A Food Science, senior assistant professor [email protected] 3)

A Review of Definition and Classification of “Biostimulants” and Their Effects on Agricultural and Horticultural Crops

Sima kavousi(1), Francesco Orsini(2) , Massimo Tagliavini(3)
1) University of Bologna , Department of Agricultural and Food Science ,International Horticultural science MSc
[email protected]
2) University of Bologna, Department of Agricultural and Food Science, senior assistant professor
[email protected]
3) University of Bologna, Department of Agriculture and Food Science. University of Bolzano, Faculty of Science and Technology , professor
[email protected]

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Abstract as the global population is rapidly increasing (anticipating up to more than 9 billion by 2050), the demand for food is growing, and food production becoming one of the most challenging world-wide issue. Till now, the food production has been kept at a high rate along with the population growth rate, through the use of different agricultural techniques with the aim of increasing plant productivity and stability. Up to now many agricultural techniques has been introduced to the market and to the farmers in order to increase the productivity. On the other hand, food production must be environment friendly. using chemical fertilizers is no more acceptable by environment neither by scientists. as a solution biostimulant can be used to increase crop productivity and stability. Biostimulants can be introduced as natural or/and synthetic products that are able to stimulate plant growth , crop yield, quality, vigor and stability to biotic and abiotic stress. The global biostimulants market consists of a wide verity of products including acid based and extracts based those which are Acid based are further classified into humic acid, amino acids and fulvic acids. And those who are based on Extracts contains seaweed extract. In this review we discuss how different categories such as humic substances, seaweed extracts, hydrolyzed amino acids and proteins, inorganic slats and microbial based biostimulants, can increase the crop productivity and sustainability through different mechanisms.

Keywords: (plant biostimulant, definition, categories, humic substance, , seaweed extracts, hydrolyzed amino acids and protein, inorganic salts, microbial biostimulants).

Introduction

Along with the population growth and global warming, using appropriate agricultural techniques becoming more important. Such techniques that are able to keep the food production at a high level according to the rate of population growth and without being environmental harmful. To have a higher level of food production either we have to use more agricultural land or using developed techniques. as we have a limited source of agricultural land using more agricultural land is becoming impossible. On the other hand, using chemical fertilizer is no more acceptable neither by environment nor by scientists. most often fertilizers are washed away from the field and penetrate into the runoff (Daverede et al., 2004). Releasing Huge amount of harmful chemical fertilizer annually to the environment , caused many irrecoverable damages to the both marine and terrestrial ecosystems. it is important to mention that, the production process of the chemical fertilizers is a highly energy consuming process, as it is known to significantly contribute to the global CO2 emission into the atmosphere (Vance, 2001). New research is now strongly focused on the use of appropriate agroecological approaches, those are able to minimize harmful chemical inputs and improve the soil quality along with having ecological friendly outcomes. Finally, with the aim of having higher level of agricultural production along with environment sustainability. during past decade we have seen the emergence of developed technological tools to promote sustainable agroecosystems. Biostimulants can be introduced as new natural or/and synthetic products that are able to stimulate plant growth , crop yield, quality, vigor and stability to biotic and abiotic stress as an environment friendly substitutive for chemical fertilizers. Biostimulants include living microorganisms, namely plant growth promoting fungi and rhizobacteria (PGPR) (Bhattacharyya et al., 2012). Furthermore, the use of biostimulant based products in agricultural field, would increase the nutrient uptake efficiency by crops (King and Torbet, 2007). As they are able to enhance the root growth and biomass. One method by which it is possible to overcome nutrition uptake deficiency is to grow crops with more robust root system and higher nutrient uptake efficiency, to ensure that crops receive enough nutrients and when they need them. However, nutrients can be more available to the crop by promoting a certain type of organisms within the soil microbial communities (Vessy, 2003). Both approaches can be achieved based on introduction of biostimulants to crop levels, seeds or soil with the aim of stimulating root growth and nutrients uptake enhancement (Khan et al., 2009; Zandonadi et al., 2007).

