Biostimulants are changing modern agriculture, as they have the potential to secure healthy and sustainable food production while preserving the environment. They have two main biological effects: growth promotion and stress protection. Both effects can lead to enhancement of the yield and improvement of the marketable grade of the produce in crops, without compromising crop quality. Their use increased exponentially in the past decade, as they are highly efficient, ecologically friendly (non-toxic, biodegradable), and applicable to all major crops. While exponential data on the physiological mechanisms of stress protection is accumulating in recent years, the information as to how biostimulants act at the molecular level is still rather limited.
Introduction
Abiotic stresses such as drought, salinity, extreme temperatures, and pollutants cause significant reductions in growth, yield, and marketable grade of crops, which can account for up to 80% of global crop yield losses. These adverse environmental conditions not only damage plants directly, but they also increase crop susceptibility to pests and diseases.
Biostimulants are naturally occurring molecules or microorganisms with growth promotion and stress protection activities. The most common groups of biostimulants on the market are derived from seaweeds (33% of the global market), followed by humic acids, fulvic acids, protein hydrolysates, amino acids, and microorganisms.
Seaweed biostimulants can activate the defense mechanisms of plants, a process referred to as molecular priming, and protect from adverse environmental conditions. For example, biostimulants produced by the brown algae Ascophyllum nodosum can protect Arabidopsis thaliana, pepper, rice, and tomato from oxidative stress; Arabidopsis, Brassica juncea, maize, okra, olive, strawberry, sugarcane, and tomato from drought; papaya and raspberry from heat; tomato from cold stress; and rice from heavy metals and salt stress (Table 1). Moreover, seaweed biostimulants can stimulate growth, enhance yield, and improve the marketable grade of fruits and vegetables without compromising crop quality, therefore contributing to a sustainable agriculture. This has recently been documented for berry, cereal, and vegetable crops such as grapevine, raspberry, strawberry, sweet cherry, and tomato (Table 1). The advantages of the seaweed biostimulants-induced molecular priming technology can be summarized as follows: (i) Seaweed biostimulants are highly efficient: small amounts of seaweed-derived biostimulants can trigger protection from abiotic and oxidative stresses such as drought, heat, and salinity and significant yield enhancement (42% in raspberry, 33% in strawberry, 70% in tomato, compared with untreated plants) (Table 1); (ii) Biostimulants can improve marketable grade and nutritional properties: larger, more uniform berry and vegetable fruits; raspberries and strawberries richer in essential nutrients such as magnesium; (iii) The molecular priming technology based on biostimulants is ecologically friendly and sustainable. As seaweed biostimulants derive from natural products (e.g., brown algae such as Ascophyllum nodosum), they are non-toxic, biodegradable, and do not pollute the environment. Furthermore, they reduce the use of traditional agrochemicals. They are obtained by controlled harvesting, preserving the natural habitats and resources; (iv) Biostimulants are easily applicable to all crops (berries, cereals, vegetable crops) by adding them into the soil, by foliar spray, or by seed priming.
