The selection of microorganisms for bioformulation development is an essential step for the startup process of bioformulation. Primary selection of microbial strains occurs on the grounds of their plant growth promoting (PGP), antagonistic, degradation potential, and any other useful properties (Wong et al., 2019; Singh et al., 2020). In order to select potential candidates, various microbial sources such as soil, water, and any other specific substances are being used for isolation, they were tested for various properties such as siderophore and lytic enzymes production, nutrient solubilization, production of phytohormones and antibiotics, xenobiotics degradation or heavy metal bioremediation, etc. (Vassilev et al., 2015) which make them a potential candidate for bioformulation development. Further, these microbes were tested for In vitro and In vivo plant growth promotion and bioremediation properties assessment. In addition, some desirable characteristics must be met with the microbial strains, including genetic stability, physiological adaptability with the host environment, extended self-life, survival capability under harsh conditions, efficient colonization with the host plant, non- pathogenicity, etc (Chakraborty, 2020). Sometimes, instead of a single microbe, more than one microbial strain is used for bioformulation preparation. Species of diverse genera i.e. Acetobacter, Arthrobacter, Azotobacter, Azospirillum, Bacillus, Burkholderia, Clostridium, Enterobacter, Flavobacterium, Frankia, Hydrogenophaga, Kluyvera, Microcoleus, Phyllobacterium, Pseudomonas, Serratia, Streptomyces, Rhizobium, Trichoderma, etc. have been already reported for splendid PGP potential and being considered for bioformulation preparation (Soni et al., 2017; Alawiye and Babalola, 2019; Suyal et al., 2019; Jeyakumar et al., 2020; Comite et al., 2021). Further, many microorganisms such as Penicillium bilaiae, Rhizobium leguminosarum, Bradirhizobium japonicum, Bacillus amyloliquefaciens, Trichoderma virens having multiple plant growth and biocontrol properties have been commercialized as biofertilizers through various organizations.

A suitable carrier is an important constitute of bioformulation preparation. It acts as delivery material for live microbial strains during the processing from laboratory to field. Individually or compositely, suitable inorganic/organic or synthetic carriers viz. peat, coal, clays, talc, vermiculite, charcoal, cellulose, sawdust, wheat bran, alginate beads, rice husk, polyacrylamide gel, calcium sulfate, silica gel etc. can be used to support microbial growth and effective delivery of desired microbes into the field (Vishwakarma et al., 2018; Table 1 ). Different carrier materials demonstrate multiple effects on microbial viability and the efficacy of delivery. Carriers used for bioformulation preparation can significantly impact the shelf life, bioavailability, release rate, and overall performance of the bioinoculants-based bio-formulation. Therefore, compatibility with the microorganisms, potential toxicity or immune response from the carrier should be prioritized during the selection of the carrier material. The carrier should also protect bioactive compounds from deterioration brought on by external elements including heat, light, and moisture. The carrier should be able to permit controlled release of the bioactive chemicals depending on the intended application in order to increase their bioavailability. Additionally, the carrier material’s particle size merits attention because smaller particles typically give faster dispersibility and dissolving rates. It’s critical to assess if the chosen carrier can be processed and scaled up effectively while remaining ecologically friendly in the context of commercial manufacturing (Singh et al., 2020; Aloo et al., 2022; Rojas-Sánchez et al., 2022);. During bioformulation preparation, the sterilization of carriers is an essential step. For this, gamma irradiation at a dose rate of 4.0 kGy for 1 h or autoclaving at 121°C for 20-30 min, is the most suitable way of carriers sterilization, and being used for selected carriers viz. rice husk, wheat bran, clay, peat moss and the mixture of peat moss and vermiculite (El-Fattah et al., 2013; Sahai et al., 2019). The sterilized carrier is mixed with actively grown microbial strains and air dried overnight to retain 15-20% moisture content, which is essential to lower down the microbial metabolic activities (Samavat et al., 2014). Furthermore, the mixture is packed and sealed in pre-sterilized i.e. autoclaving or gamma irradiation, polypropylene bags and stored at room temperature with 80% relative humidity (Arora et al., 2010; Suryadi et al., 2013; Namsena et al., 2016; Bargaz et al., 2018). A good bioformulation must contain at least ~ 10⁷ cfu/g of microbial cells (Jambhulkar and Sharma, 2014; Samavat et al., 2014).

