharma, 2013). Similarly,
Pseudomonas aeruginosa is also capable of siderophore and HCN production in chickpea
(Verma et al., 2013). In
Pisum sativum, use of endophytic
Pseudomonas strains (L111, L228 and L321) solubilized the insoluble phosphates by producing gluconic acid and also promoted the plant growth
(Oteino et al., 2015). In chickpea, seed inoculation with
Mesorhizobium ciceri synthesized indole-3-acetic-acid (IAA), 1-amino cyclopropane 1-carboxylate (ACC) deaminase, siderophores, exopolysaccharides (EPS), hydrogen cyanide, ammonia and solubilised inorganic phosphate. Moreover, microbial seed inoculation also had the ability to tolerate fungicide by decreased the levels of stressor molecules (proline and malondialdehyde) and antioxidant defense enzymes
viz. ascorbate peroxidise, guaiacol peroxidise, catalase and peroxidises of plants
(Shahid et al., 2021). It can be concluded that biofertilizers stimulate the synthesis of growth promoting substances such as indole acetic acid, gibberellins, auxins, NH
3, HCN and siderophore production.
Effect on plant growth, symbiotic traits and productivity
In plants, nitrogen and phosphorus play an important role in growth, development and finally determine the yield of the crops. Nitrogen is an important nutrient to plant growth and development as it is building blocks of protein. Phosphorus is a fundamental component of the substances that are building blocks of genes and chromosomes. Application of biofertilizers positively affects the plant growth, nodule number, nodule weight and grain yield. Biofertilizers make available nitrogen and phosphorus to the plants, as discussed above and thereby influence the plants.
In chickpea,
Rhizobium inoculation significantly improved symbiotic traits, number of pods and grain yield over uninoculated control
(Singh et al., 2011). Seed inoculation with
Mesorhizobium ciceri significantly increased the length of plants (41%), total dry matter (18%), carotenoid content (9%), leghaemoglobin content (21%), root nitrogen (9%), shoot phosphorus (11%) and pod yield (15%) over control in chickpea
(Shahid et al., 2021). Similarly, microbial inoculant
Anabaena laxa biofilm formulation significantly enhanced leghaemoglobin content of nodules, plant growth, yield parameters, grain yield and nutrient uptake as compared to uninoculated control
(Bidyarani et al., 2016).
Inoculation of seed with
Mesorhizobium and plant growth promoting rhizobacteria (PGPR) alone and their combination significantly enhanced shoot and root dry matter
(Singh et al., 2017b), symbiotic traits, chlorophyll content and grain yield of chickpea than uninoculated control
(Sharma et al., 2013). Similarly, combined and alone inoculation of
Rhizobium, phospho-solubilizing bacteria and arbuscular mycorrhizae (AM) significantly improved growth parameters, yield attributes and grain yield of chickpea over uninoculated control (
Praminik and Bera, 2012). In another study also, PGPR alone or in combination with bioinoculants (
Mesorhizobium and PSB) reduced plant mortality and increased grain yield of chickpea. Combined inoculation of chickpea seed with PSB+
Rhizobium gave significantly higher number of nodules, fresh and dry weight of nodules, grain, straw and biological yields compared to uninoculated and PSB or
Rhizobium inoculation alone
(Singh et al., 2014). Similarly, significant increases in nodule number, dry matter and nutrient content of chickpea were recorded in co-inoculation of
Mesorhizobiumsp. BHURC02 and
Pseudomonas fluorescens BHUPSB06 followed by co-inoculation of
Mesorhizobium sp.,
Azotobacter chroococcum and
Bacillus megatrium BHUPSB14 over uninoculated control
(Verma et al., 2012). Co-inoculation of
Mesorhizobium sp. and PGPR (
Pseudomonas aeruginosa) was found significantly better for nodulation (62 and 86%), plant growth [dry weight of root (44 and 57%) and shoot (26 and 45%)] over uninoculated control in pot and field condition, respectively and grain yield (32%) and straw yield (41%) of chickpea over uninoculated control in field condition
(Verma et al., 2013). The recommended dose of fertilizers (RDF) with 1 g ammonium molybdate/kg seed+
Rhizobium +PSB, RDF with 2 g ammonium molybdate+1 g FeSO
4/kg seed+
Rhizobium and PSB recorded the highest number of nodules/plant, pods/plant and grain yield of chickpea (
Gangwar and Dubey, 2012). The inoculation of seed with biofertilizers such as
Rhizobium and PSB increases the yield because the soil bacteria and fungi possess the ability to bring insoluble phosphate in soil into soluble forms by secreting various organic acids namely formic, acetic, propionic, lactic
etc. These acids lower the pH and bring about the dissolution of bound form of phosphate. Hence, supplementation of micronutrients along with
Rhizobium and PSB inoculation in chickpea may increase biological nitrogen fixation and phosphorus availability and thereby its productivity. In the controlled environment and in the field trials, single, dual and triple inoculations of chickpea seed with
Rhizobium, N
2-fixing
Bacillus subtilis (OSU-142) and phosphorus solubilizing
Bacillus megaterium (M-3) significantly increased plant height, shoot, root and nodule dry weight, pod number, grain yield and total biomass yield compared with the control, equal to or higher than N, P and NP treatments. In the field integrated use of 12.5 kg N/ha along with Rhizobium inoculation was better for nodulation than the use of
Rhizobium alone in mungbean
(Singh et al., 2021a). Seed inoculation with
Mesorhizobium also enhanced grain yield and nitrogen use efficiency of chickpea
(Doaei et al., 2020). Seed inoculation with
Mesorhizobium sp.
