Biological Nitrogen Fixation

Nitrogen is an essential component of all living organisms including plants. It is an important constituent of amino acids, nucleic acids, proteins, and energy currency (ATP, GTP, ADP), etc. Therefore, it is known as a building block of cells. Although N2 is present in about 78% of the atmosphere, it is unavailable for plants and animals due to its complex structure, i.e., the triple bond between two nitrogen atoms [93]. PGPB can convert this gaseous form of nitrogen into a usable form, i.e., ammonia, by the use of an enzyme system, nitrogenase, and make it available to plants. Microorganisms that fix nitrogen belong to diverse genera such as *Azotobacter* spp. [94], *Bacillus polymyxa* [95], *Gluconoacebacter diazotrophicus* [96], and *Burkholderia* spp. [97]. There is also a report that inoculation of mixed inoculants of *Gluconacetobacter diazotrophicus, Burkholderia tropica, Azospirillum amazonense, Herbaspirillum rubrisubalbicans,* and *Herbaspirillum seropedicae* has been very effective in promoting N fixation in sugar cane [98]. *Azospirillum* is the most studied nitrogen fixer among these bioinoculants as reported by Calvo et al. [8]. It has been reported that there is a major increase in nitrogen content in plants when some plant species are inoculated with *Azospirillum* strains. For instance, *A. lipoferum* and *A. brasilense* show 7–12%, and *A. diazotrophicus* gives a 60–80% increase in wheat and sugarcane, respectively.

#### Solubilisation of Phosphate

Phosphorus is also a chief nutrient for plants next to nitrogen. In agricultural soils, the total concentration of phosphorus generally varies between 400 and 1200 mg/kg−<sup>1</sup> but only 1 mg/kg−<sup>1</sup> is available in the forms of dihydrogen phosphate (H2O4P−) and hydrogen phosphate (HO4P<sup>−</sup>2) [14]. Soil contains P in inorganic and organic forms which are insoluble [14]. The inorganic form of P contains about 20–50% of total P [99] and is generally available in the form of PO4<sup>−</sup> ions, which are sparingly soluble due to the adsorption of positively charged ingredients of soil and also are precipitated with some metals such as Al, Fe and Ca [99]. Insoluble organic P is also available in the form of inositol phosphates, phosphate esters, and uncharacterized large organic molecules and contains 50–80% of total P [99]. In agriculture systems, the low availability of P in soil is a significant problem [8]. PGPB can increase the nutrition of plants through the process of solubilization of P [14]. To solubilize insoluble inorganic and organic P, bacteria use several approaches. There are two mechanisms for the solubilization of P by bacteria that are predominant, i.e., through the production of organic acids or through the production of phosphatase and phytase enzymes [100,101]. Due to organic acid production, insoluble inorganic P gets transformed into soluble form due to the presence of carboxyl and hydroxyl groups that chelate the cations bound to phosphates [102] and transform them into soluble forms. The pH of immediate soil is also decreased by the production of organic acids and the solubility of P is improved by releasing phosphate ions [103]. PGPB are also known to solubilize

organic P by the production of phosphatase and phytase enzymes and converting them into soluble forms [104] which plants can easily uptake from the soil.

The predominant rhizospheric bacteria and others that have the ability to solubilize P belong to the genera *Burkholderia*, *Pseudomonas*, *Bacillus*, *Rhizobium*, *Agrobacterium*, *Achromobacter*, *Streptomyces*, *Micrococcus*, *Erwinia*, etc. Most phosphate-solubilizing bacteria that show good results under laboratory conditions, may not work well in soil conditions [99]. Therefore, rigorous field studies are ongoing to successfully characterize field-compatible phosphate-solubilizing potent bacteria.