Bacterial desiccation is a natural abiotic stress condition, usually occurring in the environment by freezing, heating, or drying and rewetting conditions of soil due to low precipitation or irrigation. Hence the bioformulation must show sustenance against this for efficient effect. Several spore-forming microbes could survive under desiccation, but the condition becomes lethal, especially for non-spore forming bacteria. Although, multiple physiological mechanisms have been observed behind the desiccation tolerance in non-spore forming microbial strains, which include synthesis of a compatible solute such as disaccharide trehalose or hydroxyl pyrimidine hydroxyl ectoine (Roder et al., 2005; Narvaez-Reinaldo et al., 2010; Khan and Singh, 2021), production of heat-shock proteins, enzymes and exopolysaccharides modification or repair of DNA mechanisms (Berninger et al., 2018). Hence, desiccation tolerance has great biotechnological interest in microbial cellular stabilization which allows the long-term storage of formulated products for commercial uses. Microbial inherent desiccation tolerance could be improved during the bioformulation process by applying some strategies including drying methods i.e. freeze-drying, vacuum-drying, spray-drying, fluidized bed-drying, and air-drying, the addition of external protectants, triggering of stress adaptation, triggering of exopolysaccharide secretion, and indirect protection by “helper” microbial strains (Berninger et al., 2018). Drying method is well known efficient way for long term storage. By following suitable drying conditions, the quality and self-life of the products can be enhanced equally. Further, disaccharide such as trehalose is an example of such protectant. During desiccation, trehalose forms hydrogen bonds with other proteins in the absence of water, preventing protein denaturation (García, 2011). Moreover, the implication of sublethal stress, including the variation of pH, temperature, depletion of nutrients, anoxic conditions, and salt stress conditions has been suggested before the desiccation to activate the cellular protective mechanisms (Liu et al., 2014).

The determination of the storage ability of bioformulation is an essential and critical factor in bioformulation efficacy. Usually, the self-life of the product and its microbial stability can be expected from 6 to 12 months (Berninger et al., 2018). The additives and low temperature storage are essential factors for the survival and stability of bioformulation. The stability test is performed through serial dilution plating at different time intervals and colony forming unit (CFU) estimation is done, which should not be less than 10⁴ CFU per gram sample (Wong et al., 2019). Several encapsulation materials, a wide temperature range, and different environmental conditions are being used to test the bioformulation sustenance capacity, which determines the shelf life of bioformulation. Further, bioformulations packaged into suitable bags to carry efficient microbes and to maintain humidity.

After bioformulation preparation and its successful demonstration in fields, the bioformulation is further processed for validation, in which the same bioformulation is tested through multi-locational field trials. After validation, the bioformulation needs registration through patent and risk-related approval before commercialization.

The success of any bioformulation mainly depends upon the carrier or bulking agent, which is the 2^nd most prominent component used in the preparation of bioformulation. The carrier material provides a protective environment and energy source for microbial growth and development and guarantees the successful release of the bacterial cells after the application. Carriers used in seed treatment should have good adhesion capacity with seeds to get better efficacy (Hegde and Brahmaprakash, 1992). The characteristics of the carrier include being cost-effective, easy to be processed, chemically stable, good moisture absorption and buffering capacity, non-toxic for both plant and microbes, and ensuring bacterial cell viability after a specified period of storage (minimum 2-3 months). There are varieties of carriers used nowadays according to the physical form of bioformulation. Solid carriers commonly are derived from soil materials like peat/plant soil, coal, clays, and lime (Hartley et al., 2004), some are derived from organic materials (saw-dust, composts, charcoal, chitosan and alginate (Bashan et al, 2002; Power et al, 2011), or some are inorganic material like talc, vermiculite, bentonite and kaolin (Smith, 1992). Liquid bioformulation can be produced in broth medium, carbohydrate, mineral or organic oil, emulsions and microbial suspensions. Some examples of carrier-based bioformulation applied on various crops have been listed in Table 1 .

Currently, different types of carrier material are available but the selection of a suitable one is a must, because it is the carrier’s material that supports the survival of bioagents. The degree of support of carrier material depends upon the nutrient and moisture holding status of the carrier. The high moisture retaining carriers having a low C:N ratio and pH near 7 is considered to be the best for increasing the shelf life of the bioformulation (Arora et al., 2014; Sohaib et al., 2020). Arora et al. (2014) tested the capacity of survival of different carrier materials such as sand, begasse, saw dust, wood ash, and coriander husk and found that higher moisture retaining carrier i.e., Coriender husk which retains 7.5 times moisture is the best for sustaining the bacterial survival. However, another experiment conducted by Sohaib et al. (2020) found that carriers having a low C:N ratio i.e., Compost and Biogas slurry are more effective in increasing the shelf life as well as plant growth and development of wheat over carriers having high C:N ratio. Therefore, the water holding capacity and C:N ratio of the carrier must be taken into consideration for the selection of an effective carrier.