1) Biostimulant terminology and definition

For the first time the word biostimulant, in the scientific literatures was introduced by Kauffman, which described them as materials different than fertilizers, those are able to promote plant growth when they are applied in low quantities at crop level (Kauffman et al., 2007). Later on, the word biostimulants has been increasingly used by scientific literature during the following years, containing different range of substances and different modes of action (Calvo at al., 2014; Halpern et al., 2015). Plant biostimulants are such materials which contain substances and/or microorganisms, which are applied at different crop levels such as rhizosphere and whose function is to stimulate natural beneficial nutrients uptake. Plant biostimulants improve beneficial nutrition uptake, nutrient efficiency , crop quality traits and tolerance to abiotic and biotic stress. In fact, plant ‘biostimulant’ appears to be as a versatile descriptor of any kind of, beneficial substances to crops without itslef being nutrients, fertilzers, pesticides or soil improvers ( www.biostimulants.eu).

.Plant biostimulant hase been de?ned as: substances or/and materials, without being nutrients, fertilizers or pesticides, which, when they are applied to plants, seeds or growing substrates with specific formulations, have the capacity for modifying physiological processes of plants in a way that provides potential enhancement to increase growth, productivity, and abiotic/biotic stress response. (Du Jardin, 2012). Nowadays there are many kinds of biostimulant based products such as: peptides, amino acids, polysaccharides, humic acids, phytohormones. among the biostimulant categories, it is always included to be as the one formed by specific combination of molecules, with the capacity to effect on plant physiological response (Du Jardin, 2012). The biostimulants are organic or synthetic products, which are composed of different substances such as peptides, amino acids, polysaccharides, humic acids, phytohormones. for immediate uptake enhancement and increased nutrients availability within the plant. because they are directly absorbed by the plant their absorption does not depend on the photosynthetic activity, which it results in lower energy consumption for the plant. in fact, the aim of application of these products is not to supply nutrition, but to stimulate and favor the metabolism activity in the plant and to decrease plant biotic/abiotic stress. they are also claimed to enhance the crop growth, yield and quality through a series of widely different mechanisms such as: stimulating and activation of soil microbial activities, promotion and augmentation of the critical soil enzymes and plant hormones activities, ( Parrado et al.,2008). However, adoption of a clear definition of biostimulants for regulatory purposes is inevitable, any definition of biostimulant should be based on scientific principlesas well. So far, several concepts have been proposed to define plant biostimulants. Basak (2008), proposed that the classification of biostimulants could be basedg on their mode of action and the origin of their active ingredients. Bulgari et al. (2015), proposed that plant “biostimulants should be classified based on their action within the plants or base on the physiological plant-responses rather than, on their composition. Du Jardin (2015), proposed that any definition of plant ‘biostimulants’ should focus on the major agricultural functions of the biostimulants, neither on the nature of their constituents nor on their modes of actions. all of mentioned concepts, reflect the importance of providing a clear definition of ‘plant biostimulants’ along with providing them as a discrete class of agricultural products. However, biostimulants also could be defined by their demonstrated modes of action and their origin, or by their beneficial effect on plant productiy.

2) Biostimulant classification by reviewing the history of biostimulant based products, it has appeared that there are, noticeable different classification of these products that clarifying insight into the diversity of these products and the development of this field of study. “There are probably 30 up to 40 different classification of biostimulant based products that you could rattle off. but would be probably easier to aggregate them into the four big segments or categories. including: acids, extarcts , microbials and other material such as (proteins, chemical salts, vitamins, other elements and small molecules from organic sources). Table 1 demonstrates the evolution of biostimulant classifications by several authors. one of the first classification of the plant biostimulants was provided by Filatov (1951), when grouping them into 4 different classes. Ikrina and Kolbin (2004), provided 9 different categories of natural raw materials which are used to derive biostimulants. Basak (2008) proposed that biostimulants could be grouped into the basis of single or multicomponents formulations or to be classified depending on their origin of the active-ingredients, and the mode of action of their active ingredients. Du Jardin (2012), proposed a scientific rationale of classification which considering them into 8 different categories. In fact, Du Jardin (2012), was explicit in his exclusion the microorganisms from categorization primarily to avoid any further conflicts with other existing categorization of microorganisms as biopesticides or other sources of plant-hormones. Later Bulgari et al. (2015), proposed a biostimulant classification based on their mode of action instead of on their composition. Within this review we mostly focus on the last classification of biostimulants which has been proposed by Torre et al., (2016). and the effect of each group on plants.

