Biostimulants are natural or synthetic products derived from plants and/or microbes, and have the potential to promote the physiological processes of plants benefitting nutrient uptake and translocation, as well as enhanced tolerance to several abiotic stresses. These products can be directly applied to seeds, seedlings and plants or can be introduced into the rhizosphere of crops with the goal to improve plant productivity and growth in a sustainable way under environmental stresses. Plants continuously face stressful events such as drought, salinity, extreme temperature, high rainfall, etc., at every stage of their growth and development starting from seed germination to completion of the entire life cycle. The abiotic stress factors may alter the plant's physiological processes and/or thereby increase their susceptibility to pests and pathogens and serve as a limiting factor for plant's survival, productivity and growth (West et al., 2012; Maharshi et al., 2021). A diverse agro-chemical and traditional approaches are being adopted to mitigate the adverse environmental effects but one of the most promising and eco-friendly ways recognized to achieve this goal is to use biostimulants to address such concerns (Yakhin et al., 2017). It is because of the fact that in most cases it was observed that application of biostimulants can diminish the demand of synthetic fertilizers and pesticides as the biostimulants have the potentiality to enhance the crop's nutrient use efficiency (NUE) for both micro- and macro-nutrients (Calvo et al., 2014; Van Oosten et al., 2017). However, the plant biostimulants (PBs) are not a direct substitute of fertilizers and pesticides. But their application can very well reduce the need for fertilizers and pesticides and thereby reduce their potential impact on environmental hazards in a crop production system. In earlier articles, biostimulants are discussed mostly in the context of plant-based products that have potentiality to stimulate plant growth under various stresses. However, in majority of the articles the microbe-based biostimulants were not discussed or compared with the plant-based biostimulants. Therefore, the current article presents a holistic insight into the various plant- and microbial-biostimulants, their uses under different environmental conditions and a mechanistic view of the biostimulants.

Many definitions of phyto- and bio-stimulants are available in scientific literatures. According to Zhang and Schmidt (2000) biostimulants are the materials used in trace amounts to promote plant growth. The authors used the words “tracer amount” for biostimulants to distinguish the biostimulants from other nutrients, which also promote plant growth when applied in larger amounts. Later on Kauffman et al. (2007) defined biostimulants with little modifications and according to them “biostimulants” are materials, other than fertilizers, that promote plant growth when applied in low quantities. However, biostimulants are still a “moving target” in the European Union (EU). From the industrial perspective, the definition of biostimulants as given by the European Biostimulants Industry Council (EBIC) in 2012 is “plant biostimulants contain substance and/or micro-organisms whose function when applied to plants or the rhizosphere is to stimulate the natural processes to enhance/benefit nutrient uptake, nutrient efficiency, tolerance to abiotic stresses, and crop quality, with no direct action against the pests” (www.biostimulants.eu). The concept of PBs has continuously been investigated since 1933 but has received considerable focus only in the last two and half decades as a potential mixture to alleviate the negative effects of climate change (Craigie, 2011; Sharma et al., 2014; Ricci et al., 2019; Jindo et al., 2020). Based on the previous considerations the Environmental Protection Agency (EPA) in 2019 defined biostimulants as “naturally-occurring substance or microbe that is used either by itself or in combination with other naturally-occurring substances or microbes for the purpose of stimulating natural processes in plants or in the soil in order to improve nutrient and/ or water use efficiency by plants, help plants tolerate abiotic stress, or improve the physical, chemical, and/ or biological characteristics of the soil as a medium for plant growth.” Since biostimulants are derived from diversity of sources like plants, humic substances, protein hydrolysate, seaweed extracts, macro- and micro-alga, living microbial cultures and plant growth-promoting microbes (PGPM), etc., these compounds are therefore poorly characterized and their mode of actions are also undefined at cellular and molecular levels. Thus, due to the current knowledge gaps the concepts of PBs are still evolving (Calvo et al., 2014; Li et al., 2021), which is somewhat indicative of the diversity of inputs that can be accepted as PBs.