| Crop | Biological effect on the crop resulting from the biostimulant | Biostimulant |
| Apricot | Enhanced secondary metabolism, higher polyphenol, flavonoid, and anthocyanins. | Seaweed and yeast extracts |
| Barley | Improved nitrogen use efficiency. | Ascophyllum nodosum-derived biostimulant |
| Brassica juncea | Better performance under drought. | Ascophyllum nodosum-derived biostimulant |
| Cotton | Tolerance to drought stress. Detoxification of cadmium, higher yield. | γ-polyglutamic acid; Combined biochar, microbial, and algal biostimulants |
| Cucumber | Growth promotion and defense against pathogens. | Oligosaccharides |
| Grapevine | Enhancement of berry quality: Increase of antioxidant activity, as well as increase in anthocyanins and other phenolics. | Ascophyllum nodosum extract, glycine betaine |
| Kiwifruit | Improved growth and rooting. | Ascophyllum nodosum-derived biostimulant |
| Lavender | Enhancing growth and essential oil components. | Humic acid and extract from Malva parviflora |
| Lettuce | Increased growth and biomass under salt stress. | Ascophyllum nodosum-derived biostimulant |
| Maize | Enhanced biomass and drought tolerance. Increased biomass, photosynthetic pigments, and antioxidant enzyme activities under cadmium stress. | Chitosan, fulvic acid, and their combination; Humic acid |
| Okra | Improved biochemical and physiological functions under drought. | Ascophyllum nodosum-derived biostimulant |
| Olive | Improved growth and photosynthesis under drought. | Ascophyllum nodosum-derived biostimulant |
| Papaya | Better growth under heat. | Ascophyllum nodosum-derived biostimulant |
| Pepper | Tolerance to oxidative stress. | Ascophyllum nodosum-derived biostimulant |
| Purslane | Tolerance to salt stress. | Putrescine |
| Raspberry | 42.1% increase in yield of the primed plants, compared with the unprimed, and higher Mg content. Mitigation of heat stress. | Ascophyllum nodosum-derived biostimulant |
| Rice | Enhanced salt tolerance. Mitigation of heavy metal (arsenic, cadmium) and oxidative stress. | Ascophyllum nodosum-derived biostimulant |
| Soybean | Enhanced growth and increased salt tolerance. | Chitosan-fulvic acid |
| Strawberry | Enhances salinity tolerance; Increases the yield and Mg content in fruits; Tolerance to drought. | Putrescine; extracts from Ascophyllum nodosum |
| Sugarcane | Improved performance under drought. | Ascophyllum nodosum-derived biostimulant |
| Sweet cherry | Higher sugar and protein content, enhanced antioxidant activity and phenolic content. | Calcium and seaweed extracts |
| Tomato | Mitigation of osmotic and cold stress; Increased fruit number, enhanced drought tolerance; Enhanced salt stress tolerance and 70% yield enhancement. | Humic acid, protein hydrolysates, seaweed extracts |
| Wheat | Tolerance to salinity. Improved photosynthesis, nitrogen use efficiency, and Na+/K+ ratio. | Humic acid, Ascophyllum nodosum extracts |
The other groups of biostimulants, such as humic acid, fulvic acid, glycine betaine, polyamines such as putrescine, spermidine, and spermine, and microbial biostimulants, can exert similar beneficial effects on plant growth and stress tolerance. Some of them are used either alone or in combination with Ascophyllum nodosum extracts or other biostimulants. Humic acid, for example, protected wheat from salt stress by enhancing chlorophyll and photosynthesis, increasing the activities of antioxidant enzymes superoxide dismutase (SOD), peroxidase (POD), and catalase (CAT), upregulating salt stress-related genes such as TaNHX1, TaHKT1,4, TaAKT1, TaPRX2A, TaSOD, and TaCAT1, and reducing the Na+/K+ ratio. Humic acid enhanced photosynthetic pigments, antioxidant enzyme activities, and biomass in maize grown on Cd contaminated soils. In tomato, humic acid protects from osmotic stress. Furthermore, the combination of humic acid together with extracts from Malva parviflora enhances the growth and essential oils of Lavandula latifolia. Combinations of two or more biostimulants proved synergistic on other occasions as well.
Chitosan nanoparticles releasing fulvic acid enhanced growth and mitigated salt stress in soybean. This was concomitant with higher chlorophyll under both control and salt conditions, activation of antioxidant enzymes, lower stress parameters such as malondialdehyde and H2O2 contents, and increased expression of the Na+ detoxifying genes GmSOS1, GmSOS2, GmNHX1, and GmP5CS1. Chitosan and fulvic acid alone, as well as chitosan-fulvic acid nanoparticles, were successfully employed to enhance drought tolerance in maize, and the largest effect was observed when chitosan and fulvic acid were together.
