Biostimulants for Crop Production and Sustainable Agriculture

1 Biostimulants in Sustainable Agriculture

Bornita Bose and Harshata Pal*

Amity Institute of Biotechnology, Amity University, Kolkata, India.

Abstract

In an attempt to understand the role of biostimulants in sustainable agriculture, the concerns that arise are matters regarding the possible need for sustainable agriculture and their exact efficacy in promoting sustainable agriculture. Although conventional agricultural practices were initially aimed at increasing plant growth, it gradually became a reason for soil ecology exploitation as a result of the heavy application of fertilizers and various synthetic chemicals such as pesticides (e.g. DDT, carbamate), herbicides (e.g. atrazine), insecticides (e.g. endosulfan, phorate), fungicides (e.g. mancozeb), etc. This very reason led to the introduction of sustainable agriculture that has, so far, proven to be beneficial in balancing the soil ecology by utilizing minimal fertilizers and synthetic chemicals. Hence, sustainable agriculture can be thought of as a promise for a better future for eco-friendly farming. The introduction of sustainable agriculture allowed biostimulants to bring about changes in physiological, structural and metabolic processes to influence plant growth via reduced use of synthetic chemicals and fertilizers, improved tolerance to various biotic and abiotic stresses, efficacy in nutrient uptake and use, and improved, good quality yield. Moreover, biostimulants have also increased plant resistance to pests, biological contaminants or diseases by stimulating their natural defense systems. This chapter focuses on the definition of biostimulants, types of biostimulants, formulations of biostimulants, effects of biostimulants on plant physiology and metabolism, advantages and disadvantages of biostimulant application, challenges faced in this field and the future of biostimulants in sustainable agriculture.

1.1 Introduction

From the beginning of human civilization, agriculture, in disguise, has potentially served as a mere means of survival for living beings walking on this planet. Even though the practice of ‘agriculture’ existed from a long time, the term itself came into existence much later. The history of basic agriculture dates back to times when people lived in caves. In other words, it has been a developmental criterion in the advancement of human civilization. For decades, development in the agricultural field has been quite slow; however, farmers from Asia, Africa and Europe came up with new techniques, including open field cultivation, which allowed them to harvest edible crops (Vasey, 2002). Crop cultivation began independently in South and North America. While the initial evidence of crop cultivation has been found to be approximately 10,000 years before present in Mexico and South America, earliest evidence of crop production in North America has been observed to have begun between 5000 and 4000 years before present.

However, with time, many hurdles baffled traditional farmers which eventually led to the discovery of machines having potential to perform processes, farming tools, use of natural fertilizers, and pesticides and water pumps accompanied by electricity. By the 1900s, a farmer was able to harvest enough food crop to sustain a family of five members and another hundred population. All of this was possible after a major discovery that took place in 1866, when Gregor Mendel paved the way to major breakthroughs in the agricultural sector through the discovery of genetics. However, the real issue occurred with the invasion of the agricultural fields by pests that ranged from insects to animals such as mice and rabbits, disease-causing organisms including viruses, bacteria and fungi along with the presence of weeds in the fields. For a long time, farmers have traditionally tried to get rid of these issues by physically handpicking the insects from the plants, using natural poisons to kill the insects, cultivating the crops in an alternative manner to reduce the presence of insects and by producing high-quality bred crops. Though these physical methods helped to protect crops from the pests, it did not take long before the need to develop some kind of chemical that could control these crop-destroying pests and provide nutrition to increase crop yield. Farmers continued to rely on natural fertilizers including manures, ground bones, animal debris such as bird and bat waste known as guano, wood ash, and fish or fish parts, for providing and replenishing soil with essential nutrients. However, the scientists soon discovered the essential elements and their importance that influence the effective and rapid crop growth and that was when they started producing chemical fertilizers and pesticides containing phosphorous, nitrogen and potassium, considered as the essential elements. There was a large-scale production of these chemicals in the US and Europe by the end of 20th century. Thereafter came the production of chemical fertilizers and pesticides containing nitrates and phosphates which initially helped to increase the total crop yield but over years of usage, they lead to a decline in soil quality and degradation of the environment (Vasey, 2002). Gradually, the world began to face several challenges leading to a global demand to increase the annual crop productivity rate, while maintaining the quality, nutrition, efficacy and shelf-life of the crops to feed the growing population adequately (Rouphael and Colla, 2020).

All of these challenging circumstances impelled scientists to divert from the conventional methods and bring about holistic changes in the field of biotechnology and agricultural sciences laterally. This eventually led to the concept of sustainable agriculture, which required the invention of such eco-friendly substances that could support the idea of sustainability, alongside proving beneficial to the environment and maintaining the ecological balance. Scientists discovered one such group of substances that was not only capable of replacing chemical fertilizers, but could also promote plant growth and development, and increase nutrient uptake efficiency, annual crop productivity, overall plant metabolism and crop resistance to environmental stresses. This group are now called biostimulants (Calvo et al., 2014).