Flurry of researches were conducted in order to evaluate the PBs for improving growth and development of seedlings and subsequently the plants after subjecting to several abiotic stresses like drought, saline environment, extreme temperature, heavy rainfall, etc. (Carillo et al., 2019). Additionally, different kinds of raw materials were also used along with non-microbial biostimulant components such as humic-substances, protein hydrolysate, plant-based products, seaweed extracts, etc. and/or microbial components comprising of PGPMs such as arbuscular mycorrhizal fungi, fluorescent Pseudomonas, Trichoderma spp., Bacillus spp. etc. They all have shown tremendous potentiality to enhance plant growth, flowering, crop productivity, nutrient use efficiency (NUE) and translocation, as well as enhancing tolerance to a wide range of abiotic stresses by modifying physiological, biological and biochemical processes of the crop-plants.

Abiotic stresses like drought, salinity, extreme temperature, high rainfall, etc. are becoming most important threats to food security resulting from constant changes in climate. To cope with these stresses, plants respond by changing several molecular and physiological processes or initiating a number of defense responses to adapt such stresses. It has been highlighted in several studies that application of biostimulants even at very low quantities can promote plant's performance by re-programming the natural processes of plants under such harmful environments (Huang et al., 2013; de Vasconcelos and Chaves, 2019). Some major categories of biostimulants that are currently recognized by scientists are asserted to stimulate the plant defense responses and provide stress tolerance capabilities to them by both non-microbial and microbial means (Halpern et al., 2015; Gupta et al., 2021a). A graphical representation is presented in Figure 1 for these biostimulants according to their classes.

Abiotic stresses, including drought, high rainfall, salinity, extreme cold or heat, etc. are responsible for about 50–70% of yield loss by affecting the plant productivity (Carillo et al., 2019). To overcome the harmful effects from the abiotic stresses, PBs have been used as an efficient agricultural tool for stress tolerance. However, the activities of PBs depend on several factors, such as application techniques, targeted crop as well as timing of inoculation (Bulgari et al., 2019). These abiotic stresses cause several physiological and biological disorders including membrane disfunctioning, reduction of photosynthesis and protein synthesis rate, hormonal imbalance, etc. Most detrimental stress of salinity is ionic imbalances and hyperosmotic condition, which caused by excess production of reactive oxygen species (ROS) and the anti-oxidant defense mechanisms (Di Mola et al., 2020). These ROS like oxygen molecule (O₂), superoxide (O2-), hydrogen peroxide (H₂O₂), hydroxyl radical (HO⁻) are strong oxidizing and reducible ROS, that generated in plant cells to maintain the homeostasis and stability of the stem cells (Das and Roychoudhury, 2014; Zeng et al., 2017). However, an excess level of ROS is reason for oxidative damage of the cells that cause cell death of the crop plants (Al-Ghamdi and Elansary, 2018). And the drought stress condition may lead to low water potentiality due to high solute concentration, which makes the plant cells to become turgid resulting in wilting and desiccation in plants. Scarcity of water may also cause soil mineral toxicities and can make plants more susceptible to damage from high temperature and high irradiance. Similarly, high temperature affects metabolism, respiration, and other physiological processes of the crops causing retardation in functioning and growth of the plants. This type of heat injury may lead to death of whole plants. Cold temperature stress is also one of the most limiting factors for the crops. Like other abiotic stresses, it can also cause several metabolic and physiological dysfunctioning of the crop such as degradation of chlorophyll, plasmolysis, amylolysis (conversion of starch to sugar), photosynthetic reduction, etc. Waterlogging is another environmental stress that leads to saturation of the soil with water. The major cause of waterlogging is heavy rainfall. Excess of water replace the air from soil pores, causing deficiency of oxygen and accumulation of carbon dioxide that hampers the growth of roots and plants. Therefore, these environmental stresses affect the plants in several ways and ultimately may cause threat to food security throughout the globe (Rouphael et al., 2018a).