ciceri (LGR-33) alone or in combination with PGPRs [RB-1 (
Pseudomonas argentinensis) and RB-2 (
Bacillus aryabhatta)] improved the growth, symbiotic properties, yield attributes and yield of chickpea due to its P solubilizing property
(Singh et al., 2021b).
In lentil,
Saini and Khanna (2012) reported that inoculation of seed with
Rhizobium alone and its combination with different strains of PGPR enhanced the symbiotic parameters and grain yield of lentil to the tune of 13.6% (
Rhizobium+P-1), 14.6% (
Rhizobium+KB-133) and 14.9% (
Rhizobium+B-40) over the control. Treatment of integrated use of seed inoculation with PSB+PGPR along with recommended fertilizers+25 kg ZnSO
4/ ha
-1+1.0 g ammonium molybdate/ kg seed provided the highest grain yield of lentil (34.0% higher over control)
(Singh et al., 2019).
It also gave the highest and significantly more nodule occupancy at 45 and 90 days after sowing (DAS) than
Rhizobium sp. alone inoculation and also recorded a significant increase in grain yield and straw yield over
Rhizobium sp. and PSB alone inoculations. The symbiotic traits and productivity
(Singh et al., 2018), gross returns, net returns and BC ratio in lentil
(Singh et al., 2017a) increased significantly with
Rhizobium inoculation over uninoculated control, which further increased with the dual inoculation of
Rhizobium+PGPR.
In pigeonpea, application of 80 kg/ha P
2O
5 and 60 kg/ha sulphur with PSB inoculation significantly improved growth and yield attributes and grain yield. Combined inoculation of
Rhizobium, PSB and PGPR significantly increased grain yield of pigeonpea (
Goud and Kale, 2010). Similar beneficial effect of PSB on grain yield of pigeonpea was also reported by
Sathe et al., (2011). Inoculation of pigeonpea seed with
Rhizobium alongwith 50% RDF significantly increased branches, pods/plant and grain yield
(Reddy et al., 2011). Rhizobium strain and the dual inoculation with either
Pseudomonas putida,
Pseudomonas fluorescens or
Bacillus cereus resulted in a significant increase in plant growth, nodulation and enzyme activity over
Rhizobium inoculated and uninoculated control plants of pigeonpea
(Tilak et al., 2006).
Inoculation of greengram seed with
Rhizobium and PSB alone and their combination recorded significantly higher nodules/plant, yield attributing characters and grain yield than control
(Kumawat et al., 2010). Patel et al., (2013) also recorded significantly higher grain yield of greengram inoculated with PSB than uninoculated and it also gave the highest net returns. The seed yield increased with the inoculation of biofertilizers, however, combined inoculation of
Rhizobium and PGPR produced significantly higher seed yield over single inoculations and control in summer mungbean (
Kaur and Sharma, 2016). Single or dual inoculations of bacterial strains (
Pseudomonas fluorescens BAM-4,
Burkholderia cepcia BAM-6 and
Burkholderia cepcia (BAM-12) enhanced the growth of greengram plants in comparison with uninoculated control
(Jha et al., 2012a). The seed inoculation of cowpea with
Rhizobium and PSB alone and their combination increased grain yield significantly
(Khan et al., 2013). Seed inoculation with biofertilizer containing
Brady rhizobium strains (B.
japonicum SAY3-7 and B.
elkanii BLY3-8) and
Streptomyces griseoflavus improved plant growth, nodulation and seed yield of soybean and mungbean
(Htwe et al., 2019).
