**1. Introduction**

Plant biostimulants, as the name implies, are substances or microorganisms, which stimulate plant growth. Horticulturists coined the word biostimulant as a substance promoting plant growth that does not belong to the group of nutrients, soil improvers, or pesticides. It includes a diverse collection of compounds, substances, and microorganisms that are applied to plants to improve the crop yield, quality, and tolerance to biotic and abiotic stress [1]. According to the European Biostimulants Industry Council (EBIC) [2] "Plant biostimulants contain substance(s) and/or microorganisms whose function, when applied

**Citation:** Kumari, M.; Swarupa, P.; Kesari, K.K.; Kumar, A. Microbial Inoculants as Plant Biostimulants: A Review on Risk Status. *Life* **2023**, *13*, 12. https://doi.org/10.3390/ life13010012

Academic Editors: Hakim Manghwar and Wajid Zaman

Received: 1 November 2022 Revised: 12 December 2022 Accepted: 13 December 2022 Published: 21 December 2022

**Copyright:** © 2022 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/).

to plants or the rhizosphere, is to stimulate natural processes to enhance/benefit nutrient uptake, nutrient efficiency, tolerance to abiotic stress, and crop quality. Biostimulants have no direct action against pests, and therefore do not fall within the regulatory framework of pesticides" [2]. Other terminologies can also be used to define biostimulants such as biogenic stimulators [3], organic biostimulants [4], plant strengtheners [5], phytostimulators [6], and agricultural biostimulants [7]. It has been reported that the global market for biostimulants reached \$2241 million by 2018 and Europe was the largest market for biostimulants in 2012 [8]. Biostimulants were later elaborated by EBIC as "Biostimulants foster plant growth and development throughout the crop life cycle from seed germination to plant maturity in several demonstrated ways, including but not limited to: improving the efficiency of the plant's metabolism to induce yield increases and enhanced crop quality; increasing plant tolerance to and recovery from abiotic stresses; facilitating nutrient assimilation, translocation, and use; enhancing quality attributes of produce, including sugar content, color, fruit seeding, etc.; rendering water use more efficiently; enhancing certain physicochemical properties of the soil and nurturing the development of complementary soil micro-organisms" [9]. Thus, biostimulants are organic-based plant growth promoters and regulators. They provide nutrients to plants and enhance crop productivity in an eco-friendly manner. The word 'biostimulant' was first defined by Kauffman et al. [10] as materials, other than fertilizers that promote plant growth when applied in low quantities. They are mostly organic products and comprise amino acids, plant hormones, polysaccharides, and humic substances and are easily available for uptake by plants. They may not only deliver nutrients to the plant but also stimulates plant metabolism and alleviate biotic and abiotic stresses [11]. Parrado et al. [12] reported various mechanisms of biostimulant action through which they enhance crop yields, such as stimulation of soil microbial activity, intensification of vital soil-enzyme activities, or phytohormone production. In November 2012, the first world congress was held on the topic of the use of biostimulants in agriculture in Strasburg, France, which was participated in by 30 countries. The main intention of this congress was to bring together people working on the features of biostimulants in academia, industry, and regulatory agencies. Therefore, the uses of these biological substances became commercialized [8].

In this review, we are mainly emphasizing plant biostimulants and their role in agriculture. Besides these, we also discuss microbial inoculants as plant biostimulants, associated risk, and their biosafety regulations when applied in the agricultural field to promote plant growth.

#### **2. History of Classification of Biostimulants**

Classification of biostimulants is widely documented by many scientists, stakeholders, and regulators [8,13,14]. In 2012, du Jardin classified biostimulants into eight classes which were based on bibliographic analysis of plant biostimulants and microbial inoculants, and they were not included in these categories [13]. Calvo et al. [8] reviewed five different categories of biostimulants based on a critical review of selected scientific publications related to biostimulants. Furthermore, based on practical and theoretical knowledge of agricultural and horticultural biostimulant products used, du Jardin categorized them into seven categories containing substances as well as microbes [1]. According to the Agriculture and Horticulture Development Board (2017), they were further classified into two main groups, non-microbial and microbial [15]. Recently, Pascale et al. [16] classified plant biostimulants based on enhancing plant nutrition into five categories including microorganisms. The chronological order of classification of plant biostimulants and the basis of their categorization is summarized in Table 1.


**Table 1.** Chronological order of classification of biostimulants.

HS—Humic substances, PHs—Protein hydrolysates, SWE—Seaweed extract, PGPR—Plant Growth Promoting Rhizobacteria, AMF—Arbuscular Mycorrhizae Fungi.