To cope up with these problems plants activate several defense mechanisms against different abiotic stresses and PBs can play a significant role in triggering plant's metabolic activities and stimulating performance under such adverse conditions (Rouphael et al., 2018b). There are various microbial (PGPR, AMF and other beneficial microbes) and nonmicrobial (seaweed extracts, protein hydrolysates and humic substances) biostimulants reported that take part in activation and regulation of several types of defense mechanisms by various modes of actions to promote plant growth. They also play a major role s in abiotic stresses by altering the plant's responses via different physiological and biological mechanisms including ROS scavenging activity, osmoprotection, membrane stability, stomatal guiding and xylem conductance, metal chelation, nutrient and water availability and phytohormonal signaling in plants (Van Oosten et al., 2017). The functional and mechanistic roles of different types of biostimulants in crop performance under stress conditions are given in Table 1. Different techniques for inoculating biostimulants are available. Some of those include seed treatment, seedling dip method, and foliar spray or soil application via soil drenching, incorporation of soil marine products and addition of extracts to hydroponics. Mechanisms of actions of these biostimulants vary according to the nature and characteristics of the biostimulants. It has been investigated that the protein-based biostimulants directly penetrate plant tissues of the applied area and enter into the cells, while penetration of the protein hydrolysates in a plant cell takes place through the diffusion process through membrane openings/pores. It is an energy-dependent process (Kolomazník et al., 2012). Other biostimulants like microbial inoculants interact with the plants by penetration of the hyphae into the plant tissues and making the symbiotic or mycorrhizal associations. When these biostimulants enter into the treated leaves and root their translocation starts toward the most distant part of the plants (Pecha et al., 2012). However, for understanding the mode of action, it is necessary to first identify the biologically active compounds (Henda and Bordenave-Juchereau, 2013). Inside the host plants these biostimulants helps in activation of stress-responsive genes by the signal transduction and transcriptional regulations. Schematic representations of physiological effects by different biostimulants under adverse conditions are shown diagrammatically in Figure 2.

Among the seaweed extracts used as biostimulants, Ascophyllum nodosum extract (ANE) has been used extensively to impart tolerance to plants from number of biotic as well as abiotic stresses. It has been previously studied that foliar spray of ANE regulates the genes expression of photosynthesis and ROS scavenging activities under stressed conditions. The mechanism of ROS scavenging involves both enzymatic antioxidants including Superoxide dismutase (SOD), ascorbate peroxidise (APX), glutathione peroxidase (GPX), and catalase (CAT) as well as nonenzymatic antioxidants such as ascorbates, glutathione, proline, glycine betain, tochopherol (Miller et al., 2010; Elansary et al., 2017; Sharma et al., 2019). Further, the accumulation of MDA (malondialdehyde) content in plant cells/tissues is considered as an important indicator for drought-induced peroxidative damage (Shukla et al., 2011). It was also reported by Carvalho et al. (2018), that ANE applications in bean (Phaseolus vulgaris) reduces the ROS-induced MDA production by improving CAT activity and thereby making the plants more tolerant to drought. Similarly, soil drenching with ANE in S. nipponica and P. eugenioides growing soils mitigated drought effects by overproduction of antioxidants and higher lipid peroxidation, thereby reducing the ROS content and particular stresses (Elansary et al., 2016) (Figure 3).

The bioactive constituents isolated from A. nodosum extracts were found to regulate various steps of post-transcriptional and post-translational regulation of different TFs family proteins, such as AP2/ERF, NF-YA, WRKY, COR15A, MYB, LHY1 and some others which provide stress tolerance to crops (Goni et al., 2018; Jithesh et al., 2019). Shukla et al. (2018) reported that binding of AtGRF7 to the promoter element of AtDREB2a down-regulates its expression, which leads to salinity tolerance. It was reported in Arabidopsis that, plants treated with ANE lowered the levels of AtGRF7-led overexpression of AtDREB2a and AtRD29 genes and thereby provided better salinity tolerance (Shukla et al., 2018). ANE is also involved in regulation of cold or freezing stress tolerance by inducing the expression of cold-responsive genes such as COR15A, CBF3 and RD29A (Rayirath et al., 2009). Further, transcriptome studies by Nair et al. (2012) revealed that the lipophilic components of ANE regulate the expression of more than thousands of genes in response to freezing stress. They also concluded that seaweed extract components have the tendency to positively regulate P5CS1 and P5CS2 genes involved in proline biosynthesis and negatively regulate the expression of proline catabolic genes. The accumulation of proline inside the plant increases the ability to tolerate different types of environmental stresses.