It can be concluded that seed inoculation with biofertilizer treatments influence the nodulation, growth and grain yield significantly over no inoculation. Furthermore, dual or triple inoculation of
Rhizobium, PSB and PGPR improves these parameters over single or dual inoculations only.
Nutrient uptake and nutritional quality
Biofertilizers improve the quality of produce. Co-inoculation of
Mesorhizobium sp. and
Pseudomonas aeruginosa showed significant uptake of nitrogen (65%) and phosphorus (58.9%) by grain of chickpea over control. The co-inoculation of
Mesorhizobium sp. and
Pseudomonas aeruginosa enhanced the acquisition of phosphorus and iron in chickpea
(Verma et al., 2013). Inoculation of chickpea seed with
Rhizobium and PSB alongwith RDF and ammonium molybdate improved the uptake of N and P by the crop and also increased the protein content in the chickpea seed (
Gangwar and Dubey, 2012). Combined inoculation of chickpea seed with PSB+
Rhizobium recorded significantly higher nutrient uptake compared to uninoculated and PSB or
Rhizobium inoculation alone
(Singh et al., 2014).
Seed inoculation with
Rhizobium+PSB recorded significantly higher nutrient uptake (N and P
2O
5) over
Rhizobium alone and uninoculation in pigeonpea (
Goud and Kale, 2010). As
Rhizobium+PSB inoculation increased the availability of N and P in the soil, it resulted in higher N and P uptake and improved the content (N and P nutrient) in the seed. This is mainly due to the reason that
Rhizobium inoculation fixes nitrogen through nodules of the plant and PSB solubilizes native phosphorous, bringing more phosphorus to the soil solution. Combined inoculation of
Rhizobium+PSB+PGPR recorded the highest and significantly more N, P, K and S uptake than the other treatments due to their additive effects
(Prasad et al., 2002). Uptake of N, P, K and S also increased with PSB inoculation in pigeonpea
(Sathe et al., 2011). N, P and K content in the grains and straw were significantly enhanced where recommended nitrogen was applied in combination with seed inoculation, basal application of ZnSO
4 and seed treatment with 1 g ammonium molybdate than their single applications in lentil
(Singh et al., 2019).
Dual inoculation of
Glomus fasciculatum and
Rhizobium enhanced the chlorophyll, nitrogen and phosphorus contents of pigeonpea (
Bhattacharjee and Sharma, 2012). Inoculation of pulses with
Rhizobium increases the nodulation, thereby causing more nitrogen fixation and making it available for the plants. Moreover, it also increases rhizospheric microflora
viz. acid producers and phosphate solubilizers, causing more availability of phosphorus. In soybean also, dual inoculation of
Bradyrhizobium and PGPR significantly increased the population of
Bradyrhizobium and PGPR in soil after harvest
(Virk et al., 2017a).
The improvement in nodulation with inoculation using phosphorus-solubilising organisms may have resulted in higher nitrogen fixation and hence higher nitrogen availability to plants, whereas solubilisation of fixed phosphorus by PSB probably resulted in increased content and uptake of phosphorus by plants.
Marko et al., (2013) reported significantly higher nutrient uptake by blackgram under dual and individual inoculation of
Rhizobium and PSB than control.
Bahadur and Tiwari (2014) recorded significantly higher N and P uptake and quality parameters like sulphur content, protein content and methionine content in mungbean under dual inoculation with
Rhizobium+PSB than control. Seed inoculation with biofertilizer containing
Bradyrhizobium strains (B.
japonicum SAY3-7 and B.
elkanii BLY3-8) and
Streptomyces griseoflavus P4 enhanced nitrogen, phosphorus and potassium uptake in soybean and mungbean
(Htwe et al., 2019).
The increased leaf N uptake might have increased the amino acid synthesis and thereby could have improved the grain protein content via their translocation to grains. Further, the inoculation with P biofertilizer might have increased P availability and its subsequent utilization for the formation of carbon skeletons and the synthesis of amino acids and adenosine triphosphate (ATP). That, in turn, might have led to an enhanced synthesis of protein during grain development.