#### Production of an Iron Chelating Compound

In the biosphere, iron is the fourth most abundant micronutrient. In aerobic conditions, iron is mostly found in ferric ions or Fe+3 which is insoluble and not easily accessible to plants and microorganisms [105]. In calcareous soil, Fe is not available for plants due to alkaline conditions making it less soluble [106]. Microorganisms, especially PGPB have a mechanism for producing low-molecular-weight iron-chelating compounds known as siderophore [93]. These compounds help in transporting iron into the bacterial cells and also make it available to plants. Siderophores also act as a biocontrol agent as they can create iron-deficient areas near the plant roots by inhibiting plant pathogens [105]. There is a significant increase in Fe uptake in some plants such as sunflower and maize in nonsterile calcareous soils as compared to sterile soil. This occurs due to the action of soil microorganisms that help in the uptake of Fe to plants [106]. It has been shown by Sharma et al. [107] that there is a significant increase in iron content in rice when inoculated with the strain of *Pseudomonas* because of the production of siderophores and also enhanced nutritive value of rice grains due to the increased levels of iron. Thus, siderophore production is an important trait of PGPB and enhances the iron uptake of plants.

### Phytohormone Production

Microbial inoculants such as PGPB are also known to produce a number of plant hormones or plant-growth regulators that alter the architecture of roots and the growth of plants [108–110]. These plant hormones are gibberellins, auxins, ethylene, cytokinins, and abscisic acid [111]. A number of physiological processes can be regulated by these hormones, including root elongation, formation of root hairs, and root initiation [8]. Indole-3-acetic acid (IAA) has been widely reported as a natural auxin produced by microbial inoculants [112]. Many plant functions are influenced by IAA such as root initiation, differentiation of vascular tissue, expression of many plant genes, and mediation of tropic responses [8]. Cytokinins also play an important role in plants, including delaying leaf senescence and promoting mitotic cell division in roots and shoots [16]. Flower and fruit production, seed germination, and dormancy of vegetative organs are affected by gibberellin hormones [113]. Furthermore, abscisic acid is mainly involved in responses to environmental stresses such as high salinity, and drought along with plant development [114]. In addition, ethylene is well known as a ripening hormone, but there are other roles that have been reported such as cell expansion, flower and leaf senescence, and seed germination. Ethylene is also known as a stress hormone because it is produced under abiotic as well as biotic stress [115]. An inhibitory effect on root growth has also been reported due to the production of high concentrations of ethylene, which ultimately reduces plant growth. To overcome this problem, PGPB are also known to produce a vital enzyme, L-aminocyclopropane—1-carboxylate deaminase (ACC deaminase), which catalyzes the formation of the intermediate precursor of ethylene, ACC (1-amino cyclopropane-1-carboxylic acid), into α ketobutyrate and ammonia and regulates the biosynthesis of ethylene [8]. Moreover, PGPB also produced some low-molecular-weight volatile organic compounds (VOCs) such as ketones, alcohols, hydrocarbons, and aldehydes, which have generally high vapor pressure and enter into the atmosphere [116]. These compounds are collectively termed microbial volatile organic compounds (mVOCs) [117]. Initially, Fernando et al. [118] and Vespermann et al. [119] reported some biocontrol activity of these

VOCs of some rhizospheric microorganisms, but later promotion of plant growth also reported the role of VOCs, for instance, the growth promotion in *Arabidopsis thaliana* by VOCs of PGPB strains containing acetoin and 2,3 butanediol [120]. PGPB also affects the morphology of roots and provides nutrition to plants.