Glycine betaine and extracts from Ascophyllum nodosum were used to improve the quality of grapevine. Foliar preharvest application of 0.2% glycine betaine resulted in berries with higher anthocyanin and other phenolic levels, as well as with increased antioxidant activities. This was concomitant with the induction of genes involved in the synthesis and transport of anthocyanins (CHALCONE SYNTHASE (CHS), FLAVANONE 3-HYDROXYLASE (F3H), FLAVONOID GLYCOSYLTRANSFERASES (UFGT), and GLUTATHIONE-S-TRANSFERASE (GST)).
Putrescine, a small polyamine, has important roles in plant development and stress responses. Recently, it was successfully used to enhance salt stress tolerance in Arabidopsis and strawberry. Putrescine is synthesized by the activity of arginine decarboxylase (ADC; EC: 4.1.1.17). It can be acetylated by enzymes such as SPERMINE/SPERMIDINE N-ACETYLTRANSFERASES (SSATs) or N-ACETYLTRANSFERASE ACTIVITIES (NATAs), which may be necessary for catabolism or membrane transport. Interestingly, loss of function of the Arabidopsis AtNATA2 enhances putrescine biosynthesis through AtADC2 and triggers putrescine molecular priming, which protects Arabidopsis from salt stress.
Microbial biostimulants, including plant growth-promoting rhizobacteria, arbuscular mycorrhizal fungi, nitrogen-fixing bacteria, and endophytes, gain more popularity as well. They are totally different from the other types of biostimulants as they contain live microorganisms. Moreover, such biostimulants can contain even mixtures of different microorganisms. For example, a biostimulant derived from a mixture of mycorrhizal microorganisms and rhizobacteria exhibited a positive effect on the growth of tomato seedlings under standard conditions and under salt stress. There are also examples of combined application of both plant and microbial biostimulants. For example, a biostimulant derived from the medicinal plant Adathoda vasica, together with plant growth-promoting rhizobacteria, mitigated drought stress in maize. Whereas there is no doubt that the microbial biostimulants can be beneficial for plant growth and stress tolerance, their safety should be rigorously assessed as they are intentionally released into the environment.
Biostimulants have different degrees of compositional complexity. Whereas some biostimulants have simple chemical compositions, such as chitosan, fulvic acid, or glycine betaine, other biostimulants, like the protein hydrolysates, are more complex, and the plant extracts and the microorganisms are the most complex of all. The composition of plant-based biostimulants depends largely on the plant species but also on the technology of extraction, which is often proprietary to the producing company. The most commonly used seaweed biostimulants contain polysaccharides such as fucoidan, laminarin, alginates, as well as various phenolics, minerals, and other bioactive compounds. In such complex mixtures, it is difficult to determine the most bioactive compound, and often there is a synergistic effect of several bioactive components from the complex mixture. For example, an oligosaccharide mixture (Oligo-Mix) derived from plant cell walls and fungal cell walls stimulated growth in cucumber and provided protection against fungal pathogens. Microbial biostimulants, being the most complex of all, may act both through such oligosaccharide signals and through creating a beneficial growth-promoting rhizosphere.
These positive effects of the biostimulant treatment are good for both the farmers and the end users, who will benefit from lower consumer prices and health-stimulating properties of the fruits from primed crops. Due to the above-mentioned advantages, the use of seaweed biostimulants is growing exponentially and its market exceeds 2.6 Bln EUR, with projections to further increase in the future. The perspectives of using biostimulant-based molecular priming are enormous and attract attention both from fundamental and applied research points of view. Despite the surge in using this technology and its success, there are limited studies on transcriptome and metabolome reprogramming during seaweed-based molecular priming, and there are no functional studies, which confirm the participation of any of the regulated genes as part of the presumably complex biostimulant signaling network.
Biostimulant-Induced Molecular Priming Triggers Global Transcriptome Reprogramming
Whereas the large majority of studies demonstrate mainly the physiological aspects of biostimulants-induced growth and stress protection, a growing number of publications in recent years focuses also on the transcriptome and metabolome reconfigurations during the biostimulants-induced molecular priming.