1.2 Biostimulants

The discovery of biostimulants had a positive impact for farmers who, previously, had suffered from crop loss, production of low-quality crops and destruction of crops by pests. Moreover, the biostimulants helped to minimize global hunger and brought about serious changes in the world economy. Although it might appear that the concept of biostimulants was clear from the beginning, it was quite the opposite. Biostimulants, also known as natural plant biostimulants, were initially described as a substance without some of its essential functions such as fertilizers, growth factors and products of plant protection (du Jardin, 2012). Following this, many scientists attempted to provide a proper definition of biostimulants until 2011, when the European Biostimulant Industry Council (EBIC) was formed. According to EBIC, ‘biostimulants can be defined as the substance(s) or compound(s) and/or micro-organisms which, when administered on plants or the rhizosphere, function as a stimulant in the naturally occurring physiological processes in plants that improve nutrient uptake and efficiency, increase tolerance of the plants to abiotic stresses along with quality and yield of the plants’ (du Jardin, 2015). The Association of American Plant Food Control Officials also defined biostimulants as ‘any compound or substance except the primary, secondary or micronutrients, which proved to be beneficial by scientific approach, when naturally administered to one or more species of plants exogenously’ (Gupta et al., 2021). An impromptu study was conducted by the European Commission in 2012 to understand the nature of substances and materials involved in the formation of plant biostimulants for a better definition. This study was published by du Jardin in 2012 as an ad hoc study report for the European Commission, entitled ‘The Science of Plant Biostimulants-A Bibliographic Analysis’, from where it could be concluded that plant biostimulants are, rather, simply heterogenous substances. He also proposed in his study that there are eight categories of substances that have the ability to act as biostimulants:

•organic materials that are commonly procured from urban and agro-industrial wastes, composts, sewage sludge end products and manures;

•substances derived from humus;

•extracts of seaweeds, mainly obtained from brown, red and green macroalgae, and phosphite that form the inorganic salts;

•chemically beneficial elements, such as Al, Co, Na, Se and Si;

•substances like kaolin and polyacrylamide that act as anti-transpirants;

•chitin and its derivatives;

•free amino acids; and

•substances containing nitrogen, such as polyamines, peptides and betaines.

However, that meant that none of the existing microbial components were considered to be biostimulants (Rouphael and Colla, 2020). After three years of extensive work, a special issue on ‘biostimulants in horticulture’ was in Scientia Horticulturae, and plant biostimulants were given a more specific definition that included the nature, mode of action and type of effects on horticultural and agricultural crops with the support of scientific evidence. Du Jardin then again modified the definition of plant biostimulants as ‘micro-organism(s) or substance(s) which, when applied to plants, effectively enhances crop quality, nutrient-uptake efficiency, tolerance to abiotic stress, irrespective of the nutrient content.’ The addition to the definition to include ‘plant biostimulants also form commercial products that consist of such micro-organisms or substances’ provides a complete and meaningful definition (du Jardin, 2015). In Scientia Horticulturae special issue, Colla and Rouphael (2015) proposed categorizing plant biostimulants into six non-microbial and three microbial categories, which are as follows:

•humic and fulvic acids ( Canellas et al. , 2015 );

•phosphites ( Gómez-Merino and Trejo-Téllez, 2015 );

•seaweed extracts ( Battacharyya et al. , 2015 );

•beneficial bacteria, such as plant growth-promoting rhizobacteria (PGPR) ( Ruzzi and Aroca, 2015 );

•beneficial fungi, such as arbuscular mycorrhizal fungi ( Giovannini et al. , 2020 );

•inorganic compounds, such as silicon ( Savvas and Ntatsi, 2015 );

•chitosan and its derivatives (Pichyangkura and Chadchawan, 2015);

•protein hydrolysates and other compounds containing nitrogen ( Colla et al. , 2015 ); and

•Trichoderma spp. ( López-Bucio et al., 2015 ).

Over the years, there have been a lot of arguments regarding a proper description that could define plant biostimulants in the best way possible. Recently, a new definition has been formulated pertaining to plant biostimulants, under a new regulation (EW) 2019/1009, that not only defines biostimulants, but also provides a specification about its functions. According to this new regulation, ‘Plant biostimulants are EU fertilizing products which, when administered on the rhizosphere or the plants, functions to stimulate the nutrition processes of the plants irrespective of the nutrient content of the product with only the sole purpose of enhancing one or more characteristics of the plants or rhizosphere that are as follows: (1) resistance tolerant to (a)biotic stress, (2) efficiency of nutrient use and uptake, (3) availability of nutrients confined in the rhizosphere or the soil, or (4) characteristics of the quality of the soil.’ Following this definition, several naturally occurring bioactive substances and chemically active derivatives of synthetic and natural substances along with the beneficial micro-organisms (bacteria and fungi) were categorized under plant biostimulants that include:

•environmentally derived substances such as humic and fulvic acids;

•products of seaweed extracts obtained from macroalgae;

•animal- and vegetal-based protein hydrolysates;

•chemically beneficial elements such as silicon;

•beneficial micro-organisms:

°fungi that includes arbuscular mycorrhizal fungi (AMF); and

°bacteria including strains of N-fixing bacteria that belongs to the genera of Azotobacter , Azospirillum and Rhizobium .

1.3 Why biostimulants in sustainable agriculture?

Various studies have indicated that biostimulants have positive impacts and advantages on crop quality and yield. Biostimulants, as defined earlier, are substances that improve the quality traits, vitality, physiological processes, metabolic pathways, root and shoot growth and development, protection against biotic and abiotic stresses and also diseases, thus improving the annual crop productivity. The various substances cataloged under plant biostimulants such as humic substances (including humin, humic acid and fulvic acid) help in turning minerals into organic compounds for easy absorption by plants along with providing protection against toxic substances. These substances regulate water infiltration and induce the growth of healthy roots.

Amino acids belong to major phytohormone origin that influences the metabolic activity of plant systems. These stimulate early germination and division of seeds, and cell division rate. They enhance mature development of fruits, flowering, fruit setting and pollination in crop plants.