#### 4.6.2. Arbuscular Mycorrhizal Fungi (AMF)

Fungi are also found in soil and are associated with plant roots in the following two ways: through mutualistic symbiosis and parasitism. In mutualistic symbiosis, both organisms live together and establish beneficial relationships, whereas, in parasitism association, one partner benefits and the other is harmed [121]. A beneficial and heterogeneous group of fungi that establishes symbiotic relationships with more than 90% of plant species is known as mycorrhizal fungi [1]. Mycorrhizal fungi can be categorized into different groups, but arbuscular mycorrhizal fungi are a prevalent type of endomycorrhiza and are commonly associated with horticultural and crop plants. Early in the history of land plants [122], arbuscular mycorrhizal fungi (phylum Glomeromycota) appeared first and were associated with diverse plant taxa [123]. A special branched structure formed during the penetration of fungal hyphae of Glomeromycota species in root cortical cells of plants is called arbuscules [121,124]. Today, there is great interest in the use of these mycorrhizal fungi in sustainable agriculture, which have been established to provide enhanced nutrients (macro and micronutrients) and water uptake and also help plants survive biotic and abiotic stress [125–130]. There is a recent report that not only is there interconnection between fungi and plants established by the hyphal network, but also connecting individual species of plants within a community and helping in signaling among interplant species [131,132]. AMF plays an important role in stimulating plant growth through several mechanisms [133]: (i) enhancing the uptake of water; AMF increases the surface area of the root through which plants can easily take up water; (ii) availability of nutrients, especially phosphorus, under nutrient-deficiency condition; (iv) modifications of root architecture; (v) changes in enzymatic and physiological activities, especially for plants that are involved in antioxidative responses; and (vi) induction of ABA plant hormones, which are mainly involved in stress conditions [134]. Auge, Brundertt, and Begum [125,135,136] reported some ameliorating effects of drought due to mycorrhizal symbiosis in some plant species, including wheat, onion, soybean, lettuce, and corn. This occurs due to increased root growth resulting in enhanced tolerance to drought. It also maintains high water efficiency and increased growth when plants are colonized by AMF [8]. Furthermore, the water potential of plants may also be affected by the changes in the structure of soil by the production of a soil-binding material such as glomalin, a glue-like substance that is insoluble in nature by the hyphae of AMF [125]. Protection of plant roots from the toxicity of heavy metals by the use of AMF has also been reported by Leyval et al. [137]. There are also some reports available and reporting that drought tolerance of plants is augmented by the application of co-inoculation with AMF and PGPR. For example, improved plant growth, stomatal conductance, the efficiency of water use, as well as increased photosynthetic rate, being reported in lettuce plants when co-inoculated with AMF *Glomus mosseae* and *G. intraradices* and *Bacillus* spp (PGPR). A better result was obtained in co-inoculation with AMF and *Bacillus* spp. as compared to individual organisms. This occurred due to PGPR, i.e., *Bacilus* spp., enhancing the growth and colonization of AMF [138]. But there are some limitations on the use of AMFs as biostimulants, which may result from their biotrophic character; they have difficulty for propagation on a large scale, and researchers have been unsuccessful in understanding the determinants of host specificities and other population dynamics of mycorrhizal fungi in agricultural ecosystems [139]. Some fungi which are distinct from mycorrhizal species are also reported such as *Trichoderma* spp and *Sebacinales*, which are able to colonize roots and provide nutrients to their hosts, but the mechanisms are not well studied [121]. However, these fungi can be used as bioinoculants to improve the nutritional status of plants. *Trichoderma* spp. is well known for its biocontrol and biopesticidal activities, but Colla et al. [40] and Shoresh et al. [140] also reported some

stimulatory effects on plants such as enhanced efficiency of nutrients, morphogenesis, and organ growth along with increased tolerance to abiotic stress. These fungal endophytes may be considered biostimulants as well as biopesticides based on these effects on plants as reported by researchers [40,140]. Therefore, microbial inoculants including beneficial bacteria as well as fungi are a promising tool in sustainable agriculture. They not only enhance plant nutrition but also assist plants in tolerating a number of environmental stresses. They improve our agriculture system without any deleterious effects. The overall mechanisms of action of different plant biostimulants in the plant are represented below (Figure 1):

**Figure 1.** Schematic representation of the different types of plant biostimulants and their beneficial mechanisms in plants [1,8].