Initially, in 1896 gelatin was first used as a carrier in the commercial production of Nitrogen bioformulation in the United States of America. Later ‘peat’ replaced all carriers and was named as a “gold” carrier until the 1990s (Williams 1984). The success of peat-based formulation can be varied according to the physical state of peat in bioformulation (Solid/powder, pellet, liquid/slurry). In a study, granular peat-based bioformulation greatly enhanced plant growth compared to powder and slurry-based bioformulation (Clayton et al., 2004). Peat in combination with either chitin or chitin-like materials, enhances the biocontrol efficiency of bioformulation along with the growth of microbes and promoted seed germination and plant biomass (Manjula and Podile, 2001). Lignite, charcoal, sawdust, various composts, organic wastes, and vermiculite are the other popular alternatives to peat. Inorganic material like talc-based formulations is very popular in India, as it is economical and easily available. Despite its limitations, this talc-based formulation has shown to be beneficial in various crops as biological control and enhancer of plant growth (Saravanakumar et al., 2009). Moreover, in a comparative study of different materials such as talc, kaolinite powder and bran of wheat, barley, and soybean used as a carrier for Pseudomonas fluorescens isolate RRb-11 based fertilizers, talc powder based microbes and has a maximum shelf life of 150 days after storage and is also best to manage bacterial leaf blight disease in rice (Jambhulkar and Sharma 2014). Whereas some Pseudomonas strains in peat bioformulation could stabilize for two years at ambient temperature (Georgakopoulos et al., 2002).

Bioformulations with easily degradable high carbon containing carriers like biochar based Bradyrhizobium japonicum lead to higher bacterial survival efficiency and better nodulation in soybean (Głodowska et al., 2017). Charcoal-based carrier, i.e., biochar, enhances the survivability of bioformulation and is environmentally benign as they don’t have any hazardous impacts. Another advantage of employing charcoal is that it may be kept without being sterilized owing to its low water content. In addition, Alginate is a nontoxic biodegradable synthetic polymer and is also used in the encapsulation of microorganisms. Alginate-based carrier provides longer shelf life to microbes and provides constant and slow delivery of inoculums to their target site (Bashan, 1986). A study has shown that dried alginate beads could sustain microbial survival for up to 14 years (Bashan and González, 1999). Bioformulations using Bacillus subtilis and Pseudomonas corrugate in alginate-based formulations produced incredible outcomes compared to charcoal and liquid-based bioformulations (Trivedi et al., 2005).

Adjuvants/adhesives are natural or synthetic polymers or polysaccharides, polyalcohol derivatives, or caseinate salts that increase the stabilization of microbes, enhance the adhesion potential, help in handling and mixing, and reduce the amount of dust in bioformulation (Jambhulkar et al., 2016; Pedrini et al., 2017). Adhesive application in bioformulation also prevents the dispersion of inoculants during sowing. Nowadays, carboxymethyl cellulose (Zhou et al., 2017), methyl cellulose (Lopisso et al., 2017), gum arabic (Ehteshamul-Haque et al., 2007), pelgel (Ugoji et al., 2006), skim milk (Power et al., 2011), humic acids (Schoebitz et al., 2013), PVP (Polyvinylpyrrolidone) (Surendra and Baby, 2016), glycerol (Anitha et al., 2016) and trehalose (Surendra and Baby, 2016) are generally used as an adjuvant in bioformulation preparation. Carboxymethyl cellulose (CMC) is a non-ionic water-soluble polymer, which is the most common or widely used adjuvant because of its easy availability and cheap economical value. Stimulatory effects of CMC have been demonstrated in various studies for increasing the shelf life and efficacy. Application of CMC supplemented saw dust carrier-based Rhizobium inoculants with M. pruriens demonstrated fighting against M. phaseolina pathogen (Aeron et al., 2012). In one study of chickpea (Cicer arietinum L.) seed treatment with CMC based Pseudomonas jesenii MP1 and Rhodococcus qingshengii S1010 bioformulations results in increased overall crop growth and soil nutrition (Joshi et al., 2019). Gum arabic is a complex polysaccharide, that protects the microbes from desiccation and increases their survivability (Wani et al., 2007). Poly vinyl pyrrolidone is a synthetic polymer that helps the survival of Bradyrhizobium japonicum in formulation (Singleton et al., 2002). PVP also protects against desiccation and provides the defense to inoculated microbes against toxic phytochemicals secreted by seed coats during germination. The additional adhesive layering of seeds with superfine calcium salts has decreased seedling mortality and increased plant growth. Here, calcium salts promote plant growth by balancing the acidic nature of the soil (Murata et al., 2008). The use of humic acid as an additive with Ca²⁺ amended alginate-based encapsulation of Bacillus, resulted in high bacterial survival and a positive impact on plant growth. The advantage of the inclusion of humic acid in this formulation is its function as a carbon source for the bacteria, which may lead to the survival of microbes during long storage (Rekha et al., 2007).

Generally, it has been seen that solid carrier-based bioformulations exhibit low shelf life and cannot retain bacterial load during the crop cycle (Chaudhary et al., 2020). To answer this problem, the use of liquid-based bioformulation is a better option. They provide long shelf life to microbes and maintain the survival of bacteria during the whole crop cycle. They also provide temperature and stress tolerance to bioinoculant (Chandra et al., 2018). The use of various adjuvants/adhesives in liquid bioformulations can improve the survival of microbes in a stressful environment, which results in better establishment with host interactions (Mugilan et al., 2011). The amendment of glycerol imparts long shelf life and stress tolerance to Pseudomonas against high temperature and desiccation via increased water holding capacity (Taurian et al., 2010). In another liquid bioformulation, Azospirillum in 16mM trehalose and phosphate solubilizing strain in 3% PVP maintain very high microbial density (10⁸ CFU/ml) as PVP protect microbes in toxic and stressed circumstances because of their water retention capacity (Surendra and Baby, 2016). Therefore, it can also be concluded that glycerol, PVP or trehalose amended liquid bioformulation can be more reliable and have high potential in the agricultural field. The survivability of microbes depends upon the physio-chemical properties of the carrier. So carriers must be selected based on microbial multiplication and survival during storage and the general method of planting. In summary, each carrier and adjuvant have some advantages and disadvantages. So, the selection of a carrier for bioformulation production is an essential step which majorly depends upon the cost, effectiveness, and need of the grower.