Table 1: proposed biostimulant categories
Ikrina and Kolbin, 2004
Microorganisms (bacteria, fungi), Plant materials (land, freshwater and marine), Sea shellfish, animals, bees, Humate- and humus-containing substances, Vegetable oils, Natural minerals, Water (activated, degassed, thermal)

Kauffman et al., 2007
Humic substances, hormone containing products (seaweed extracts), amino acid containing products
Du Jardin, 2012
Humic substances, complex organic materials, beneficial chemical elements, inorganic salts (such as phosphite), seaweed extracts, chitin and chitosan derivatives, antitranspirants, free amino acids and other N-containing substances
Calvo et al., 2014
Microbial inoculants, humic acids, fulvic acids, protein hydrolysates and amino acids, seaweed extracts
Halpern et al., 2015
Humic substances, protein hydrolysate and amino acid formulation, seaweed extracts, plant growth promoting microorganism (including mycrorrhizal fungi)
Torre et al., 2016
Humic substances, seaweed extracts, hydrolyzed protein and amino acids, inorganic salts, microorganisms

3) Biostimulants categories

3.1) Humic substances
HS (Humic Substances) or humates have significantly positive effects on the beneficial macro and micro nutrients uptake , which consequently would improve the plant metabolism, growth, yield, and relevant agricultural quality traits (Bronick and Lal 2005; Ferrerasetal.2006; Nardi et al. 2009). The positive effects of HS on the plant metabolism are well recognized as hormone-like activity such as: auxin, gibberellin or cytokine activities within the plant, by induction of some positive changes in root architecture through the lateral roots, root hair and increased root biomass production (Trevisan et al. 2010; Canellas et al., 2011; Moraet et al. 2012; Pizzeghello et al., 2013). on the other hand, HS increase root plasma membrane and consequently the H+-ATPase enzyme activity. increasing nitrate and other, benefiacial nutrient uptake, also contributing to cell wall loosening and cell enlargement and finally organs growth (Zandonadi et al.2007; Jindo et al., 2012). furthermore, application of HS containing substances on agricultural crops has a significantly positive effect on improvement of TCA cycle, phenylpropanoid metabolism, and uptake and metabolism of nitrate (Quaggiotti et al. 2004; Vaccaro et al., 2009). However, the effects of HS on plant growth cannot be overgeneralized, because of their different, origins (for example they can be from volcanic soil, compost or brown coal), dosage (it can differ from types of culture media), as well as plant species (Nardi et al., 2009; Roseetal. 2014). Among different HS, can mention to leonardite, which is an oxidized form from lignite with a medium brown coal like appearance. Which it is ussually found at shallow depth over the more compact coal in various coal mines (Stevenson 1979), and all around the world, mostly in the USA (Fernandez et al., 1996), nowadays this brown coal, particular lyenriched is used to manufacture a wide range of commercial HS products. Akinremi et al., (2000), demonstrated that when leonardite is applied to Canola it can increase the biomass and dry matter, yield and beneficial nutrient uptake. Nutrients such as (N, P, K, S). HS from leonardites enhanced the resistance of tomato plants under salinity stress and within the greenhouse conditions (Casierra Posadaet et al., 2009). Arnica montana L. which has been treated with HS from leonardite show a higher floral stems number, flowerheads numbers and yield compared to the control plants (Sugier et al. 2013). It has been registered that a low molecular weight fraction of HS from leonardite can enhance the seedling-root surface area, root length, and total roots number of ‘snap bean’ (Qian et al. 2015). David et al., (2014), demonstrated that potassium humate salts extracted from lignite, and potassium humate regenerated from lignite with two oxidizing agents (nitric acid and hydrogen peroxide), positively influenced root growth and division, starch and protein contents in treated Zea mays seedlings. Leonardite is thus referred to as a benchmark humic material with respect to responses on plant growth. Although the effects of leonardite on crop production, resistance to stress, and soil microbial activity have already been reported, (Bulgari et al, 2015). Arnica montana L. which has been treated with HS from leonardite show a higher floral stems number, flower heads number, and yield compared to the control plants(Sugier et al., 2013). David et al., (2014), has demonstrated that potassium humate-salts that are extracted from lignite, and potassium humate regenerated from lignite with two oxidizing agents including: nitric acid and hydrogen peroxide, positively has influenced root growth, root division, starch and protein contents in treated Zea mays seedlings.

nitrogen and phenylpropanoid metabolism, (3) proteins,
sugars, and total phenols content in roots and leaves.
nitrogen and phenylpropanoid metabolism, (3) proteins,
sugars, and total phenols content in roots and leaves.