#### **3. Properties of Plant Biostimulants**

A new category of agricultural chemicals derived from biological sources and applied as foliar applications or as seed treatments to improve the productivity of crops and overall growth and yield is known as plant biostimulants [17]. They mostly act as biofertilizers in agriculture and horticulture. There are various properties reported by many scientists of plant biostimulants which are mentioned below:


#### **4. Plant Biostimulants and Their Mechanism**

#### *4.1. Humic Substances (HS)*

Humic substances (HS) are diverse organic molecules that are formed during microbial and chemical degradation of organic matter in soils and are found most abundantly in nature [21–23]. They also contain a total of 60% organic molecules in the world's soils [23,24]. HS was earlier called "gelbstoff", a yellow-colored organic compound generally found

in marine water, freshwater, and soil, and made of linked polymers of amino acids, fatty acids, and carbohydrates which are resistant to further degradation by microbes [24,25]. But today, it is believed that HS is composed of small organic molecules linked together by hydrogen bonds and hydrophobic interactions [23,26,27].

Aiken et al. [28] defined HS as "a general category of naturally occurring, biogenic, heterogeneous organic substances that can generally be characterized as being yellow to black in color, of high molecular weight (MW), and refractory". Humic substances obtained from freshwater and terrestrial ecosystems contain lignin, but it is absent in marine ecosystems [25]. Many scientists have reported that humic substances play an imperative role in the function of soil and plants [29]; for instance, by exchange of carbon and oxygen between the soil and atmosphere, and regulating the availability of nutrients [8], they enhance the physical and chemical properties of soil [16] and transform toxic materials and transport them [29,30]. In addition to this, humic substances also affect the chemical and functional properties of rhizospheric microorganisms [31]. One important feature of humic substances is the formation of complexes, although the solubility is a function of pH and the molar ratio of the complex, with micronutrients (iron) avoiding leaching, and in turn, making them available in the soil for plant nutrition [32,33].

Based on their solubility and molecular weight, humic substances can be divided into three groups: humic acids; humins; and fulvic acids. Humic acid is a relatively highmolecular-weight compound and is soluble in alkaline media. It can be easily extracted from soil by treating with dilute alkali and gets precipitated in acidic media, whereas fulvic acid is a low-molecular-weight compound. It is soluble in acidic and alkaline media and cannot be easily extracted [29,34]. Humin is a humic-containing substance instead of a humic substance because it is made up of humic and non-humic materials [35,36]. HS is known to increase the fertility of soil and also alleviate heavy-metal stress. Heavy metals can bind with the carboxyl and phenolic groups (binding site for heavy metal) of the humic substance, resulting in heavy-metal-deficient areas and plants which are unable to take up these metals [37]. Lead toxicity can be minimized with the application of humic and fulvic acid, which reduces the transfer and contamination of Pb2+ in the food chain [38,39]. Thus, humic substances have multiple roles and can be applied to stimulate the physical, chemical, and biological activity of soil and plants.

#### *4.2. Protein Hydrolysates and Amino Acids*

Stimulation of plant growth and tolerance to abiotic and biotic stress can also be reported using a variety of protein-based products which are different from nitrogen sources. These protein-based products can be categorized into two main groups—protein hydrolysates and amino acids. A combination of peptides and amino acids of either plant or animal origin that are manufactured from partial hydrolysis of a protein source are known as protein hydrolysates [8,16]. Some specific amino acids also function as plant stimulators such as glutamine, glycine betaine, proline, and glutamate. Protein hydrolysates are commercially available in different formulations in the form of powder, granules, and liquid and they may be applied to plants near the root system or as foliar sprays [40]. Various processes are involved in the preparation of protein hydrolysates, e.g., chemical, enzymatic, or thermal hydrolysis of plant residues (carob-germ protein, alfalfa residues, algal proteins, and wheat-condensed distiller solubles) and animal residues (connective or epithelial tissues, collagen, and elastin of animals) [8]. Protein hydrolysates are commercially available in the market of various countries with diverse names such as Aminoplant or Siapton (Italy), Macro Sorb foliar (Spain), and ILSATOP (Italy). The concentration of free amino acids and protein/peptides generally present is 2–18% and 1–85%, respectively, in protein hydrolysates preparation. Free amino acids such as arginine, alanine, valine, leucine, glutamate, proline, and alanine are the major components usually present in protein hydrolysates. In addition to protein/peptide and free amino acids of hydrolysates, some non-protein components also influence plant-growth stimulation. For instance, carob-germ extract, a plant-based product comprising carbohydrates, fats, macronutrients, and micronutrients and also containing phytohormones [8]. Another group of protein-based products is individual amino acids which include non-protein amino acids that are found extensively in a few plant species, such as glycine betaine, glutamate, histidine, and proline, render anti-stress properties [8,41]. There are many stimulatory effects of protein hydrolysates on plants such as improvement of soil respiration, increase in biomass, and activity of microorganisms because plants and microorganisms can easily utilize these amino acids and peptides as a source of nitrogen and carbon [42]. They also provide macronutrients (Ca, Mg and K) and micronutrients (Fe, Mn, Zn, and Cu) to the plants because they can chelate these metal nutrients present in soil and make them available to plant roots [1,40]. Some industries use this strategy for making biofertilizers having high nutrient efficacy. Moreover, protein hydrolysates are also known to induce the defense mechanism of plants and also increase tolerance to a range of abiotic stresses such as drought, salinity, oxidative conditions, and temperature [43–47]. Several higher plants that include soybean, alfalfa, rice, barley, and maize can tolerate a wide variety of abiotic stress due to the exogenous supply of these compounds [44,48,49]. Corte et al. [50], in their study, found that there was the absence of any kind of genotoxic effects shown by animal-based protein hydrolysates on soil microflora and fauna, yeast, and plant bioassay systems [49,50].