After field applications, solid bioformulations provide the protective and nutritive platform for desired microbes. It reduces contamination chances and enhances storage efficiency. It includes granules, powdered, and water-dispersible granular formulations containing active ingredients, binders, and carrier material. Based on applications, the solid bioformulation materials include soil-derived carriers i.e. charcoal, fine clay, turf, organic carriers i.e. sawdust, wheat, soy and oat bran, vermicompost, sewage sludge, animal manure, cork compost and inert carriers i.e. talc, peat, perlite, vermiculite, alginate, bentonite, kaolin, silicates, polyacrylamide beads, charcoal, etc. (Mishra and Arora, 2016). Further, solid bioformulation is characterized by the following:

Liquid formulations are aqueous suspensions and consist of biomass suspensions in water, oils or both (Schisler et al., 2004). It contains 10 – 40% microorganisms, 1 – 3% suspender, 1 – 5% dispersant, 3 – 8% surfactant, and 35 – 65% carrier liquid (Brar et al., 2006). Liquid bioformulations are helpful in stabilizing organisms throughout production, distribution, and storage. It protects from abiotic environmental factors and increases persistence. Liquid bioformulations can be categorized as suspension concentrates (Tadros, 2013), oil-miscible flowable concentrate, ultralow volume suspension (Singh and Merchant, 2012), and oil dispersion (Mbarga et al., 2014). The liquid carriers may be water, broth, fruit juices, jaggery syrups, and polyvinylpyrrolidone. The suspension concentrates have been prepared by mixing solid active ingredients with poorly soluble in water and stable to hydrolysis (Tadros, 2013). This mixture is non-dusty, measurable, and easily poured for spray. The oil-miscible flowable concentrate is a suspension with active ingredients in organic liquids. The ultralow volume suspension is used in their respective equipment. This equipment is aerial or ground spray for fine spray purposes (Singh and Merchant, 2012). Oil dispersion formulation comprises one active ingredient suspended in the oil phase and is chiefly used as herbicide and insecticide. NagaChandrabose (2018) has been found efficient liquid bioformulation of P. fluorescens, Purpureocillium lilacinum, and Trichodermaviride against the natural population of root-knot nematode Meloidogynehapla. Recently, Prakash and Arora (2020) developed a liquid bioformulation to enhance the growth, nutrient uptake and stevioside content of Stevia rebaudiana by using paneer-whey. Moreover, oils of groundnut, pongamia, and sunflower with nutrient broth and water have been used as a carrier, which retains the survival of B. subtilis, Brevibacillus borstelensis, Brevibacillus sp, Lysinibacillus xylanilyticus, and consortium (Jayasudha et al., 2018). Hence, liquid bioformulations help to enhance the shelf life of products and act as an excellent carrier to stabilize the bioinoculants throughout production, distribution, and long-duration storage.

The solid and liquid bioformulations have certain limitations like long term storage and viability of microbial spores. In such a scenario, immobilization and encapsulation have improved shelf-life and eased the field application of bioinoculants (Wong et al., 2019; Vassilev et al., 2020). Encapsulation provides controlled release of dynamic target bacterial cells and their metabolites in their rhizospheric environment (Wu et al., 2020) which gives a new strategy for soil microflora improvement and development of sustainable agriculture. Encapsulated bioformulations involve the coating of microbial cells within a polymeric material to produce beads, which are permeable to nutrients, gases, and metabolites for maintaining cell viability within the beads (John et al., 2010). Encapsulated bioformulation protects the active microbial components under unfavorable or environmental stress conditions i.e. mechanical injuries, pH, temperature, biochemical factors, ionic strength, etc. Gelatin, cellulose, starch, and some other polymers have been used in the encapsulation process (Cheze-Lange et al., 2002). There are two types of methods of encapsulation formation i.e. macro-encapsulation and microencapsulation. The macro-encapsulation involves beads of millimeters to centimeters in size, while microencapsulation is 1 – 1000 µm in size. Humic acids have significantly higher viability for encapsulation in certain bacteria. Natural polymers i.e. alginate, agarose, chitosan, cellulose, collagen, xanthan, and synthetic polymers i.e. poly(ethylene glycol), polyvinyl alcohol, polyurethane, poly(ether-sulfone), polypropylene, sodium polystyrene sulfate, and polyacrylate poly(acrylonitrile-sodium methallylsulfonate) have also been distinguished for cell encapsulation (De Vos et al., 2014). Diversity among nitrogen fixing bacteria (NFB) in symbiotic and non-symbiotic associations has revolutionized the crop yield and progress of sustainable agriculture. In this progress, microencapsulation in biofertilizers provides an alternative approach to the development of traditional nitrogen-based fertilizers. NFB and nodule-forming bacteria in association with the nodules of lupine plants of Southern Chile and their microencapsulation by spray drying using sodium alginate: maltodextrin has provided an alternative approach for Nitrogen biofertilizer (Campos et al., 2014). Recently, a novel electrospun microbial composite-based seed coat encapsulation of Canola (Brassicanapus) seeds has been developed for its rhizosphere stabilization by using a composite of poly(vinyl alcohol)/poly(vinylpyrrolidone) plasticized with glycerol and the microbial consortium of Bacillus subtilis and Serratia marcescens (Hussain et al., 2019).