3.2) Seaweed extracts
Seaweeds extracts (SWE), are form an integral part of the marine coastal ecosystems. such extracts include the macroscopic and multicellular marine algae which they usually inhabit the coastal regions of the world’s oceans, where there are suitable substrata for them. Till now, it has been estimated that there are about 9,000 species of macro algae which are widely classi?ed into three main classes, based on their pigmentation (as an example, Phaeophyta, Rhodophyta and Chlorophyta) or ( brown, red, green algae). The brown seaweeds are the second most abundant group which comprising about 2,000 species. and to reach their maximum biomass levels they need rocky shores of the temperate zones. they are the type which is most commonly used in agriculture (Blunden and Gordon, 1986). among brown algae, Ascophyllum nodosum (L.) Le Jolis, is the most researched type (Ugarte et al., 2006). besides A. nodosum, there are other brown algae such as Fucus spp., Laminaria spp., Sargassum spp., and Turbinaria spp. Which are mostly used as biofertilizers in the field of agriculture (Hong et al., 2007). Seaweed extract are a complex mixture of bioactive compounds such as: phytohormones polysaccharide, fatty acids, vitamins and mineral nutrients (Battacharyya et al., 2015). Several studies reported stimulation of rhizosphere and root growth after application of SWE on cuttings or plants (Vernieri et al., 2006; Pacholczak et al., 2016). further, Vernieri et al., (2006), propsed that the addition to the nutrient solution containing SWE can significantly increase the root growth and biomass of hydroponically grown rocket. and especially under nutrient deficiency. The bene?ts of SWE as sources of organic matter and nutrients have led to their application as soil conditioners for centuries (Blunden and Gordon 1986; Temple and Bomke 1988; Metting and others 1988). According to FAO annually about 15 million tons of seaweeds extract based products are produced (FAO 2006). when, a considerable portion of them is used for nutrient supplements and enhancement and as biostimulants to increase the plant growth, yield and vigor. There are a wide range of commercial seaweed extract products that are available for use in both agriculture and horticulture field table 2. So far, numerous studies have revealed a wide range of bene?cial effects from seaweed extracts applications on crops, such as: early seed germination , seed establishment, improved crop-performance and yield, improved resistance to both biotic and abiotic stress and postharvest, shelf-life, crop quality traits enhancement (Hankinsand Hockey 1990; Blunden 1991; Norrie et al.,2006). Seaweed extracts such as: macro and micro nutrients element, amino acids, vitamins, cytokinin, auxin, and abscisic acid (ABA) and growth substances, effect on cellular metabolism in treated plants which leading them to increase crop growth and yield (Crouch etal., 1992; Crouch et al., 1993; Reitz and Trumble 1996; Durand et al., 2003; Stirk et al., 2003; O¨rdo¨g et al.,2004). Seaweed extracts are bioactive compounds at a relatively low concentrations (diluted up to 1:1000 or more) (Crouch and van Staden1993). Although many of the various chemical components of seaweed extracts and their modes of action remain unknown, still it is plausible that tsuch components exhibit synergistic activities in the plant (Fornes et al., 2002; Vernieri et al., 2005). The application of both seaweeds and SWE triggers the growth of bene?cial microbes within soil substrata as well as secretion of soil-conditioning substances by these microbes. As mentioned, alginates effect on soil properties and consequently encourages the growth of bene?cial fungi. Ishii et al., (2000), observed that alginate oligosaccharides, that are produced by enzymatic degradation of alginic acid. and are extracted from brown algae, can signi?cantly stimulated hyphal growth, elongation and biomass in arbuscular mycorrhizal (AM) fungi also they observed their infectivity on trifoliate orange seedlings. all the extracts from various marine brown algae can be used as an AM fungus growth promoter or stimulator (Kuwada et al., 2006). Kuwada et al., (1999), previously demonstrated that methanol-extracts from brown algae which are fractionated by ?ash chromatography can promote AM hyphal growth in vitro, as well as enhanced root colonization by AM fungi on trifoliate orange and Poncirus trifoliate seedlings. also indigenous AM fungi has demonstrated about 27% improvement in both root colonization and growth, while the number of spores have increased up to 21%, over the controls. When liquid fertilizer containing tangle (L. japonica) extracts have been applied by sprinkler system in a citrus orchard (Kuwada et al., 2000). Kuwada et al., (2006), demonstrated that in vitro condition the organic fractions (25% MeOH eluates) of both red and green algae, can improve hyphal growth of AM fungi. Their results demonstrated that, the application of about 25% of MeOH eluates of both red and green algal extracts can also promote the root of papaya (Carica papaya Linn.) and passion fruit (Passi?ora edulis Sims.) while they have shown improved mycorrhizal development compare to control treatment. Kuwada et al., (2006). implied that both red and green algae have AM promoters-compounds, which play a significant role in mycorrhizal development in higher plants. some research has also been conducted on the effects of seaweed extracts on other different bene?cial AM fungi. Which consequently their effects on plant growth, root development and mineral nutrients. Which all emphasize on the effects of Seaweed products as plant growth promoters (Metting et al., 1990; Jeannin et al.,ers 1991). some research estimated that, the root growth stimulatory effect, has been more increased when seaweed extracts were applied at an early stage of growth in maize, also the responses were similar to those of auxin, which is an important root growth promoting hormone (Jeannin et al., 1991). the SWE applications on marigold and cabbage can reduce the damaged caused by transplant-shock in seedlings by increasing the root size and vigor (Ald worth and van Staden 1987), tomato (Crouch and van Staden 1992). application of SWE treatment in tomato seedlings could enhance both root/shoot-ratios and biomass accumulation through stimulating root growth (Crouch and van Staden 1992). wheat plants were treated with SWE exhibited an increase in root/shoot and dry mass ratio, which indicates that the components in the seaweed extracts has a considerable effect on root-development (Nelson and Van Staden 1986). The stimulatory activity of SWE was lost on ash, which suggesting the fact, that SWE effects are caused by the active principles within the extraction and organic compounds (Finnie and van Staden 1985). The root growth-promoting activity was observed when the seaweed extracts were applied either to the roots or as a foliar spray (Biddington and Dearman 1983; Finnie Andvan Staden 1985). The concentration of kelp-extract is a critical factor in its effectiveness. as Finnie and van Staden (1985), demonstrated for tomato plants by which a high concentration (1:100) inhibited the root growth but on the other hand, the stimulatory effects were found at a lower concentration (1:600). biostimulants in general are capable to effect root development by both improving lateral and root formation (Atzmon and van Staden 1994; Vernieri et al., 2005), and also by increasing total volume of the root system (Thompson 2004; Slavik 2005; Mancuso et al., 2006). an improvement in root system has been observed by application of endogenous auxins as well as other compounds in the extracts (Crouch et al., 1992). in general Seaweed extracts, improve the nutrients uptake through root growth (Crouch et al., 1990), improved root systems consequently enhanced water uptake and nutrient ef?ciency, thereby causing general enhancement in plant growth and vigor.