#### *4.3. Seaweed Extracts and Botanicals*

Seaweeds are also known as large marine algae including multicellular, macroscopic, and benthic organisms that inhabit the world's oceans and provide shelter and food for oceanic animals and also offer a valuable product as single cell protein for mankind [51]. In Asian countries, fresh seaweed is mainly used for food along with traditional remedies [52]. It contains a variety of constituents, i.e., polysaccharides, proteins, polyunsaturated fatty acids (PUFA), polyphenols, pigments, and plant growth hormones [53]. In coastal regions, seaweed has been used as a fertilizer to enhance the growth of plants [54].

These liquid extracts are commercially available for horticulture and agriculture [54,55]. The extracts act by adding chelators and phytohormones and by improving soil structure and aeration [56]. Seaweed extracts are commercially made from brown algae such as *Ralfsia*, *Ascophyllum nodosum*, *Padina*, *Turbinaria*, *Sargassum*, *Laminaria*, *Fucus* spp., and others [57–60]. The seaweed extract is formulated in liquid or dried form and can be blended with micronutrients and fertilizers for field application [8]. The biostimulation effects of seaweed extracts include increasing plant growth, fruit and flower production, and crop yield, helping to develop resistance against abiotic and biotic stresses, enhancing shelf life after harvest, and increasing chlorophyll levels [8,60].

Botanicals are substances that are extracted from plants and are used in cosmetic products and pharmaceuticals, food ingredients, and plant protection products [61]. The biostimulatory effects of botanicals, except seaweed extracts, are not well understood and need to be explored. In ecosystems, plant-active compounds known as allelochemicals, which are known to mediate plant interactions, receive more attention regarding sustainable crop management [1]. Recent studies exploring biostimulatory effects of higher plant botanicals on white hat cabbage and radish have led to valuable additions for the vegetable plant under study [62,63]. Further study is required to establish the biostimulatory effects of these botanicals.

#### *4.4. Chitin and Chitosan Derivatives*

After cellulose, chitin is the second most copious biodegradable polysaccharide in nature and is composed of N- acetyl-d-glucosamine groups linked by β (1–4) glycosidic bonds through the activity of chitin synthases to form a linear chain [64]. It is mostly obtained from the exoskeleton of shrimp, insects, and crabs along with the cell walls of fungi and algae [65]. Chitin is mostly insoluble in water, ia a high-molecular-weight biopolymer, and has a porous structure favoring high water absorption. Chitosan is a derivative of chitin and is produced after the deacetylation of chitin which influences its chemical and biological

properties and is also responsible for antimicrobial activity due to the protonation of its amino groups in solution [66]. Another derivative of chitin is oligochitosan (chitooligosaccharides), formed during the chemical and enzymatic hydrolysis of chitin and composed of mainly 3–10 saccharide residues of N- acetylglucosamine or glucosamine [67]. Biostimulatory effects of chitin and their derivatives have been reported by many researchers, and include the protection of plants from pests and diseases, enhancing the antagonistic action of microorganisms, improving the beneficial plant-microbe interactions, and regulating plant growth and development [65,68]. Shahrajabian et al. [69] reported various beneficial effects of chitin and its derivatives on vegetable crops such as increased photosynthetic activity, tolerance to abiotic stressors (salinity, drought, temperature), expression of defensive genes, increased antioxidant-enzyme activity, activation of plant innate immunity, induction of secondary metabolite synthesis, etc.

#### *4.5. Antitranspirants*

Chemical compounds which favor a reduction in the rate of transpiration from plant leaves are known as antitranspirants and alleviate drought stress by reducing the size and number of stomata [70]. Many chemicals reported as antitranspirants, such as chitosan, kaolin, calcium carbonate, salicylic acid, etc., are eco-friendly and increase the water-holding capacity of soil as well as reduce the rate of transpiration. Thus, the use of antitranspirants in plants increase crop yield in water- and high-temperature-stress conditions [71].