Bacteria secrete various secondary metabolites to act as bioregulators, plant growth promoters, and antagonists against phytopathogens ( Table 3 ). Moreover, such microbial metabolites are β-1,3-glucanase (Confortin et al., 2019), ACC-deaminase (Onofre-Lemus et al., 2009), Hydrogen cyanide (Olanrewaju et al., 2017), phenazines (Biessy and Filion, 2018), pyrrolnitrin (Pawar et al., 2019), 2,4-diacetylphloroglucinol (Almario et al., 2017), pyoluteorin (Keswani et al., 2020), viscosinamide, tensin, Amphisin (Nielsen et al., 1999), siderophores (Deveau et al., 2016);, pyochelin (Ho et al., 2018), tetracenomycin (Gurusinghe et al., 2019), dialkylresorcinols (Schöner et al., 2015), peptides antibiotics and rhizoxins (Gross and Loper, 2009), mupirocin (El-Sayed et al., 2001), oxyvinylglycines (Okrent et al., 2016), orfamide A and H (Ma et al., 2020), phenazine-1-carboxylic acid (Morrison et al., 2017), furanomycin (Masschelein et al., 2017), brabantamide A (Schmidt et al., 2014), obafluorin (Pu et al., 1994), eruginaldehyde (Ye et al., 2014), safracins (Santos Kron et al., 2020), syringomycins SP22 or SP25 (Bensaci et al., 2011), tabtoxin (Arrebola et al., 2011), syringopeptins (Grgurina et al., 2005), rimid (Matilla et al., 2016), kalimantacin (Thistlethwaite et al., 2017) etc. These metabolites exhibit various properties like antimicrobial activity, insecticidal properties, mobilization of nutrient elements, eliciting plant defense systems, and acting as biosurfactants ( Table 3 ). Most of the above said microbial metabolites belong to the secretions of soil rhizosphere microbial communities. Pieces of evidence support that using such a combination of metabolite-producing bacteria as bioinoculants may promote plant growth and enhance agricultural productivity (Morel et al., 2015; De Souza et al., 2017; Santiago et al., 2017; Tewari et al., 2020). The isolation, characterization, and structural elucidation of bioactive microbial metabolites have depended on high-throughput technologies of molecular biology and analytical chemistry i.e. DNA chip, UV-Vis, Ultra-high-performance liquid chromatography – diode array detector – quadrupole time-of-flight mass spectrometer (UHPLC-DAD-QToF-MS), etc. (Gurusinghe et al., 2019; Kwon et al., 2019).

The secreted products in the form of enzymes, toxins, and other metabolites from desired microbial cells can be used to prepare CFCS bioformulation. The CFCS bioformulation is prepared by separating the supernatant from the cell pellet by centrifugation and passing through a 0.22 µm filter and mixing with a suitable carrier (Tewari et al., 2020). Multiple studies have significantly isolated and implemented the CFCS to enrich soil fertility and crop improvement. Patel and Thakker (2020) have assessed the amount of soluble phosphate from the CFCS while evaluating the mineral weathering efficiency of Streptomyces nanhaiensis YM4, the rhizospheric fungi of the millet crop. Moreover, in studying biocementation process of soil by calcite and aragonite, Citrobacter freundii and Pseudomonas azotoformans have been reported highest extracellular urease activities i.e. 45.5 ± 3.4 and 54.9 ± 3.5 U/ml, respectively. The study confirms that cell-free supernatants of C. freundii and P. azotoformans have participated in the precipitation of CaCO₃ from the cementation solution of urea and CaCl₂ (Abdel-Aleem et al., 2019). Manhas and Kaur (2016) have reported the biocontrol potential of Streptomyces hydrogenans and, cell and culture supernatant against Alternaria brassicicola, the causal agent of black leaf spot and damping-off of seedlings of crucifers. Recently, Kaur et al. (2019) have worked on biocontrol and plant growth promoting properties of Streptomyces sp. MR14, the soil actinobacteria, concluded its role of supernatant and extract in suppressing Fusarium wilt disease caused by Fusarium moniliforme in tomato plants. In a similar study, Bacillus amyloliquefaciens LZN01 showed antagonistic properties against Fusarium oxysporum f. sp. niveum, which was examined by functional components of CFCS from B. amyloliquefaciens. CFCS had shown damage to cell membrane integrity, which was further confirmed by confocal laser scanning microscopy. The major metabolites in CFCS were identified as myriocin, sphingofungin E, sphingofungin F, 3-methyl-2-oxovaleric acid, gabapentin, and sphingofungin C (Xu et al., 2019). Further, Silambarasan et al. (2012) have explained the antagonistic properties of CFCS of Actinobacteria isolates from Ratnagiri hills, Tamil Nadu, against Bacillus subtilis, Klebsiella, B. cerus, Staphylococcus aureus, Escherichia coli, Curvularia sp., Candida albicans, C. trophicalis. CFCS bioformulation has been observed with higher shelf life than living cells for plant growth promotion (Tewari et al., 2020). Hence, CFCS can provide the scenario for next-generation bioformulations by enhancing crop productivity and the development of sustainable agriculture.