In tomato, molecular priming by Ascophyllum nodosum-derived biostimulant resulted in growth promotion, increased yield, and improved marketable grade of tomato fruits. This was associated with upregulation of genes encoding transcription factors from similar families (WRKY, NAC) both in leaves and the fruits. The transcriptional reprogramming involved induction of many stress-protective genes, whereas senescence-related genes were repressed. Genes encoding components from the jasmonic acid and brassinosteroids pathways were among the most induced under severe stress, the latter similar to Arabidopsis. This indicates that similar pathways (e.g., ABA, brassinosteroids) may be activated in both model and crop plants by the molecular priming.
Another brown seaweed biostimulant, conferring cold stress tolerance in tomato as well as larger and more fruits under both normal and cold stress conditions, induced genes related to proline, flavonoids, and chlorophyll. This transcriptomic data were corroborated by higher proline, polyphenols, flavonoids, and carotenoids contents in the biostimulant-treated plants.
In maize, the molecular priming by chitosan-fulvic acid nanoparticles, which enhanced tolerance to drought, induced genes encoding similar transcription factors, such as ZmDREB1A, ZmNAC28, ZmbZIP1, and the ABA-dependent ZmCIPK3. This further corroborated the role of the ABA pathway in attaining drought tolerance by biostimulant priming.
A biological stimulant derived from a mixture of mycorrhizal microorganisms and rhizobacteria had a positive effect on tomato seedling plant growth and development under standard conditions and in response to moderate salinity. The transcriptomic analysis showed differential expression of stress-related genes such as GOX3 or DIR1, as well as genes related to jasmonic acid and auxin hormones. A number of genes encoding transcription factors (TFs) from WRKY and other families, including TFs presumably involved in stress defense, were induced by the biostimulant with and without stress, suggesting that the biostimulant-treated plants have already activated their defense system before the onset of stress.
Strategies To Identify New Players Of The Genetic Network Which Controls Biostimulant-Induced Stress Tolerance And Crop Yield Enhancement
As indicated above, biostimulants influence growth and development, as well as metabolism, stress responses, fruit size and quality, and ultimately yield. These processes themselves are regulated by intricate genetic networks converging developmental and environmental signals, and utilize plant hormones, reactive oxygen species, lipids, and other secondary messengers. Whereas some of the stress signaling pathways are modulated by biostimulants, others are not related to biostimulants and act in alternative pathways. Despite the recent omics studies implicating particular genes and biochemical pathways in the molecular priming, no key genes have been identified and evaluated functionally so far. Below we describe strategies to functionally validate genes and pathways involved specifically in the biostimulant-controlled genetic networks in both model plants (A. thaliana) and vegetable crops (pepper and tomato).
Identification of genes that are part of the biostimulants signaling network in Arabidopsis thaliana
Biostimulants can protect Arabidopsis from many stresses such as drought, heat, and oxidative stress. However, when essential genes of the stress-signaling network modulated by biostimulants are mutated, the biostimulants may no longer provide protection against abiotic and oxidative stresses, and their stress-protective effect will be minimized. In contrast, mutations in genes that are not part of pathways modulated by biostimulants will not have an effect on the stress mitigation by the biostimulants. Hence, genetic studies can be designed to identify genes specifically involved in the biostimulants-modulated gene network, using, for example, the well-established system based on the protective effect of seaweed biostimulants against drought and oxidative stress (Figure 1). Two alternative strategies can be applied to identify such biostimulant-related genes:
Mutant screening to identify genes, which are part of the biostimulant network.
(a) Forward genetics approach utilizing either gene inactivation by T-DNA/transposons to inactivate unknown positive regulators (gene 1) or activation tag construct to identify negative regulators (gene 2) of the biostimulant network.