Seaweed extracts are widely extracted from red, green and brown algae. Among all the types of algae, brown algae such as Sargassum, Turbinaria, Laminaria, Ascophyllum nodosum and Fucus contribute greater advantages to crops, including nutrient-uptake efficiency, faster and quicker germination of seeds, fruit setting, development of quality fruits and flowering, resulting in the formation of healthy crops. These are applied as biostimulants for the presence of natural phytohormones such as auxins, gibberellins, and cytokinins, along with metal elements like nitrogen, aluminum, potassium, iron and manganese.

Looking at all the advantages provided by natural biostimulants in improving quality, quantity, desirable characteristics and productivity of crops, it is now believed by biotechnologists that utilization of biostimulants can, indeed, bring about a new dimension in sustainable agriculture.

1.4 Microbial and non-microbial biostimulants

1.4.1 Humic and fulvic acids

Humic and fulvic acids come under the broad category of humic substances. Humic substances can be defined as any substance that is the end product of natural decomposition of microbes, plants, animals and degradation of dead biota that are present in the soil, chemically (Lavkulich et al., 2019). Humic substances have been reported to have huge effects on the crop yield, quality characteristics, nutrient-uptake efficiency, effectiveness of gas exchange, plant physiology and biological factors. These are naturally occurring heterogenous substances that are inherently classified on the basis of their molecular weights into three major types:

•Humic acids are natural, alkali-soluble humic substances that are present in soil. These are derived from soil by addition of a dilute alkali, followed by precipitation in acidic solution.

•Fulvic acids are soluble in alkaline as well as acidic solutions.

•Humins are humic substances that cannot be extracted from soil.

The function of humic substances in various plant species is greatly influenced by their structural differences. The variations can be observed in acidity, extent of polymerization, content of carbon and oxygen, color and molecular weight. These can be accounted for by the difference in the structure of humic and fulvic acids (Fig. 1.1).

Fig. 1.1. (a) Humic acid. Molecular size range from 10,000 to 100,000 with a combination of complex carbon chains and rings that radiate a dark color. Adapted (with permission) from de Melo (2016). (b) Fulvic acid. Molecular size range from 1,000 to 10,000, consisting of nitrogen, hydrogen, oxygen and carbon. (c) Suwannee River fulvic acid model. Reprinted (adapted) with permission from Atalay, Y.B., Carbonaro, R.F. and Di Toro, D.M. (2009). Distribution of proton dissociation constants for model humic and fulvic acid molecules. Environmental Science & Technology 43(10), 3626–3631. © 2009 American Chemical Society, used with permission.

The differences in structure, function and effects on plant species are as follows (Canellas et al., 2015):

•Humic acids are large molecules with molecular size ranging from 10,000 to 100,000 Da with a complex combination of carbon chains and rings and nitrogen, oxygen, hydrogen and phosphorous. Fulvic acids are smaller molecules ranging from 1000 to 10,000 that consists of nitrogen, hydrogen, oxygen and carbon.

•Humic acids often act as chelating agents that bind to toxic metals, thus preventing their entry into the plant biome. Moreover, it helps in stimulating the activity of microbes in the soil, increasing the water retention capacity of the plants and enhancing the growth and development of roots and shoots. On the other hand, fulvic acids are the best chelating agents ever known that work by binding to the essential nutrients present in the soil and transports them into the plants for efficient physiological processes.

•Humic acids radiate a dark color whereas fulvic acids emits a golden color.

•While humic acids act as dilators to increase the permeability of the cell wall for better absorption of nutrients, fulvic acids act as railcars as they help in transportation of essential macronutrients and micronutrients from the soil to the plants.

•Due to the presence of a high content of oxygen, fulvic acids are twice as biologically active as compared to humic acids.

Humic acids are the ‘all-stars’ of the biochemical, biophysical and physiological processes of plants as they help in setting the optimum environment for optimum functioning, whereas fulvic acids act as carriers to provide vital nutrients and minerals to the plants.

According to research conducted by Iowa State University on the regulation of humic and fulvic acids on soybean, it was found that humic and fulvic acids help in enhancing the root development. Along with that, the effects of these substances on soybean plant were also investigated when a soybean cyst nematode was present as it leads to approximately $1.5 billion in loss of crop annually (McGrath et al., 2013 ). Early research had also shown that exposure of soybean plants to humic substances had led to an increase in the dry weight of roots, shoots and nodule; however, it was also noted that the nodule weight was inversely proportional to the amount of humic substances administered.

From Table 1.1, we can conclude that the initial effects of humic substances (humic and fulvic substances) on any crop species is initiating growth in the primary and secondary roots, followed by shoot growth. Moreover, it has enhanced the uptake efficacy of various macronutrients and micronutrients by the plants and has thus increased the rate of flowering of buds and development of fruits with high levels of carbohydrates, soluble sugars and other nutrients. It has also increased the H+-ATPase activity along with providing protection against abiotic stress. Therefore, it can be concluded that administration of humic substances on various crop species has, indeed, proved to govern a positive effect on the biological, biochemical and physiological processes of the plants along with increasing the total crop yield.

Table 1.1. Different effects of humic substances on various species of plant or crops. Adapted (open access CC-BY) from Calvo et al. (2014).