#### **5. Risk Status of Microbial Inoculants (Plant Growth Promoting Bacteria)**

PGPB, which are considered potent candidates for plant growth, should be safe for mammals. Some of the microbial inoculants commonly used as biostimulants, and their risk groups are listed in Table 2. Despite their array of beneficial effects on plants (Table 2), they may pose a risk to other living organisms, especially human beings. Although most PGPB do not have a negative effect, some genera are involved in causing infections in animals and humans. Bacteria belonging to the genera *Serratia, Acinetobacter, Bacillius cereus*, *Stenotrophomona, Enterobacter, Herbaspirillum, Ochrobactrum,* and *Pseudomonas* are not only powerful candidates for plant-growth promotion but may also cause disease in humans [141,142]. *Pseudomonas,* besides being a potential candidate as PGPR, is also responsible for many types of opportunistic infections in humans who are aged, immunocompromised, or suffering from conditions such as cancer, severe burns, or cystic fibrosis. Some common pathogenic species of *Pseudomonas* are *P. cepacia, P. aeruginosa, P. putida, P. fluorescens,* etc [143]. Although *Bacillus* sp. is commonly known for its wide variety of applicability in agriculture, industry, and the pharmaceutical sector, it still is associated with many types of illness in humans and animals. It can cause disease in immunocompromised as well as in healthy individuals. Some species may cause minor infections, but some species may be associated with severe or lethal infections. *B pumilus, B licheniformis, B coagulans,* and *B thuringiensis* are examples of *Bacillus* species that are associated with various infections [144]. *Aeromonas* sp. *is* used as PGPR but also causes diseases in immunocompetent and immunocompromised people such as septicemia, gastroenteritis, and wound infections [145]. Another potent PGPR, *Comamonas* spp., is also associated with many life-threatening illnesses such as endocarditis, and septicemia in immunocompetent individuals [146]. *Streptomyes* sp. can cause changes in tissue structure in humans leading to diseases such as cancers, mycetomas, and actinomycetomas [147]. In spite of such immense positive impact of *Trichoderma* sp. on plant health, it is now emerging as a human pathogen causing diseases such as peritonitis, subcutaneous infections, and hematologic disorders [148]. Although *Enterobacter* sp. has a variety of uses as a plant growth stimulator, is also known to cause nosocomial infections and is involved in an array of ailments such as skin infections, inflammation in the respiratory system, and meningitis in neonates, immunocompromised individuals and hospitalized patients [149]. But there are no national or international rules or regulations to assess the risk associated with the commercial use of these plant-beneficial microbes [150]. Even commercial biofertilizers such as Biosubtilin, Nitrofix, and Bioderma. (Table 2) do not mention risks associated with the respective inoculants in their packets.

Risk groups and biosafety levels (BSL) are two terminologies used to describe and categorize microbes as per the level of hazards they can cause [93]. According to the World Health Organization (WHO 2015), microorganisms that are categorized under various risk groups (RG) are based on certain criteria such as their pathogenicity and virulence, host range, mode of transmission, availability of vaccines for effective prevention, availability of medications, etc. Thus, the classification, e.g., from RG-1 to RG-4 articulate the level of hazard a particular microorganism causes. RG-1 refers to a group of microbes that do not cause or are not associated with any type of illness in healthy animals (including humans). Microbes under RG-2 group are associated with a disease that is generally mild and there are medications readily available to treat the disease. RG-3 microbes are concomitantly associated with a serious and lethal disease that may or may not be treatable. Microbes belonging to RG-4 category have the ability to cause fatal and deadly diseases for which treatment is rarely found. Biosafety level (BSL), e.g., from BSL-1 to BSL-4, is a precautionary procedure and protocol used to avoid or prevent risks associated with these risk groups while handling them. Organisms belonging to BSL-1 are nonpathogenic in nature and can be easily handled in the laboratory through general laboratory guidelines. Microorganisms under BSL-2 have the ability to cause disease in a healthy individual, but there are ample medications and vaccines available to easily cure such diseases. Proper laboratory guidelines and special training are required to handle BSL 2 organisms. Specialized safety measures and containment facilities are required to handle microbes that come under the BSL-3 group because such microbes can cause fatal infections but do have effective remedies and anticipatory treatments available. BSL-4 encompasses high-risk-associated organisms, which have aerosol-transmission ability and for which effective treatment is not available. Laboratory personnel handling such organisms must have special training and should know the primary and secondary containment of BSL-4 organisms. The literature suggests that many bacteria isolated from the rhizosphere, soil, and water, besides having PGP activity, are also involved in causing diseases in immunocompromised and healthy individuals [151–153]. Hence there is an immense need to develop a systemic and polyphasic approach through which we can check the disease-causing ability of microbes isolated from an environmental niche in addition to checking their PGP activity and bioinoculant development [154]. A study by Vílchez et al. stabilized a polyphasic protocol called EHSI (environmental and human safety index) to check the biosafety level of plant-growthpromoting bacteria. EHSI articulates the overall effect of PGPB on soil microflora, beneficial macroflora and fauna, and animal and human health. In this study, according to EHSI, both being potent PGPB, *Pseudomonas putida* KT2440, is relatively safe as compared to *Burkholderia cepacia* CC-Al74. In another study by Kim et al. [155], it was suggested to assess or check for the presence of genes involved in the virulence or pathogenicity of novel bacteria isolates to determine their safety level concerning humans and plants. Keswani et al. [150] suggested that whole-genome sequencing of a bacterial isolate is the best way to obtain a complete understanding of its phylogenetic categorization and pathogenic behavior. Hence, research organizations and institutions which are involved in isolating novel microbial isolates and bioinoculant development, after thorough polyphasic characterization, should use isolates that belong to BSL-1 and Risk group-1 for bioformulation because they will pose minimum risk to the environment and human health [150].