Table 2: commercial seaweed extracts used in agriculture and horticulture industries (J Plant Growth Regul (2009) 28:386_399)
Product name Seaweed name Company Application
Acadian Ascophyllum nodosum Acadian agritech Plant growth stimulant
Acid Buf Lithothamnium calcarum Chance and hunt limited Animal feed
Agri Gro Ultra Ascophyllum nodosum
Agri Gro Marketing Inc
Plant growth stimulant
Agro Kelp Macrocysist pyrifera Algae Bioderivados Marinos, S.A. de C.V
Plant growth stimulant
Alg-A-Mic Ascophyllum nodosum BioBizz worldwide N.V. Plant growth stimulant
Bio-Genesis High Tide Ascophyllum nodosum Green air products, inc Plant growth stimulant
Biovita Ascophyllum nodosum PI industries Ltd Plant growth stimulant
Emerals RMA Red marine algae Dolphin sea vegetable company Health product
Espoma Ascophyllum nodosum The Espoma company Plant growth stimulant
Fartum Unspecified Inversiones Patagonia S.A. Biofertilizer
Guarantee Ascophyllum nodosum Main Stream Organics Plant growth stimulant
Kelp Meal Ascophyllum nodosum Acadian Seaplants Ltd Plant growth stimulant
Kelpro Ecklonia maxima BASF Plant growth stimulant
Kelprosoil Ascophyllum nodosum Productos del pacifico, S.A. de C.V. Plant growth stimulant
Maxicrop Ascophyllum nodosum Maxicrop USA, Inc. Plant growth stimulant
Nitrozime Ascophyllum nodosum Hydrodynamics International Inc. Plant growth stimulant
Profert Durvillea antarctica BASF plant biostimulnat
Sea Winner Unspecified China ocean university product development Co., Ltd plant biostimulnat
Seanure Unspecified Farmura Ltd. plant growth stimulant
Seasol Durvillea potatorum Seasol international Pty Ltd plant growth stimulant
Soluble Seaweed Extract Ascophyllum nodosum Technaflora plant product, Ltd plant growth stimulant
Stimplex Ascophyllum nodosum Acadian Agritech plant growth stimulant
Synergy Ascophyllum nodosum Green air products, Inc. plant growth stimulant
Tasco Ascophyllum nodosum Acadian Agritech Animal feed