1.4.2 Seaweed extracts obtained from microalgae

To begin with, seaweeds can be defined as the brown, green and red marine algae that are mostly found anchored to a solid support, near the seashore, by means of a rootlike ‘holdfast.’ These extracts of seaweeds are chemically composed of some very important constituents that mainly include complex polysaccharides, phytohormones, vitamins, minerals, essential nutrients and fatty acids that play an integral role in traditional agriculture. These extracts impart valuable inputs as plant biostimulants, and their application extends to horticultural crops where they mainly serve as plant-growth promoters and as an ameliorating factor to induce tolerance against abiotic stress, such as extreme temperatures, drought, flood, salinity, nutrient deficiency, etc. Recent research has highlighted the effects of seaweed extracts on activation of certain mechanisms involved in plant processes that are still being studied. Moreover, they have also been observed to increase shelf-life, productivity and yield of the crops on administration (Battacharyya et al., 2015).

It has been reported by several scientists that certain seaweeds such as Ecklonia maxima, Ascophyllum nodosum or Pterocladia capillacea have the ability to (Kocira et al., 2019):

•improve the settling of fruits in eggplants ( Pohl et al. , 2019 ); and

•enhance the agronomically evolved performance of potato ( Wadas and Dzuigiel 2020 ), bean ( Kocira et al. , 2020 ), and Jew’s mallow ( Ashour et al. , 2020 ).

The effects of seaweed extracts on various plant species are still being studied. One such example is the Bio4Safe project an ongoing project that is funded through the European Interreg 2 Seas Programme. It is being conducted in the 2-Seas region coordinated by PCS Ornamental Plant Research (Belgium) and other six partners: Ghent University (BE), North Sea Farmers (The Netherlands), Vertify (The Netherlands), NIAB-EMR (UK), Junia (France) and Pôle Légumes (France) (Interreg 2 Seas Mers Zeeën Bio4safe, https://bio4safe.eu/about. accessed 9 April 2022). This project is based on a tenure of four years with a total budget of €3.2. This project was started with the aim of increasing the efficiency of water and nutrient use by crops by the application of biostimulants in the form of seaweed extracts. The objectives of this project are as follows:

•Reduction of the water consumption by horticultural crops by 20%.

•Reduction of the fertilizer consumption by horticultural crops by 10%.

•Development of a protocol dedicated to the policy makers for tracking the impacts and effects of biostimulants on fertilizers and water uptake efficacy of plants.

The trials under this project are performed by the North Sea farmers in the four countries within the 2 Seas region – France, Netherlands, United Kingdom, and Belgium. They are using commercially available seaweed-derived biostimulants on various economically viable crops that include strawberry, lettuce, tulip, tomato, raspberry, hydrangea, chrysanthemum and surfinia. This project aims to determine the effect of seaweed extracts on the horticulture and agriculture industries in Europe. The following impacts have been observed so far:

•Provide a detailed market study for the seaweed-based companies of the region that would include the calculation defining the economic potentiality of seaweed-derived biostimulants.

•There has been an improvement in the water retention capacity of the soil, water uptake by the plants and increased content of chlorophyll in the leaves.

•Foliar application of seaweed extracts has helped to increase the depth and 50% of the surface area of the root system, thus allowing crops to absorb more water and nutrients and increasing the resilience of the crops.

•A 10–30% increase in the dry weight of the plant system has been observed.

•There has been an average increase of 30% of the leaf sizes that has enabled an increase of the chlorophyll content. With the increase of chlorophyll content, the fresh look of floral crops is preserved.

•The increase in the chlorophyll content also regulates the doubled production of flowers in each plant along with an increased vase life.

•Results have shown a remarkable increase of approximately 43% of the total crop yield, even under stressful conditions such as high salinity or drought.

It has also helped in increasing the production of flavonoids, carbohydrates, phenols, antioxidants and proteins, hence improving the overall quality of the crops.

It is predicted that the application of seaweed extract-based plant biostimulants will, indeed, bring about a major shift in the ecological provision, safeguarding the food requirement for the future, and lessen a farmer’s fight against odds.

1.4.3 Animal-based and vegetable-based protein hydrolysates and other compounds containing nitrogen

Protein hydrolysates can be broadly categorized as protein-derived products that, on application, can stimulate plant growth and resistance to (a)biotic stresses. These protein-derived products are classified into two types of protein hydrolysates: (i) a combination of amino acids and peptides originating from plants and animals as its constituent; or (ii) those composed of amino acids alone such as proline, glutamine and glutamate (Kauffman et al., 2007; Ertani et al., 2009; Kunicki et al., 2013; Cavani et al., 2021), along with some major amino acids, including glycine, alanine, arginine, valine and leucine (Ertani et al., 2009). Chemical, enzymatic and preparation of protein hydrolysates involve hydrolysis of a wide range of plant and animal residues, such as connective tissues and epithelial cells (Ertani et al., 2009; Cavani et al., 2021), alfalfa residues (Schiavon et al., 2008), elastin and collagen fibers (Cavani et al., 2021), glycoproteins present in the cell wall of Nicotiana (Apone et al., 2010), proteins derived from carob seeds and protein obtained from algae (Lucia and Vecchietti, 2012).

Application of protein hydrolysates has a direct influence on the microbial activity and biomass, cellular respiration and fertility of the soil. It is also evident from experiments that certain amino acids serve as chelating agents (e.g. proline) that can bind heavy metals within the soil, thus preventing plant damage. Protein hydrolysates also help in increasing the mobility and the addition of micronutrients to the plants. This indirectly contributes to the development of roots and nutrient availability to all the parts of a particular plant (Lavkulich et al., 2019).