The main application of biofertilizers in agriculture is to ensure food security and the nutritive value of plants for the good health of consumers like humans. After the green revolution, the continuous use of chemical fertilizers was able to fulfill food quality, but it is diminishing the nutritional value of plants and soil. Nitrogen (N), phosphorus (P) and Potassium (K) are essential macronutrients for proper plant growth and act as major limiting factors in terms of crop production as these elements play a vital role in plant metabolism, growth, and development. N, P, and K are present in different forms in soil, but the plants do not take the majority forms (Khan et al., 2019). Hence, most of the soil land in the entire world lacks plant-available nutrients (Karamesouti and Gasparatos, 2017). Therefore, in agriculture practice, the use of chemical fertilizers to increase the NPK content in soil increased, resulting in the leaching of excessive minerals into the soil environment. Plants uptakes nitrogen, phosphorus, and potassium through their roots from the soil, so the application of N-fixation bacteria, phosphate solubilizing bacteria (PSB), and potassium solubilizing bacteria as biofertilizers will increase the available NPK in soil and influence the plant nutritional status along with yield ( Figure 2 ). “BioGro” inoculant is a mixture of microbial strains isolated from rice crop soils. The application of this inoculant increases the grain yield and nutrients like N and P content in rice (Nguyen et al., 2017). Colla et al. (2015) reported a significant increment in the growth of shoot, root biomass, and leaves number by 23%, 64%, and 29%, respectively, and an increase in yield (8.3% to 32.1%), depending on the growing season and high nutritional grain quality along with enhancement in protein, K, P, Fe, and Zn concentrations after direct treatment with consortium of arbuscular mycorrhizal (AM) fungi (R. intraradices and F. mosseae) and T. atroviride as compared with untreated. The seed inoculation with the liquid formulation of Pseudomonas flouorescens increased the plant growth, biomass, and grain yield, and reduced the recommended dose of N fertilizer in maize (Sandini et al., 2019). A study to identify the best combination of bioformulation and chemical fertilizers for maximum chickpea production in hilly areas found that bioinoculants (N-fixers and PSB) with 20 Kg N/ha urea concentration resulted in high crop yield in chickpea and enhanced the rhizosphere and soil nutrition in comparison to alone biofertilizer, chemical fertilizer, and untreated control, as bioformulation increased the survivability of microbes (Joshi et al., 2019). This combinational approach for applying bio and chemical fertilizer to improve production with economic efficiency was also found applicable in sugarcane (Pereira et al., 2018). These studies showed that the correct combination of appropriate doses of chemical and biofertilizers could boost plant growth, which will help reduce the amount of chemical fertilizers.

Bio-control agents (BCA) and inducers of induced systemic resistance (ISR) have been widely studied to reduce the use of chemical fungicides in agriculture crops. In most cases, BCA can control plant pathogens directly or indirectly by developing a non-physical relationship with host-pathogen ( Figure 2 ). Another way to prevent the plant from biotic stresses is the competition for micronutrients and space to colonize and survive in the rhizosphere (Upadhayay et al., 2021). BCA colonization at pre-empty infection sites allows them to consume available plant resources and leaves the pathogen for nutrient and space scarcity. In a study of Lindow (1987), plant foliar colonization of Pseudomonas syringae strain on pear plants resulted in less infection caused by Erwinia amylovora than untreated plants.

Another way to control plant infection against pathogenic microorganisms and insects is to induce an Induced systemic response (ISR) defense system in plants (Pieterse et al., 2014). Bacillus spp are reported to produce cyclic lipopeptide compounds that result in plant ISR mechanism elevation through jasmonic acid (JA)/ethylene and salicylic acid (SA) pathways against phytopathogens. Chitin amended talc-based bioformulation of Pseudomonas fluorescens Pf1 reduced the disease effect of Macrophomina root rot in Moongbean by inducing the expression of the defense-related proteins and phytochemicals accumulation at the site of infection, which decreased the colonization of pathogens in the root (Saravanakumar et al., 2007). In this study, chitin amendment increased the growth and survival of chitinolytic microbes through acct as a carbon source in bioformulation (Bell et al., 1998).