3.3) Hydrolyzed amino acids and proteins
Amino acids and small peptides both are absorbed by roots and leaves, then later they are translocated into the plant, as has been reported by (Watson and Fowden, 1975; Soldal and Nissen, 1978; Matsumiya and Kubo, 2011). However, the availability of amino acids and small peptides at root level can be strongly reduced by soil microbial activity (Wilson et al., 2013). Studies show, the measuring of amino acids consumption by soil microorganisms estimated about a half-life of 1–6 h for the soil amino acids (Moe et al., 2013), although a longer half-life for the amino acids (up to 32 d) has been also reported for forest soil (in a taiga) during incubation at 10?C (Jones and Kielland, 2012). Several studies indicated, that soil microorganisms use approximately, about 30–40% of amino acid-C for the respiration and also remaining amino acid-C for the production of cell biomass and cell maintenance (Jones et al., 2009). Moreover, about 30–40% of the N associated with the respired amino acid-C was consistently excreted into the soil as ammonium-N, which can be taken up by both plants and microbes or further be oxidized to produce nitrate. Foliar application of PHs can also increase the availability of amino acid and peptide for plant uptake, by reducing the competition between plants and microorganisms. Stiegler et al., (2013), measured the amount of leaf N-uptake on creeping bent grass up to 48% ,when labelled N (N15) was sprayed 8 hours before analysis with proline, glutamic acid and glycine, respectively. In another analysis, the amount of leaf amino acids uptake in peach fruit has been reported up to 26% after foliar application, along with 14, 10, 25 % for alanine, glutamic acid, glycine and lysine, respectively (Furuya and Umemiya, 2002). the rate of foliar N absorption has increased as the molecular weights of amino acids decreased. However, such relationship was not always found, and the foliar absorption rates of N from arginine and lysine were signi?cantly higher compare to the other amino acids which having the same molecular weights. Following the root and foliar absorption , the amino acids and peptides are later transported through cell to cell and over long distances through the plant vascular system (xylem and phloem) to support both plant metabolism and development. Several classes of integral membrane proteins are involved in amino acid and peptide transport through cell membranes in plants. For instance, members of the lysine–histidine-like transporters family, amino acid permease family and proline transporter family play a direct role in amino acid uptake through the roots (Tegeder, 2012). agricultural and horticultural crops are frequently cultivated under unfavorable environmental, chemical and soil conditions, such as: salinity, drought, thermal stress, adverse soil pH and nutrient deficiency. Which all these factors leading plants to stunted growth and loss in productivity. application of PHs and speci?c amino acids can induce plant defense responses and increase plant tolerance to a variety of abiotic and abiotic stress and also oxidative conditions (Chen and Murata, 2008; Kauffman et al., 2007; Apone et al., 2010; Ertani et al., 2013; Calvo et al., 2014). Small peptides and amino acids derivatives have been extensively studied because of their different effects inducing: plant defense responses, plant tolerance to abiotic and biotic stress, thus enhance plant tolerance to a wide range of stresses (Tuteja, 2007). The application, of exogenous glycine betaine, improves growth and tolerance to a wide variety of plants under environmental stress conditions (Cuin and Shabala, 2005; Park et al., 2006). Application of glutamate, ornithine, precursors of proline, can also increase tolerance to salt stress (Chang et al., 2010; DaRocha et al., 2012), while arginine, that plays a critical role in plant N storage and transport, has been shown to accumulate under abiotic and biotic stress (Lea et al., 2006). Also, the positive effects of proline against both abiotic stresses and the toxicity due to heavy metal, have been well demonstrated (Sharma and Dietz, 2009). However, significant accumulation of proline due to the increased synthesis and decreased degradation, under a wide range of stress conditions, such as: salt, drought and toxicity of heavy metal ions. on the other hand, a decrease in the level of accumulated proline in the hydrated plants due to both down-regulation of proline biosynthetic pathway enzymes and up-regulation of proline-degrading enzymes has been demonstrated in many plants after biostimulant addition (Lucini et al., 2015). Ertani et al., (2013), reported the capacity of a PH derived from alfalfa plant can positively stimulate short-term growth in the presence of sodium chloride in maize plants. PH treatment can signi?cantly stimulate the growth of sodium chloride treated plants, mostly due to its contents of plant growth regulators, such as: indoleacetic acid and triacontanol. Following table show the effects of protein hydrolysates (PHs) on horticultural crops.