In an experiment conducted on tomato plants at extreme climatic conditions, regulation of peptide-derived products or amino acids in the form of non-microbial protein hydrolysates have provided tolerance against a wide range of stresses, which mainly include hypo-toxic, salinity, excessive nutrient and heat stresses and unfavorable environmental conditions (Francesca et al., 2020). Administration of protein hydrolysates on maize seedlings that were grown under hydroponic conditions alleviated the effects of multiple (hypoxia + salinity + nutrient stress) or single (hypoxia or salt deficiency) stresses that could have been detrimental to the health of the maize plants. It also upregulated the primary genes responsible for transport of nitrates and detoxification or oxygen reactive species which consistently improved the growth of the shoot system, thus eliciting the structure and architecture of the newborn maize plants (Trevisan et al., 2019). Application of vegetal-based biostimulants, especially those that are legume based or tropical plant-derived protein hydrolysates, has significantly increased the functional and nutritional quality of tomato (Caruso et al., 2019) as well as lettuce (Cozzolino et al., 2020).

Some amino acids (e.g. glycine betaine, which is the substituted N-methyl derivative of glycine and proline) are known to act as osmolytes or osmo-protectants, enzymes, stabilizing proteins and membranes as a result of denaturing effects of high salinity conditions and extreme temperatures (Chen and Murata, 2008; dos Reis et al., 2012; Ahmad et al., 2013;). Hence, proline and glycine betaine have come to be related to stressful conditions since administration of these have shown to induce increased abiotic stress tolerance in a wide variety of crop plants such as maize, soybean, barley, rice and alfalfa (Chen and Murata, 2008; dos Reis et al., 2012; Ahmad et al., 2013). Along with certain other amino acids (e.g. ornithine and/or glutamate), proline precursors, when exogenously applied, can increase the resistance against abiotic stress (Chang et al., 2010; da Rocha et al., 2012). When plants are exposed to stressful conditions, accumulation of arginine within the plant has also been observed (Lea et al., 2007). Some non-protein amino acids, including gamma-aminobutyric acid (GABA) and beta-aminobutyric acid (BABA), function as endogenous molecules of signaling and enhancers of stress tolerance (Zimmerli et al., 2008). Regulation of GABA has, indeed, helped in curing the postharvest chilling injury in Prunus persica (peach) (Schwartz, 1978).

1.4.4 Chemically beneficial elements and inorganic compounds

There are certain chemicals that are not required by all crops for normal sustainability; however, the presence of such essential elements can enhance crop development. There are five important compounds that are particularly beneficial as biostimulants: Na, Si, Al, Co and Se. These elements are present in the form of inorganic salts in the plants, as well as in the gramineous form of non-crystalline silica (SiO2.nH2O) in insoluble forms. These inorganic salts have constitutive functions, including strengthening the cell wall or expressing themselves under stressful environment, such as Na+ osmotic stress or during attacks of Se. Hence, the functions of these inorganic salts are not only dependent on their unique chemical structures, but also dependent on the external environmental conditions. Under such conditions, they have been observed to have promoted plant growth and development along with resistance to such stress. On the other hand, the activity of some biostimulants such as residues of crop or crop waste, or seaweed extracts, may provoke the expression of physiological functions of these inorganic elements.

The derivatives of these inorganic salts or beneficial compounds such as phosphite anions, phosphates, chlorides, silicates and carbonates, have been known to have functioned as fungicides under certain conditions. Although all the functions of these compounds are not yet known, it has been evident that they help in maintaining the redox homeostasis and pH, hormonal signaling, and act as enzymes. Moreover, they improve the activity of the co-factor–enzyme complex, osmoregulation, enhance the nutrient uptake, symbiont interactions, and provide protection against toxic heavy metals (Lavkulich et al., 2019).

1.4.5 Chitosan and its derivatives

Chitosan can be defined as a de-acetylated polysaccharide that is naturally and industrially derived from chitin, one of the world’s most abundant polysaccharides. It contains amino acids in its structure which makes it viable for getting easily protonated, thus converting it into an acid-soluble polysaccharide. It is known that the cell membranes are negatively charged, so characterization of chitosan by the presence of positive charges allows it to interact with the cell membranes efficiently. This character also allows it to bind to various components of cells, for example DNA and constituents of the cell wall and membrane. It also has the ability to bind to particular receptors, thus, regulating cell signaling.

Due to the characteristic properties of a chitosan, including natural origin, abundant availability, biodegradable nature, reactivity, etc., it has been broadly associated with plant growth, abiotic stress tolerance (drought, salinity, drought, waterfall) and production of primary and secondary metabolites (Lavkulich et al., 2019).

1.4.6 Beneficial fungi

Over the ages, fungi have interacted with the plant system by colonizing around the roots of the host plant. They have been either directly involved through symbiotic relationship or indirectly involved through parasitism. In either way, they have shown to have increased the capability of nutrient and water absorption by the host plant system, while on the other hand, the plants provide shelter and food in the form of carbohydrates (produced from photosynthesis) to the fungi. Mycorrhizae is one such symbiotic relationship between plants and fungi. It has been predicted that almost 90% of land plants live in close association with the mycorrhizal fungi (Herbarium and Matthew, 2021).

Among all the forms of mycorrhizal fungi, the most essential and popular form of endomycorrhiza that is related to crop and horticultural plants is the arbuscule-forming mycorrhiza (Rouphael et al., 2015). This type of tripartite association allows the unique species of fungal hyphae to invade the cortical root cells, thus forming branch-like structures called arbuscules. Considering the known facts about the increased efficiency of water, mineral and nutrient (micro- and macro-) uptake by the plant system in the presence of AMF, they have been considered to function as biostimulants. Through experiments it has been evident that AMF has helped in balancing the water content and increased resistance against (a)biotic stress. Application of AMF on plants has shown responses that include promotion of morphogenesis and organ growth, efficient usage of nutrients and overall enhancement in the crop yield (Lavkulich et al., 2019).