(b) Reverse genetic approach, in which genes regulated by biostimulants (e.g., SuperFifty, SF) are mutated or CRISPR-Cas9 genome edited. If a gene is involved in the biostimulant pathway, its inactivation will result in a sensitive phenotype upon SF-drought exposure (mutant 1), as opposed to the inactivation of a gene which is not part of the biostimulant pathway (mutant 2).
Forward genetics screening of mutants to identify genes which are modulators of the biostimulant network
Large mutant collections of A. thaliana with genome coverage, containing mutants in nearly all Arabidopsis genes, can be screened to identify mutant lines which are sensitive to drought or/or oxidative stress after biostimulant application (Figure 1).
This is a labor-costly but unbiased forward genetics approach, which will identify many new genes of the biostimulants stress-protective network. After treatment with the biostimulant, mutated genes which are positive regulators of the biostimulant pathways will render plants sensitive to drought and oxidative stress (Figure 1). The mutant collections to be utilized can be both from chemical (EMS) or insertion (T-DNA, transposons) mutagenesis. Negative regulators of the biostimulant pathways, on the other hand, can be identified by activation tag mutant collections, in which the activation tag constructs inserted near the negative regulators will activate them and consequently the activated repressors will suppress the biostimulant pathway (Figure 1).
Reverse genetics approach as a tool to validate genes from the biostimulant network
In this approach, comprehensive transcriptional analysis of biostimulants-modulated gene expression, conducted by RNA sequencing, can identify genes that are regulated during biostimulant-induced molecular priming. Selected biostimulant-induced genes, presumably involved in the biostimulant network and contributing to the observed stress tolerance, will be inactivated by insertion mutagenesis (e.g., T-DNA knockouts or transposons) or CRISPR-Cas9 genome editing, and the resulting mutant and genome-edited lines will be functionally evaluated for altered responses to biostimulants. The corresponding mutated/genome-edited lines will be subjected to drought and oxidative stress to evaluate the functionality of the studied genes (Figure 1). Inactivation of genes that are positive modulators of the biostimulant pathways will presumably abolish the stress-protective effect of the biostimulant on stress tolerance.
This reverse genetics approach offers a more targeted way to identify directly new genes that are essential players of the drought or oxidative stress-signaling network. As oxidative stress is a consequence of many other abiotic stresses such as extreme temperatures and salinity, the mutant and genome-edited lines will be evaluated for the whole spectrum of most prominent abiotic stresses (drought, heat, salt stress etc.). Altogether, the two approaches proposed by us have the potential to reveal the majority of the key biostimulant network genes in Arabidopsis.
GWAS analysis of biostimulants response in major crops
The reverse genetics approach can be applied not only for Arabidopsis but also for major crops. Genes commonly regulated by the biostimulant in cereal, berry, and vegetable crops can be identified, and a selected number of them functionally validated (e.g., by CRISPR-Cas9 genome editing in maize and tomato or VIGS in pepper). In this line, comprehensive transcriptome analyses by RNA-seq of SuperFifty-induced stress protection have very recently been conducted in maize, raspberry, and tomato. This will allow identification of key genes that contribute to agronomically important traits in these crops, including stress tolerance, corn and seed cob weight, fleshy fruit size and weight, and yield.
In an alternative approach, there is an exciting prospect to apply GWAS on the response of large panels of crop lines to biostimulant treatment, in order to identify genetic polymorphisms and corresponding genes that regulate processes such as biostimulant-induced drought stress tolerance (Figure 2). The natural genetic variation has already been linked to different levels of stress tolerance and other agronomically important traits in tomato and other crops.
GWAS analysis pipeline for identification and verification of genes modulating the biostimulant pathway in vegetable crops.
Genetic resources: hundreds of pepper and tomato lines and cultivars will be primed with biostimulants and evaluated under normal and stress (e.g., drought) conditions. Agronomically important traits such as growth, stress tolerance, fruit nutritional properties (metabolites, essential elements, vitamins, and yield) will be studied, and candidate genes identified by GWAS analysis will be verified using CRISPR-Cas9 genome editing.