**Table 2.** Microbial inoculants in agriculture and horticulture systems and their indicative risk status in the risk group databases.



Risk group \* (classification of prokaryotes into risk groups under Biological Agents Ordinance: RG 1 refers to prokaryotes that generally do not cause infectious disease in humans; RG 2 refers to those microbial groups which do not pose a significant risk to laboratory workers but may cause disease if there is exposure and for which there are therapeutic interventions available), RG—Risk group, BSL—Biosafety level as per ATCC; #—The exact name of the organisms in the original concerned database of risk group; (-) indicates that it has not been commercially formulated.

In addition to posing health risks to animals, unprecedented use of PGPR also affects other biotic communities of an ecosystem, especially soil resident flora. As it is already known that newly introduced microorganisms change the microenvironment of soil, creating their niche which can have an immense effect on the structure and composition of resident microbes [217]. The interaction of PGPR with soil flora may be negative, positive,

or neutral depending upon the nature of the PGPR introduced into the soil [218]. The main concern is the introduction of antimicrobial-producing PGPR in the soil milieu [219]. A study by Walsh et al. [220] revealed that there was a reduction in the diversity of the rhizobacterial population due to the introduction of 2,4-diacetylphoroglucino (an antibiotic substance) producing bacteria in the rhizosphere. Some type of perturbance in the resident-flora population is also possible as found in the study by Albright et al. [221].

#### **6. Safety Determination of Microbial Inoculants**

Several microbes belonging to *Pseudomonas, Bacillus, Acinetobacter, Burkholderia, Staphylococcus, and Stenotrophomonas* have been used as inoculants for plant-growth promotion and biocontrol of plant pathogens; however, these also include microbes identified as opportunistic pathogens and that cause human pathogenesis [150]. It has also been reported that the invasion and colonization mechanisms involved in the pathogenesis of PGPR on plant and human tissues are similar [151,222,223]. Therefore, the safe application of PGPR to protect human health and the environment is needed, which involves collaborative efforts of different expertise, and technological advancements. Microbial inoculants need to be identified and well characterized to unveil their hidden risks to humans and the environment. Several physiological and molecular approaches are now used to check the virulence and pathogenicity of infectious microbes. These methods can also be employed to detect the pathogenicity level of PGPB. The following are some important detection methods that can be taken into consideration.

#### *6.1. Morphological and Biochemical Methods*

To detect the pathogenicity level of bacteria, it is necessary to identify the species of bacteria which can be done through cultural studies and fast biochemical tests. For example, growth on blood agar will indicate that the bacteria are pathogenic in nature. The use of differential and selective media will enhance the probability of isolating microbes that have a pathogenic nature. Various biochemical tests such as tests for enzyme detection of catalase, urease, deaminase, decarboxylase, deaminase, β galactosidase, hydrolase, etc., are helpful in the polyphasic characterization of bacteria. These enzymes can also be detected using chromogenic media that contains specific chromogenic substrates which are hydrolyzed and produce a particular color in the media indicating the presence of enzymatic activity in bacteria. Nowadays various biochemical kits and their detecting instruments are available commercially, which enables the rapid detection of microbes [224–226].