Studies have also inferred that the activity of AMF can be maximized by adopting agriculturally beneficial practices, involving various useful strains of AMF and, most importantly, careful selection of the host plant. One such experiment was the application of four types of microbial biostimulants (AMF, Trichoderma, and enriched rhizosphere with seaweed extracts and amino acids) on common green bean. The results showed an elevated yield of seeds and pods, along with increased chemical and nutritional composition (Petropoulos et al., 2020).

1.4.7 Beneficial bacteria

There are two major groups of beneficial bacteria: (i) plant growth-promoting bacteria (PGPR) rhizobacteria; and (ii) Rhizobium of endosymbionts. Rhizobium and its associated taxa are commercially applied as biofertilizers, whereas the PGPRs are used for its multifunctional properties that influence each and every physiological process of plants, including the morphogenesis and organ development, plant growth and nutrient-uptake efficacy, tolerance against biotic and abiotic stress, and interaction with the ecosystem, including organisms and external environment. Bacillus thuringiensis, an important PGPR, is also used as an efficient biostimulant to boost the crop yield and pave a way for sustainable agriculture. Concerning the improvement of quality traits of crops, application of Bacillus subtilis CBR05, a strain of PGPR, to tomato plants significantly increased the quality and content of carotenoids (lycopene and β-carotene) (Chandrasekaran et al., 2019). Another experiment conducted by inoculating Rhizophagus intraradices, or a combination of R. intraradices and Funneliformis mosseae into saffron, grown without soil, showed an improvement in the antioxidant activity, synthesis and accumulation of bioactive elements, such as picrocrocin, crocin II and quercitrin, and molecules like polyphenols, anthocyanins and vitamin C that promote plant health (Table 1.2; Caser et al., 2019a, 2019b).

Table 1.2. The effect of various biostimulants on crop species. Source (with permission): du Jardin (2015).

Although biostimulants are natural-derivates and environmentally beneficial, sometimes these may not be effective in its native state. There is therefore a need to formulate these natural biostimulants to produce more economically and environmentally beneficial product forms.

1.5 Biostimulant formulations

Biostimulant formulation can be simply defined as the processed forms of those natural substances considered as biostimulants, that can be easily stored, transported, produced and be more efficient in its function, therefore, creating a safe and convenient path for practical use. These are generally produced based on the physiological characteristics of the already existing materials in respect to market demand. Biostimulants, based on different principles, are of various formulations including a diverse range of molecules like amino acids (Colla et al., 2017), seaweed extracts (Battacharyya et al., 2015), microbial elements (Mire et al., 2016), phytohormones (cytokinins, gibberellins, auxins, ethylene, brassinosteriods and abscisic acid) (Pacifici et al., 2015), polyamine (Fuell et al., 2010), protein hydrolysates (Colla et al., 2017), nitrobenzene, etc. They are also available in the form of granules, powders, or solutions given to soil or as foliar applications in liquid or dried form.

1.6 Types of biostimulant formulations

The various types of biostimulants in use, e.g. fitofortificants, supplements, plant strengtheners and conditions, soil improvers, depends on individual countries and their regulatory laws.

1.6.1 Old formulations

As this type of biostimulant formulation has been used time and again for various purposes, including the increase in bio-efficacy, rapid growth of crops within a stipulated time and others, it is termed an ‘old’, ‘traditional’, ‘classical’ or ‘conventional’ biostimulant formulation. Biostimulants in the developing countries of the Pacific region and Asia, exist in the form of solutions or wettable powder, dust or emulsifiable concentrations.

Over decades, old formulations of biostimulants have been strictly restricted to natural, homemade preparations that had significant incorporation of extracts derived from organic solvents and crude oil for improving the crop yield and efficacy. There have been many such natural formulations of biostimulants that have been prepared by various scientists and have proved to be effective in various ways.

For the regulation of plant growth, a composition of abscisic acid and a plant-growth regulator such as 1-naphthyl acetic acid, triacontanol, gibberellic acid, nitrohumic acid, maleic hydrazide, fulvic acid, brassinolide, nitrohumic acid, oligosaccharins, salicylic acid, chitosan, etc. was formulated that claimed to be suitable for certain crop plants such as rice, soybean, cotton, tobacco, corn, sugarcane, cereals, sugar beet, corn and rape (Tan et al., 2002).

Recently, a novel plant-growth stimulating composition consisting of heteroauxin indole-3-acetic acid, gibberellins and cytokinin 6-(4-hydroxy-3-methyl-2-trans-betenylamino) purine, has been developed by Jones and Gates (2019). All the components of this composition act actively thus producing a synergistic effect on the plants that regulate shortening of the dormancy period in seeds and early seed germination, increasing the fruiting and flowering period, and improving crop yield. Not only this, but it has also been observed to prevent lodging, help in the recovery of damaged crops, and has enhanced root and shoot proliferation (Jones and Gates, 2019).

There are four main types of old formulations of biostimulants:

•Wettable powders (WP) are a form of old formulations that can be made by using 50% or more dry concentrates of micronized active biostimulant that is mixed with a dispersing agent, a wetting agent and a finely ground diluent. The dispersing agent acts by inhibiting the agglomeration of biostimulant particles, while the wetting agent provides assurance of effective wetting of the active biostimulant in water. The most commonly used solid diluent in the production of wettable powders is clay because of its certain unique properties. The properties are as follows:

°It has a naturally occurring small and fine particle size ranging from 5 to 10 μm.