Singh et al. (2014) found that seed coating of chickpea with a bioformulation using gum arabic as an adjuvant led to higher plant growth and an elevated amount of phenolic compounds in fungal pathogen Sclerotium rolfsii infected chickpea, in comparison to untreated control and single inoculations. Similarly, Saravanakumar et al. (2007) studied a mixture of three Pseudomonas fluorescens Pf1, TDK1, and PY15 strains to reduce the rot disease in rice with an increase in grain yield (Saravanakumar et al., 2007). In both studies, these consortia led to the activation of the plant host defense mechanism by elevating the level of defense-related enzymes, proteins, and phenolic content in the plant, which causes the ISR mechanism activation in the host to deal with biotic stresses. While in another application of Trichoderma strains with two synthetic fungicide agents (acibenzolar-S-methyl and thiamethoxam) decreased disease indices of phytopathogen Pyrenophora tritici-repentis in wheat by inducing plant defense system and activating pathogenesis-related enzymes which directed for ethylene signaling (Perelló and Bello 2011). The combination of microbial-based bioformulation with chemical compounds has resulted in more growth and caused less disease occurrence, so the use of the biological and chemical combinatorial approach for healthy plant and crop production will reduce the fungicide application. There is a robust future for new development and research in applying multi-strain carrier-based bioformulation in agriculture to manage biotic stresses.

The use of microbial bioformulations is often seen as a viable alternative to improve the crop yield under different abiotic pressures (Singh et al., 2021). Abiotic stress like drought, waterlogging, low or high temperature, salinity stress, and deficient or excessive mineral content negatively influence plant growth, yield, and nutritional quality of seeds. Recently, a research study documented improved cowpea’s biomass and crop yield under water-deficient conditions following treatment with silicon dioxide and starch-based- P. putida bioformulation (Rocha et al., 2019b). The study of Sohaib et al. (2020) reported that a bacterial consortium promotes high nitrogen and phosphorus content in straw and grains with better wheat plant growth and crop productivity by mitigating the salt stress and reducing ethylene production in organic compost biogas slurry based carrier bioformulation. Accelerated ethylene production is known to occur in stress conditions and induce senescence by degrading chlorophyll pigments, mineral misbalancing, and inhibiting protein synthesis under salinity stress. This result was also supported by previous research that highlighted the application of ACC deaminase containing bio-inoculants prevented ethylene’s output, which protects the plant from senescence (Zahir et al., 2011). The above-mentioned carrier-based bioformulation surges the survival of the above bacterial consortia until three months, which is best to protect the wheat plant. The same kind of effect was also reported by using PGPB like Pseudomonas fluorescens YsS6, Pseudomonas migulae 8R6 in peat-based bioformulation in tomato plants (Ali et al., 2014), and application of liquid-based alone or combination of different ACC deaminase producing microbes UW3 (Pseudomonas sp.) and UW4 (P. sp.) rhizobacterial isolates CMH3 (P. corrugata) in both barley and oats under high salt stress (Chang et al., 2014). Under abiotic stress, plant’s survival mechanisms induce through complex signaling pathways, which remarkably enhance by PGPR through the array of mechanisms (Wang et al., 2019). Under stress, plant activates signaling pathways with sensors, receptors, and ion channels. Specific protein kinases, like AtHKT1 in Arabidopsis thaliana, detect signals, triggering downstream gene activation via secondary messengers like reactive oxygen species and inositol (Gupta et al., 2022). These messengers induce calcium oscillations, driving stress-responsive protein formation (Ali et al., 2017). In a study, Bacillus subtilis priming was reported to modulate the HKT/K+ transporter gene (HKT), improving the K+/Na+ ratio by reducing Na+ uptake (Zhang et al., 2008). In another study, Pseudomonas fluorescence and P. putida regulate the At3g57530 gene, impacting calcium and calcium-dependent protein kinases (CDPKs). Rhizobacteria offer drought resilience through RIDER (Rhizobacterial-Induced Drought Endurance and Resilience). RIDER involves PGPR-induced changes like producing phytohormones, exopolysaccharides, cyclic metabolic pathways, and reinforcing antioxidant defenses with compounds like amino acids, polyamines, sugars, and heat shock proteins (Saharan et al., 2022). Additionally, the Piriformospora indica fungal endophyte was also found to enhance drought resistance by upregulating antioxidant enzymes, drought-related genes, and CAS mRNA levels in stressed leaves (Sun et al., 2010).