Table 3: effects of protein hydrolysates (PHs) on horticultural crops

crop Type of PH PH application mode Experimental condition Effects References
Banana Chicken feather dried PH Root and foliar Field trail Early flowering, increased nutrient, chlorophyll content Gurav and Jadhav (2013)
Corn Alfalfa derived PH Root Hydroponic system growth chamber Increase crop salinity, tolerance, N Ertani et al. (2013|)
Grapevine PHs from soybean Foliar Field trail Up-regulated, defense genes encoding
pathogenesis-related proteins Lachhab et al. (2014)
Kiwifruit Animal derived PHs Foliar Pot trail Increase Shoot and root biomass Quartieri et al. (2002)
Tomato Plant derived PHs Root Soilless culture in growth chamber Increase rooting and shooting Colla et al. (2014)

3.4) Inorganic salts
According to the EBIC definition of inorganic salts, they are salts that doesn’t contain any carbon. They are important nutrients that can be divided into cations and anions. Na, K, Ca, Mg, Fe, Cu, Zn, Br are the main inorganic salts. Chemical elements those promoting plant growth may be essential and belong to particular taxa but on the other hand, they may not be needed by all plants (Pilon Smits et al., 2009). five main beneficial elements including Al, Co, Na, Se, Si, are presented in soils and in plants as different inorganic salts and as insoluble forms, like amorphous silica (SiO2. nH2O) in graminaceaous species, which is an inorganic soil and usually used in semiconductor circuits in order to isolate different conducting regions because of its mechanical resistance, very high dielectric strength and its selectivity to for chemical modification. These beneficial functions can be constitutive, such as strengthening the cell walls by depositing silica, expressed in defined environmental conditions (like pathogen attack for selenium and osmotic stress for sodium). the definition of beneficial elements is not only limited to their chemical natures but also, they must refer to the special contexts, where the positive effects on plant growth and stress response may be expressed. some important effects of beneficial elements in plants include: cell wall rigidification, osmoregulation, reduced transpiration by crystal deposits, thermal regulation via radiation reflection, enzyme activity by co-factors, plant nutrition via interactions with other elements during uptake and mobility, antioxidant protection, interactions with symbionts, pathogen and herbivore responses, protection against toxicity of heavy metals, both plant hormone synthesis and signaling (Pilon Smits et al., 2009). Atonik is a biostimulant which is composed of three phenolic compounds: sodium para-nitrophenolate (PNP), sodium ortho-nitrophenolate (ONP), sodium 5-nitroguaiacolate (5NG) and water. it has been used successfully for many years in the cultivation of many important crops worldwide and has positive effects on yield and productivity (Djanaguiraman et al., 2004; Bynum et al., 2007; Grajkowski and Ochmian, 2007; Budzy?ski et al., 2008; Kositorna and Smoli?ski, 2008; Kozak et al., 2008; Malarz et al., 2008; Michalski et al., 2008; Sawicka and Mikos Bielak, 2008). studies show that Atonik positively affects a various range of processes which control plant growth, such as: plant development and productivity. as biostimulant treated plants are more advanced in growth and development compare to untreated plants (Djanaguiraman et al., 2005; Gulluoglu et al., 2006; Kozak et al., 2008; Borowskiet al., 2009), and also transpiration rate, without a reduction in relative water-content (Wróbel and Wo?niak, 2008; Borowski et al., 2009). It has been demonstrated that biostimulants play an essential protective role against of a various abiotic and biotic stress, including: extreme temperature conditions, drought and water scarcity, heavy metals toxicity and salinity (Gulluoglu et al., 2006; Gawro?ska et al., 2008; Borowski et al., 2009). beneficial inorganic salts such as: phosphites, chlorides, phosphates, carbonates and silicates have been used to control fungus and demonstrated high effective (Deliopoulos et al., 2010). Although, the modes of action of many inorganic salts are not yet fully established, these compounds can positively influence plant cells osmotic pressure, pH, hormone-signaling and enzymes involved in stress response like ( peroxidases). their function as plant biostimulant can positively effect plant growth , nutrition uptake efficiency and abiotic and biotic stress tolerance. Hence, their distinct fungicidal action from their fertilizer functions, as sources of nutrients deserves more attention.