°It has the innate ability to inhibit the biostimulant particles inside the spray tank.

°It has immense compatibility with the actives present in the biostimulants.

•The wettable powder is added to water enclosed in a spray tank before using it as foliar spray or direct application on target crop species. This achieves the minimum effective concentration. The wettable powder is quite often applied as a dilute aqueous suspension because it is easier to control its reach to the non-target areas, which is difficult in dustable powders. Even after all these advantages, there is still a major concern regarding exposure of this type of respirable biostimulant. In India, an approximate of registered biostimulant in wettable powdered form is sulfur 80% WP, TSS containing marine plant (Ascopyllum nodosum) extract 32% WP, etc. (Knowles, 2008).

•Dustable powder is a form of old biostimulant formulation that is prepared by the sorption of plant extracts or any other active particles onto a fine, inert, solid ground, for example, clay, talc or chalk. As these biostimulant particles are dry and not wetted prior to its application on the crops, so their sizes are generally higher, ranging from 25 to 35 μm. A very innovative formulation including water–water or dry flowable dispersible granules was formulated with Trichoderma strains. This formulation increased micronutrients and phosphorous metabolization which, in turn, promoted plant growth and enhanced shelf-life ( Knowles, 2008 ).

•Emulsifiable concentrates (EC) is a blend of emulsifiers, adjuvants and biostimulants mixed in a volatile oil. This formulation is stable only when it is dissolved in water inside the spraying tank. The emulsifying agents used here are usually chemicals with long chains that face toward the oil droplets and form a water–oil complex that does not allow the oil and water to get separated. Traditionally, liquid biostimulants or biostimulants with low-melting point have been formulated to produce ECs. In India, the registered concentration of emulsifiable biostimulant is nitrobenzene 20% EC ( Knowles, 2008 ).

•Soluble liquids (SL) are the simplest form among all the types of biostimulant formulations. A soluble liquid or concentrate is a biostimulant formulation that is clear in appearance and is applied only after dilution in water. This type of formulation is mainly based on either water alone or any other solvent that is completely miscible in water. A soluble concentrate or soluble liquid is a clear solution to be applied as a solution after dilution with water. Soluble concentrates are based on either water or a solvent that is completely miscible in water. Few polar compounds like humic acids, polar plant extracts, amino acids and others are very useful in the production of this formulation. Here, the process rarely needs agitation in water inside the spray tank. SLs are very effective in containing the salt form of biostimulant, which leads to an increased salt concentration in the spray tank as compared to other biostimulant formulations. Even after having advantages, this formulation is not dynamic because of the limited hydrolytic solubility and water solubility of the formulated biostimulant. This might, sometimes, give rise to flocculation of other materials that are being dissolved inside the tank such as ECs. In India, the registered soluble liquid biostimulant is Ethephon 39% SL ( Knowles, 2008 ).

As the new-age generations emerge along with their productive and innovative ideas, these formulation types are becoming ancient and conventional that notably utilize organic solvents and petroleum for their production. So, there is a speedy shift from the older formulations to newer, environment-friendly biostimulant formulations.

1.6.2 New formulations

Over the years, it has been observed that implementation of older, conventional methods of formulation (mentioned above) has caused harm to environment and human health. As an alternative, biostimulant formulations are being extensively studied and researched worldwide so that new versions of biostimulant formulations can be invented. These newer formulations are produced with the intention of reducing toxicity, increasing efficacy and safety, minimizing harmful effects on the environment and human health, ease of application, decreased need of labor and better cost effectiveness. The areas of attention include concentrated emulsions – controlled release, microemulsion, etc. and water dispersible granules.

Water dispersible granules (WDG), also termed as dry flowables, are modified forms of wettable powders that are produced by aggregation to form uniform granules. This increases the ease of handling and is also efficient in eliminating respirable particles. WDG are an alternative to wettable powders as the use of the same ingredients, including clay and dispersants, but usually have lower levels of diluent and higher levels of activity. The application process of WDG is also similar to that of WP. The ingredients used in this process allows optimum efficacy of the active particles due to their fine particle sizes, which also prevents clogging in the nozzle.

The formation of uniform granules and powder blend uses various methods, including fluid bed granulation, high-speed mixer agglomeration, spray drying, extrusion granulation and pan granulation. Out of all these methods, extrusion granulation is the most preferred method because of its versatility, safety and economy. The uniform granules produced are dissolved in water already present in the spray tank and then administered as dilute suspensions, alike wettable powders.

WDG formulations comprise materials, such as dispersing agents 5–15% (e.g. naphthalene sulphonate-formaldehyde condensates, lignosulphonates), active ingredients (e.g. seaweed, humic acid) and binder (e.g. lactone). Other components include disintegrating agents or fillers (e.g. precipitated silica, China clay), where the active ingredients constitute 50–90% of the total mass.

This latest type of biostimulant formulation is now becoming quite popular due to the advantages it provides that involve convenience in packaging and ease of use, and free-flowing, dust-free granules that have the potential to disperse immediately on adding to water in the spray tank. Some of the properties are as follows:

•There is a uniformity in particle size that ranges from 1–2 mm and is relatively hard.

•It easily disperses and disintegrates in water to result in the formation of a homogenous stable suspension.

In India, registered WDG biostimulant includes seaweed extract (Ascopyllum nodosum) 25% WG and sulfur 80% WG. When WDG formulations are compared to WP formulations, it is seen that WDG provides better outcomes, including ease of handling and measuring, complete evacuation from the container and probability of less spillage (Tan et al., 2002).