### *6.2. Antibiotic Sensitivity Method*

Sensitivity to various antibiotics will indicate whether the given bacterial isolate is safe for release into the environment or not because multiple drug-resistant PGPB bacteria that somehow cause disease in humans and animals will be difficult to treat or cure such disease through prevalent antibiotics. In addition, antibiotic resistance is generally plasmidborne and most of the plasmid can be transferred from one bacterium to another, thereby spreading the antibiotic-resistant character in the soil microbiome [227].

#### *6.3. Protein Profiling Method*

Every genus has a particular set of proteins, and protein profiling will help in identifying the bacterial genus. Even various species in one genus can be differentiated through protein profiling as they have a particular set of proteins, i.e., they contain enzymes involved in a unique biochemical pathway. In addition, it may be possible that these unique biochemical pathways enable a particular microbe to thrive in a harsh climate making them a more favorable candidate for bioinoculant production [227].

#### *6.4. Molecular Level Detection Techniques*

Studying at the genetic level is the most precise, rapid, and sensitive technique in today's era to help in the proper understanding and identification of microbial species. Detection of ubiquitous and universal sequences (containing conserved and variable regions) such as 16s rDNA/18s rDNA is the most prominent and simple way to identify microbes at the species level. Techniques based on the hybridization process are used to detect genes of interest through the use of probes tagged with fluorescent dyes. For example, the fluorescence in situ hybridization (FISH) method uses universal probes to detect a particular microbe [228,229].

Amplification of genes conferring the virulence property of a particular microbe is also an effective way to check the pathogenic nature of bacteria. Quantitative polymerase chain reaction (qPCR) and reverse transcriptase real-time PCR (RT-qPCR) are employed as amplification techniques. One such example is an *invA* gene, which is a virulence gene found in *Salmonella* sp. that is detected through PCR using compatible primers [230].

Gene chip technology or DNA microarray is yet another efficient technique that can not only identify and differentiate among various species of microbes through a variety of probes and universal or consensus primers, but can also give information regarding different resistant measures adopted by a specific microbe [231]. With the advent of the Sanger method of sequencing, a first-generation sequencing technique, it is now possible to sequence the whole genome of a particular microorganism in a very rapid and efficient way. The sequence of the whole genome will not only identify the bacterium but also disclose its pathogenic nature and resistant profile. Whole-genome sequencing also helps in the rapid designing of primers [232,233]. Nowadays, NGS (next-generation sequencing) has proven to be a powerful method for the detection of virulence factors of infectious microorganisms within a few hours. In clinical microbiology, there are numerous methods available for the detection of human pathogens, which are compiled in Table 3. These technologies, in combination with the routine characterization and evaluation of potential microbial biostimulants, can be used to guide as per Figure 2 for the safe development and enrichment of microbial stimulants for use in agriculture.


**Table 3.** Comparative table of different technological approaches for the detection of pathogenic organisms.

**Figure 2.** A graphical abstract of isolation, characterization, and identification of microbial inoculants and their commercialization as per their respective categorization into different risk groups (compiled from) [150,227,237,238].

#### **7. Legal Framework of Biofertilizer Implementation in Different Countries**

In spite of having so many advantages over chemical pesticides, the biofertilizer industry faces too many legal obstacles to overcome before entering into commercial production. Earlier the legal regulations regarding biofertilizer use were very inadequate and weak. But in today's era, as researchers have shown the great potential of biofertilizers, many countries amended and developed strong policies and legal regulations to increase the usage of biofertilizers [240,241].

Having an appropriate legal definition is a crucial part of making biofertilizers an appealing commercial product to the producers. In the USA and European Union (EU), there is no proper definition of biofertilizers that can define their actual characteristics. In the EU, biofertilizer comes under e EU Commission Regulation n. 889/2008 on organic production, which states that biofertilizers can only be used as plant protectants against pests and diseases. Hence, biofertilizer comes under the legal agenda of plant protection products. The same outline is followed by the US National Organic Program which categorizes biofertilizers as biological organisms that can only be used as plant protectants [242].