Therefore, single or dual or triple inoculation of biofertilzer(s) improve the nutrient uptake, nutrient content and protein content, thereby the quality of the grain.
Soil fertility
All the living diazotrophs fix atmospheric nitrogen in the soil and lower the C:N ratio, which in turn enhances mineralization of N. Several biofertilizers containing microorganisms secrete growth promoting substances like IAA, gibberellic acid (GA), cytokinin
etc. Single or dual inoculations of bacterial strains (
Pseudomonas fluorescens BAM-4,
Burkholderia cepcia BAM-6 and
Burkholderia cepcia (BAM-12) were able to solubilize the insoluble inorganic phosphorus in mungbean
(Jha et al., 2012a).
Seed inoculation with biofertilizers significantly influences the available N and P in the soil. Seed inoculation with
Rhizobium+PSB+PGPR and
Rhizobium alone recorded significantly higher available N in the soil over seed inoculation with PSB alone and uninoculated control (Goud and Kale, 2010). Biofertilizers enhance soil fertility by solubilizing unavailable sources of elemental nitrogen and bound phosphate into available forms in order to facilitate the plant to absorb them (
Singh and Yadav, 2008).
Khan et al., (2013) reported that seed inoculation of cowpea with
Rhizobium and PSB significantly increased organic carbon, available nitrogen, available phosphorus and available potassium than uninoculated control. In chickpea, microbial inoculant
Anabaena laxa biofilm formulation increased nitrogen fixation and soil available nitrogen
(Bidyarani et al., 2016).
Seed inoculation of
Rhizobium and PSB increased the microbial count in soil after the harvest of mungbean (
Bahadur and Tiwari, 2014). An increase in the population of total bacteria by
Rhizobium inoculation may be due to higher availability of available nitrogen and carbon, which act as food for these microbes. PSB inoculation increased the phosphorus availability by solubilizing the fixed P in the soil, which provides the energy to the microbes for their better proliferation. Therefore, seed inoculation with biofertilizers improve organic carbon, available nitrogen, available phosphorus and available potassium and microbial count over uninoculated control and thus improve soil health.
Residual effect
Seed inoculation with biofertilizers such as
Rhizobium leaves a net positive N balance in the soil due to biological nitrogen fixation. Under satisfactory N-fixation conditions, the N fixed as a result of inoculation leave a net positive N balance in the soil. It is a general observation that under such conditions, symbiotic nitrogen-fixing systems satisfy a major portion of their N requirement from atmospheric nitrogen and additional fixed N, as expected, will be contributed to soil reserves for the succeeding crops. The quantity of N fixed depends on the species of pulses, location, yield level and also the effectiveness of the specific
Rhizobium strain.
Dual biofertilizers (
Rhizobium+PSB) resulted in maximum N-balance in the soil (230.5 kg/ha) after pigeonpea
(Ahirwar et al., 2016). The biological N-fixation was also found the highest in dual biofertilizers (
Rhizobium+PSB) treatment (84.5 kg/ha)
(Ahirwal et al., 2016).
Economic benefit
Economic evaluation of crop responses to biofertilizers application is an important aspect to assess the profitability of their use. Such an assessment is carried out by studying the gross returns, net returns and the benefit:cost ratio.
Rhizobium+PSB inoculation of pigeonpea seed recorded higher net returns and B:C ratio over control
(Singh et al., 2013). The cost of
Rhizobium+PSB and
Rhizobium is very low in comparison to the added output. Similarly, combined inoculation of
Bradyrhizobium and PGPR recorded the highest gross returns, net returns and benefit: cost ratio in soybean
(Virk et al., 2017b). Reddy et al., (2011) reported that 50% RDF+seed inoculation with
Rhizobium recorded the highest net returns followed by 50% RDF+dual inoculation with
Rhizobium and PSB.
Patel et al., (2013) recorded higher net returns of greengram inoculated with PSB than uninoculated control. The net returns and B:C ratio of combined inoculation of
Rhizobium and PGPR was maximum in summer mungbean (
Kaur and Sharma, 2016) and in chickpea
(Singh et al., 2021b). Treatment of integrated use of seed inoculation with PSB+PGPR along with recommended fertilizers+25 kg ZnSO
4/ ha
+1.0 g ammonium molybdate/kg seed provided the highest gross returns (34.0%) and net returns (54.8% higher over control) in lentil
(Singh et al., 2019). Seed inoculation with biofertilizers resulted in marked yield increase and thus higher net returns over uninoculation.