1.7 Selection of the type of biostimulant formulation

The most important factors when determining the biostimulant formulation includes the following:

•potentiality;

•biological effectiveness;

•convenience of manufacturing;

•ease of handling;

•cost effective;

•environment-friendly;

•ability to enhance crop productivity and yield; and

•develop crop tolerance against (a)biotic stress.

To choose the most appropriate biostimulant formulation for a given crop species, there are certain factors that are to be taken into account. These include:

•Design of the formulation:

°compound inputs – chemical, physical, and biological properties;

°marketing inputs – safety, attractiveness, economy, user friendly, durability;

°application inputs – plant species, climatic conditions, and various ingredients; and

°manufacturing inputs – QC facilities and production equipment.

•Development of biostimulant formulation:

°preliminary studies – this includes preparation of the lab, and collection of all the chemical and physical tests;

°investigational stage – shelf-life, performing small scale trials of the field, development of analytical methods, bio-efficacy and phytotoxicity; and

°commercial – process of formulation, development of packaging, and compatibility of tank mixing.

•Requirements to design an ideal formulation of biostimulant:

°it should be biologically effective on application with no undesirable side effects;

°it should contain higher levels of active ingredients in its composition to have maximum biological effects at the minimum expense;

°it should be capable of providing reliable and effective dispersion;

°it must be favorable for large scale manufacture at a minimum cost;

°it should provide safety during manufacture, packaging, storage and transportation;

°it must have an adequate shelf-life; and

°it should be produced in such a way that it is acceptable by the registration authorities and consumer ( Lavkulich et al. , 2019 ).

1.8 Advantages of administration of biostimulants in sustainable agriculture

According to EBIC, biostimulants influence the growth, differentiation and development of a crop plant beginning from seed germination through to the entire maturity of the crop. It regulates a lot of physiological, chemical and biological functions and plant metabolism in various ways.

•It influences nodulation and nutrient assimilation which allows improvement in the efficacy of nutrient uptake and better utilization of nutrients and minerals by the crop plants.

•It increases soil fertility by nurturing the growth of soil microbes around the crop plants which also replenishes the soil with nutrients from where the crop plants absorb nutrients for themselves.

•It provides crops with enhanced tolerance to biotic and abiotic stresses. This prevents damage of the economically viable crops at the growth stage.

•It improves the efficiency of the metabolic pathways that occur in the plant systems.

•Administration of biostimulants have shown results of crops with improved quality traits, such as improved fruit seeding and color, increased carbohydrate content, vitamins, proteins and many others ( Lavkulich et al. , 2019 ).

•It has also decreased the harmful effects on the environment and consumption of biostimulant-based crops have proved to be safe for the human health.

1.9 Challenges in biostimulant administration

Biostimulants are an emerging concept which, until now, has not been socially or widely accepted by all nations. Researchers are still determining the exact effects of biostimulants on various crop species. It has not been universally defined as of yet. This involves certain challenges that include:

•Regulatory challenges – since it is an emerging concept and is not universally defined, it applications become limited to certain extent. It is an ongoing researched subject which has limited market data and availability. Due to all these reasons, this becomes very less reliable which restricts the regulatory bodies to formulate any regulatory framework for biostimulant administration specifically.

•Scientific challenges – since the physiological and metabolic effects of biostimulants on the plant species are still being researched, the extent of complexity is still not entirely known to us. Hence, this challenge really becomes a topic of immense attention. Even though we have certain results to show the response of plants toward itself and the external environment on application of biostimulants, we cannot be sure of it entirely because environmental changes are subjective and it evolves every day. Only further studies on this field can help to clarify the ongoing doubts.

•Technical challenges – these challenges mainly cover the formulation of biostimulants with other organic and inorganic compounds and plant protection products. The technical issues include the study of methods of application and their outcomes under different climatic conditions which still requires a lot of research.

1.10 Conclusion

Biostimulants are considered as an effective weapon that can bring about valuable changes in the conventional methods of agriculture and have the potential to carve a path that leads to sustainability. Today, the world is dominated by food insecurity, hunger and insufficient crop products to feed the stomachs of millions of humans and animals on this planet. Year after year, the soil quality has been observed to have been degraded by the overuse of chemical fertilizers, pesticides, fungicides and other such substances under the need to increase crop yield. However, despite giving positive results, these methods ultimately reduced the crop quality and yield. At this time of need, biostimulants have emerged as an alternative method that can provide several benefits to farmers and can help satisfy the global food crisis. So far, the research on biostimulants have reported quite good results, including improved resistance to abiotic and biotic stress, increased efficacy of nutrient uptake and utilization, increased carbohydrate content, mature flowering, improved seed germination and fruiting, and enhanced good quality traits and crop yield. They show potential in reducing harmful and hazardous effects on the environment and human health. They have helped increased nodulation that has allowed the accumulation of soil microbiome, which in turn has replenished the soil with nutrients and fertility. Biostimulants also provide the opportunity to evolve the formulations in a way that encourages the development of more efficient and resilient agricultural technologies. The studies on biostimulants have so far proved that it can bring about a major change in the field of sustainable agriculture. However, there is a long way to go for biostimulants to get widely and universally accepted within the global market. Following a systematic study and focusing on the constant evolution of biostimulant formulations, the physiological effects and plant metabolism can help in shaping a more secure future in sustainable agriculture. Moreover, trying out new formulations can help in creating a synergistic and resilient effect on crop plants in the near future.

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