Protein hydrolysates and humic substances also play crucial roles in stress tolerance. They involve in activation of antioxidant enzymes and free radicals having ROS scavenging properties and chelating ionic compounds and metals (Abd El-Mageed et al., 2017). The exogenous application of proline and betaine to Hordeum vulgare reduces the NaCl-induced efflux of potassium ions by relatively low concentrations of compatible solutes (Cuin and Shabala, 2005). Garcia et al. (2012) reported that extracts isolated from vermicompost when used for treatment of rice (Oryza sativa L.) plants it differentially regulated H⁺ ATPases located on the plasma membranes and provided tolerance to salinity and drought stresses. Similarly, when tomato (Solanum lycopersicum) plants were treated with vermicompost it has led to more than 40% of proton extrusion by facilitating the potential of acid growth, hormonal regulations and nutrient uptake (Zandonadi et al., 2016). Similarly, microbial biostimulants comprises of various kinds of beneficial endophytic, symbiotic and ectoparasitic microbes. These microbes show positive interactions with the plants or colonize in the rhizosphere and stimulate several growth responsive genes. They also involve significantly in activation of several defense responsive mechanisms including SAR and ISR. PGPR have been found to enhance the growth of several vegetables and crop plants including lettuce, beans, tomato and pepper etc. by production of 1-aminocyclopropane-1-carboxylate (ACC) deaminase and ISR in several plants against drought and other stresses (Mayak et al., 2004). ACC deaminase producing microbes induce longer roots of the host plants that provide better uptake of water and minerals even in drought like situations (Zahir et al., 2008). It was documented that a volatile organic compound (VOC) produced by a Bacillus spp. involved in ISR. Similarly, plants treated with exopolysaccharides (EPS)-producing bacteria also helped in improving the soil structure and provided resistance to water stress. EPS by interacting with the sodium ions in soil also helps in reducing the salt stress (Sandhya et al., 2009). Similarly, co-inoculation of Rhizobium with Pseudomonas in maize plants helped in accumulation of proline and uptake of potassium ions resulted in salt tolerance (Bano and Fatima, 2009). There are some other beneficial microbes that have tendency to regulate the phytohormones, viz., abscisic acid (ABA), ethylene (ET), salicylic acid (SA) and jasmonic acid (JA), Brassinosteroid (BRs) mediated singling pathways which are mostly stimulate the accumulation of antioxidant in the plant cells and engaged with the defense responses of the host plants (Sharma et al., 2019). It was also observed that wheat plants treated with Bacillus amyloliquefaciens and Azospirillum brasilense had the tendency to tolerate heat at their young seedling stage. Additionally, Rhizoglomus irregular, Glomus (AMF) or Bacillus licheniformis inoculation with the diverse communities of beneficial bacteria involved in solubilisation of phosphate and production of siderophores compounds and indole-3 acetic acid (IAA), which normally benefits the crop's performance (Saia et al., 2015; Rai et al., 2021).

In addition to the above-mentioned biostimulants, the use of their natural products or whole organisms can also have dual benefits against both biological and environmental stresses. For example, seed priming of tomato plants with Trichoderma spp. provides resistance to Fusarium spp. by accumulation of antioxidant, defense-related WRKY proteins, and quantifiable lignin in plant cells (Hyder et al., 2017). Similarly, Woo and Pepe (2018) reported that potential biostimulants comprising more than one microbe such as Trichoderma-Azotobacter and Trichoderma-Glomus like microbial formulations had proved better agricultural tools for sustainable crop management in improving harvest quality and quantity of final yield.