Compared to other countries, India has the most comprehensive and defined legal regulation and framework for biofertilizer implementation. In India, biofertilizer comes under the Essential Commodities Act of 1955, Ministry of Agriculture, and can be defined as "the product containing carrier based (solid or liquid) living microorganisms which are agriculturally useful in terms of nitrogen fixation, phosphorus solubilization or nutrient mobilization, to increase the productivity of the soil and/or crop". Seven standard criteria have been set to formulate a biofertilizer that includes viable inoculum density, the physical form, level of contamination, pH, moisture content, the particle size of carrierbased products, and efficacy level. Four groups of microbes are mainly included under the biofertilizer category i.e., *Azotobacter, Rhizobium, Azospirillum*, mycorrizal fungi, and phosphate-solubilizing bacteria [242,243].

In Poland, Polish Law on Fertilizers and Fertilization 2007 includes biofertilizers under "growth stimulators" and groups them under plant conditioners. This law defines biofertilizer as "a positive impact on plant growth or other metabolic processes of plants in other ways than plant nutrients" and shall "pose no threat to [the] health of humans or animals or to the environment after their use and storage instructions" [242].

Spain, which is one of the leading countries in organic farming, does not have a separate category and definition of biofertilizer in its legal structure. It includes microorganisms as one of the components of compost and organic amendments under Real Decreto 506/2013 [242].

China has a strict and defined legal framework for biofertilizer implementation. It has set various parameters through which it can access the quality of biofertilizer including inoculum density, water, and carbon content, outer appearance, granule size, contamination, viability, and validity. Chinese standards mostly rely on the amount of inoculum to access the quality of biofertilizer, which should range between >1.5 × 109 CFU mL−<sup>1</sup> or >0.2 × 109 CFU g−<sup>1</sup> and >0.5 × 109 CFU mL−<sup>1</sup> or >0.1 × 109 CFU g<sup>−</sup>1, for solid and liquid products, respectively. Seven categories of microorganisms are included in biofertilizers, i.e., fast- and slow-growing species of rhizobia, organic and inorganic phosphate-solubilizing bacteria, nitrogen-fixing bacteria, silicon-solubilizing bacteria, and various consortia containing multiple microorganisms [244].

### **8. Conclusions**

Plant biostimulants prove beneficial to plants by improving their growth. Microbial inoculants, single or consortia, naturally improve plant growth and performance without using any agrochemicals in the field. They can act as biofertilizers, soil improvers, growth regulators, stress relievers, and biocontrol agents. However, more research needs to explore and establish their biocontrol properties. Much research has been conducted to understand their properties and functions followed by their commercialization to promote eco-friendly and safe agriculture practices for the fortification of plants with nutrients. The global markets of biostimulants also need to be expanded in the near future so that farmers can easily buy these products at affordable prices. Furthermore, extensive characterization research emphasizing the safety issues of the inoculant microbes becomes inevitable to address recent reports of many inoculants belonging to either higher-risk groups or potential pathogens of human beings, such as *Pueudomonas*, *Klebsiella*, *Enterobacter*, *Acinetobacter,* etc., which may cause various kind of suffering, for example, septicemia, gastroenteritis, wound infections, inflammation in the respiratory system, meningitis, etc., of varied severity under different conditions of human-health status, such as immunocompromized and comorbidity with other diseases, etc. Advances in technologies including biochemical, immunological, proteomics, and genomics approach unraveling the characters and identification of microbes have enabled the research community to rapidly and accurately address safety concerns, such as pathogenicity, of biostimulant microbes following a suitable strategic plan before releasing the inoculant for field application.

**Author Contributions:** Conceptualization, M.K., P.S. and A.K.; resources, A.K.; writing—original draft preparation, M.K., P.S. and A.K.; writing—review and editing, K.K.K. and A.K.; visualization, K.K.K. and A.K.; supervision, K.K.K. and A.K.; project administration, A.K.; funding acquisition, K.K.K. and A.K. All authors have read and agreed to the published version of the manuscript.

**Funding:** This research received no external funding.

**Institutional Review Board Statement:** Not applicable.

**Informed Consent Statement:** Not applicable.

**Data Availability Statement:** Not applicable.

**Conflicts of Interest:** The authors declare no conflict of interest.