In a research endeavor, chickpea seeds were subjected to an experimental treatment involving the use of sodium alginate and CaCl₂ as carriers for Paenibacillus lentimorbus B-30488. This treatment led to a notable proliferation of beneficial bacteria in the soil and the formation of biofilms. Subsequently, this enhanced bacterial activity played a pivotal role in improving the chickpea plants’ resilience to drought stress by positively modulating their physiological responses to dehydration (Khan et al., 2011). Use of sodium alginate and calcium chloride increases the biofilm production and better seed attachment in this bioformulation and leads to overcoming the drought effect in plants. So further, these bioformulations may also be used in the phytoremediation of marine soils.

This method is more convenient for farmers because it takes less time for large areas and protects small or fragile seeds from damage, but it is expensive and generally needs a very high amount of inoculants. Soil treatment is used when many bacterial species are being applied to the soil, which enhances the probability of inoculant interaction with the rhizosphere, thus improving overall plant growth. Soil can be treated by solid, liquid, or encapsulation-based bioformulation (Malusa et al., 2012; Bashan et al., 2014). Granular forms of carriers like peat, charcoal, perlite, or other soil material have shown good effect in soil inoculation. Powder, slurries, and liquid inoculants have also been effectively added directly to the soil. In this approach, bioformulation is spread on the top surface of moist soil of the field before sowing using granular applicators, hand or mechanical sprayers. Soil treatment can also be done in the standing crop, but bioinoculants could not be distributed uniformly during these circumstances. Soil treatment is more beneficial when dealing with soil-borne phytopathogens as they protect plants from preoccupying sites. This approach has limitations in large application areas due to cost, large quantities of inoculants, and the requirement of specific equipment (Vosátka et al., 2012).

It is the direct application of biofertilizer to plants via two methods, either through root dipping or foliar spray. In this way of treatment, we can inoculate more inoculum or a concentrated amount of microbes as it allows multiple applications of bioformulations. In foliar spray, wettable or liquid bio-formulations are usually added to foliar sections of plants with the use of spray equipment which varies from hand to aircraft like mechanical equipment to combat above-ground plant pathogens, especially foliar pathogens, and provide nutrition to plants. A suppression in disease caused by Sclerotinia sclerotiorium in canola plants was reported after the foliar application of liquid bioformulation containing consortium of Pseudomonas chlororaphis (PA-23), Bacillus amyloliquifaciens (BS6 and E16) and Pseudomonas sp (DF41) (Fernando et al., 2007). For foliar spray, a major disadvantage is the need of large amount of microbial inoculant, which may be expensive, laborious, and treatment timing as it is limited to low environment temperature, high relative humidity, and turgid leaves during application (Bejarano and Puopolo, 2020). Root dipping of rice seedlings in talc-based bioformulation of Pseudomonas fluorescens suspension prior to transplantation reported a decrease in bacterial leaf blight of rice (Jambhulkar and Sharma 2014). This method decreases disease incidences because of previous inoculants colonization in the rice rhizosphere, which primarily avoids the development of the host-pathogen partnership. The root dipping method’s main disadvantage is the preparation of the nursery, which is a mandatory, laborious, and time-consuming procedure (Adholeya et al., 2005; Mahmood et al., 2016).

It is the most common bioinoculant application method as it needs a relatively low quantity of bio-formulation, which is widely used in a variety of cereals and legumes (Woomer et al., 2014). Seed inoculation or seed treatment delivers PGPM directly to the rhizosphere of the target plant, or in the case of an endophyte, these microbes enter the plant itself, which helps to develop an intimate plant-microbe interaction (Philippot et al., 2013). Seed treatment may be done using a number of methods, such as seed soaking, seed coating (seed dressing, pelleting/encrusting, film coating, slurry coating), and bio-priming, based on the size, shape, weight of treated seed and equipment availability (Joshi et al., 2019; Rocha et al., 2019a). In brief, seed coating is generally done by making a slurry of carrier-based bioformulation, with or without adjuvant, followed by uniform mixing of slurry onto the seeds, and further drying creates a thin layer of bio-inoculants over seeds (Choi et al., 2016). These inoculants can be applied with seeds by hand, low cost spinning drums, wide dough or cement mixers or hydraulic machines or automated seed coaters (Schulz and Thelen, 2008). Drying could also be performed either by natural air drying or using drying equipment. During liquid bioformulation application, inoculants are sprayed directly on seeds, followed by drying. Another advantage of seed inoculation is that it can also be used to modify seed characteristics (shape, size, weight, etc.) to make it easier to manage seed sowing and supply effective bio-inoculants to seeds (Halmer, 2008). Apart from the many advantages (such as low amount of inoculants, cost effective, fast, ready to use product), seed inoculation through bioformulation also has several drawbacks. The main drawbacks of seed inoculation are poor survival or reduced shelf life of active microbes in bioformulation and less inoculant coating over the small seed (due to lesser surface area). Sometimes seeds may be destroyed during the inoculation process, which prevents the germination of the seed. In some cases, the seed coat can be lifted out of the soil during germination, causing the death of the bacteria. So, the choice of inoculation method depends on the equipment available, the size and shape of the seed, the delicacy of seed coat and cotyledon, and the comfort and cost-effectiveness for the farmer (Deaker et al., 2004).