The soil nutrient imbalances or poor quality of soils cause chronic diseases in crop plants surviving in the nutrient-deficient area. In nutrient deficient condition the plants are unable to synthesize important molecules like chlorophyll, nucleic acids, proteins and lipids that are important for normal growth of the crops. Plants become susceptible to several diseases and many enzymes may not function properly under such situations and even complete plant failure may appear at seedling stage in nutrient-deficient conditions. Hence, it is important that plants should be very effective in nutrient use efficiency (NUE). NUE by plant is dependent on the plant's ability for efficient uptake of nutrients such as N from soil followed by its internal transport, storage and remobilization. NUE is different from biological nitrogen fixation (BNF) in the context that in BNF soil bacteria such as Rhizobia can fix atmospheric nitrogen (N₂) and convert it to the usable NH₃ form which then can be utilized by plants. According to several scientific studies on biostimulants, it was proved that biostimulant components have the ability to increase nutrient uptake with increasing crop productivity (Ebic Welcomes Compromise Reached At Trilogue Meeting on Fertilising Products Regulation, 2019) through different strategies (Garcia-Martínez et al., 2010). In fact its application may be effective in bad soil circumstances as they have the ability to change soil texture as well as increasing macro- and micro-nutrient solubility and modify architecture of the roots system for better transport of nutrients in plants (Halpern et al., 2015). Protein hydrolysates also have the tendency to form complex structures with soil nutrients and therefore they become more available to crops (Farrell et al., 2014). Di Mola et al. (2019) confirmed in their study that plants treated with protein hydrolysates and SWE-based biostimulants enhances the fresh biomass under optimal and sub-optimal nitrogen compositions (0 and 10 kg ha/1) compared to the untreated plants, but the efficiency of PBs was reduced under excesses nitrogen fertilizer (20 and 30 kg ha/1). Similarly, another study showed that synergistic application of non-microbial and microbial (Trichoderma virens) based biostimulants on lettuce plants grown with suboptimal, optimal and supraoptimal nitrogen conditions (0, 70 and 140 kg ha/1) (Rouphael et al., 2020a). The valuable impressions of PBs were less prominent under optimal nitrogen conditions, while absent under excesses N circumstances. There are so many symbiotic and non-symbiotic microbes that are present in the soil including Azospirillum, Azotobacter, Bacillus, Klebsiella, Azorhizobium, Bradyrhizobium, Rhizobium, and Sinorhizobium that widely used as plant growth promoters as they increase the amount of N, P, K and other micro-nutrients in soil (Bhardwaj et al., 2014). Recently, Gupta et al. (2021b) and Ngoroyemoto et al. (2021) reported that treatment of the onion (Allium cepa) and Amaranthus (Amaranthus hybridus) plants individually with two PGPR strains namely Bacillus licheniformis and Pseudomonas fluorescens and a seaweed extract or in combinations improved the mineral nutrition, growth and yield parameters of onion and Amaranthus.

Several experiments were also done to investigate the positive effect of PBs on the productivity of crops. For example, Koleska et al. (2017)'s report showed that foliar spray of biostimulants products on tomato plants grown under NPK nutrient deficient conditions had better yield in comparison to control plants. Similar results have been observed by Anjum et al. (2014) where growth of garlic plants were stimulated by use of PBs under nutrient deficient conditions. Sandepogu et al. (2019) have demonstrated an experiment on combined application of A. nodosum extract and humic substances on lettuce and spinach plants. A combination of 0.25% ANE + 0.2% HA was found suitable for better growth and productivity of crops as compared to the other treatments. Similarly, Fan et al. (2014) where they noticed that pre-harvest root treatment of spinach plants by ANE extract had efficiency to minimize the post-harvest losses (PHL) by reducing ROS and lipid peroxidation activity. Sandepogu et al. (2019) concluded that treatment with PBs before harvesting of crops not only improved the NUE and mineral status but also addressed the issue of reducing post-harvest losses in a sustainable way.

The biostimulants including natural substances and microbial inoculants are a novel category of agricultural inputs having great tendency to stimulate better N uptake, plant-growth and tolerance to different abiotic stresses. This review provides knowledge on the current understanding on the progress in the area of identification and utilization of biostimulants in agriculture and further highlights their effects on enhancing crop tolerance to abiotic stresses. The article also focuses on the plant defense mechanisms following application of biostimulants under different environmental stress conditions. Due to high diversity and complexity of biostimulants, characterization of their bioactive components, physiological and molecular stimulation, signaling molecules and mode of actions are still not worked out thoroughly and therefore, are important interests for future researches and commercial applications. The effect of PBs depends on many parameters including raw materials, application techniques, targeted crop and timing of inoculation as well as climatic conditions. From the previous studies and observations it was observed that synergistic application of different biostimulants or consortia of microbial inoculants can play significant roles in plant growth promotion and promote crop's tolerance to survive in challenging environments. Therefore, it would be equally useful to have products combining plant-based extracts mixed with microbial strains for greater efficacy and effectiveness.

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