The GWAS approach proposed by us will identify naturally occurring genetic variations, which alter the responsiveness of pepper and tomato to biostimulants and influence their stress tolerance. The identified candidate genes will be verified by functional studies using CRISPR-Cas9 genome editing or virus-induced gene silencing, and the molecular mechanisms of biostimulant-induced stress mitigation will be further studied by omics technologies such as transcriptomic and metabolomics (Figure 2). This GWAS approach has never been used with biostimulants and crops before. It will provide the researchers with tools to discover genes involved not only in stress tolerance but also genes, which modulate crop productivity and yield under unfavorable environmental conditions. As already mentioned before, drought and heat are the two most predominant abiotic stress factors influencing the world’s agriculture, and the identification of genes that mitigate these stresses will have a profound impact on food security.
Prospects For Achieving Stress Tolerance And Yield Enhancement
Besides the fundamental value of the biostimulants research, which will reveal essential players in processes such as stress physiology, vegetative, and fruit development, the studies on the genetic control of biostimulant pathways will have a deep impact on global agriculture. As already noted, various stresses can account for more than 80% of agricultural production worldwide. The advantages of biostimulants allow broad application of this molecular priming technology to all major crops. Data that is already available from all major berry, cereal, and vegetable crops indicate that the biostimulants-induced molecular priming has the potential to increase the revenues of farmers by either protection from unfavorable environmental conditions or/and enhancement of yield and crop quality.
Clear examples of this positive impact on agriculture include the improved nitrogen uptake by an Ascophyllum nodosum-based biostimulant in Arabidopsis, wheat, and barley, with surplus N assimilated by the biomass in the form of glutamate, glutamine, free amino acids, soluble proteins, and chlorophyll. Other examples include mitigation of heavy metal stress and salt stress in rice, as well as mitigation of salt stress and improvement of marketable grade and yield in tomato and other vegetable crops. Someone reported that the seaweed biostimulant SuperFifty increased the number of tomato fruits and improved tomato yield by 70% under normal conditions. Furthermore, tomato plants primed with SuperFifty had larger fruits than unprimed plants under salt stress. Likewise, others reported larger fruits and higher yield in SuperFifty-primed tomato, as well as mitigation of drought stress. Improving both stress tolerance and yield in tomato and other vegetable crops will have a huge impact on world agriculture and further contribute to global food security.
Furthermore, the GWAS studies on genetic resources in vegetable crops and their responses to biostimulants can reveal additional genes that modulate fruit size, metabolic content, and yield.The perspectives of such studies are not limited to berry, cereal, and vegetable crops. The growth or/and stress tolerance of other crops, such as Brassica juncea, grapevine, okra (Abelmoschus esculentus L.), olive, papaya, and sugarcane, was also positively influenced by biostimulants. The molecular priming technology developed on the basis of seaweed biostimulants is widely applicable to all crops. Overall, it can be developed into a universal eco-friendly technology that can protect all plants against the major abiotic stresses, as well as improve marketable grade and enhance crop yield.
Conclusion And Outstanding Questions
There is growing evidence that biostimulants regulate many aspects of plant growth and development, as well as stress responses. Furthermore, many publications in recent years document the positive effect of the biostimulants on nutritional properties and crop yield. Comprehensive transcriptional profiling in model (A. thaliana) and crop plants implicates genes encoding signaling compounds, plant hormones such as ABA, brassinosteroids, and ethylene, and a number of transcription factors responsible for the global transcriptional reprogramming and the positive effects of the biostimulants. However, there are still no functional studies linking specific genes to the above-mentioned processes. There is a huge perspective for research in this field to unravel the key genes of the apparently intricate biostimulants network.
Biostimulants products for agriculture, different from plant growth regulators, are biological substances that can help crops improve nutrient absorption efficiency, improve crop health, resist adversity, optimize production, and restore the true flavor of crops. The common organic biostimulants products are ‘Amino acids’, ‘Humic acids’, ‘Seaweed extract’. See more specifications on Dora’s website.