3.5) Microorganisms
There are many beneficial microorganisms with the capacity to stimulate and solubilize plant essential macro and micro nutrients, trough enhancement of nutrients uptake. These microorganisms are able to first mobilize and later uptake the nutrients at different plant levels like leaf or root. Nitrogen as one of the most essential nutrients for the plant, can be stimulate and uptake by such microorganism those are in association with plant. in fact plant through this symbiosis with microorganisms is able to uptake more nitrogen at root level from rhizosphere, using biostimulants products that containing such microorganisms that are able to enhance plant beneficial microbial association can improve the nutrient uptake efficiency and consequently can increase yield and productivity. In this symbiosis nitrogen-fixing bacteria or mycorrhizae with microbial biostimulants (MBS) are able to induce root and increase crop root system in order to improve nitrogen uptake. Bacteria fungi interacts with plant-roots from mutualistic symbioses (symbiosis means, when both organisms live in direct contact with each other and establish mutually beneficial relationships), (Behie et al., 2014). Microbial biostimulants such as arbuscular mycorrhizal fungi (AMF) and Trichoderma spp. are also considered to be as promising tools which are able to overcome the limitations due to salinity and alkalinity conditions and improve crop growth and productivity. many AMF and Trichoderma spp. strains are able to enhance vegetable tolerance to abiotic and biotic stresses through increasing nutrients uptake and creating more effective root area (for AMF) and on the other side a better solubilization of micronutrients (for Trichoderma spp.) throughout the production of small peptides, volatiles and also metabolisms of important hormones such as: indole-3 acetic acid, auxin analogs (Giovannetti et al., 2001; López Bucio et al., 2015). Furthermore, the microbial production of the auxin indole-3 acetic acid (IAA) has been extensively reported after introducing them to the plant (Ali et al., 2009). IAA have an important function in plant both growth and development via influencing in many plant functions, such as: cell wall elongation, cell division, apical dominance, root initiation and elongation, vascular tissue differentiation, biosynthesis of ethylene, moderating the environmental stress responses, and also in the expression of specific plant genes. many studies show that the use of microorganisms that produce IAA have a positive effect on IAA production, root development and morphology (Döbbelaere et al., 1999; Aloni et al., 2006). Plants and fungi have been co-evolved together since the origin of terrestrial plants. here using the concept of mutualism is useful to describe the extended range of relationships that are developed over the evolutionary times (Bonfante and Genre, 2010; Johnson and Graham, 2013). Experiments on several vegetable crops, based on co-inoculation of AMF with Trichoderma atroviride at transplanting, have shown increased plant growth under non-stress conditions (Colla et al., 2015). recent review of López Bucio et al., (2015), demonstrated plant growth enhancement by Trichoderma through a multilevel root–shoot communication. The phyto-stimulation effect of Trichoderma applications has reported several direct and indirect effects on plant growth, via releasing substances with auxin activity like (indole-3-acetaldehyde, indole-3carboxaldehyde, indole-3-ethanol). small peptides as well as volatile organic compounds, are able to improve root system (positive effects on total root initiation, branching, length, density) along with assimilation and solubilization of macro and micro nutrients like (P, Fe, Mn, Zn) thus, increasing plant growth and consequently crop productivity (Harman et al., 2000; Howell et al., 2003; Harman et al., 2004; Contreras-Cornejo et al., 2009, 2010; Hermosa et al., 2011; Lorito et al., 2012; Lorito et al., 2015; Rouphael et al., 2017). endophytic fungi including Trichoderma spp. interacts with other members of the microbial community in the plant rhizosphere. therefore, it is essential to assess and manage the ecological impacts of different soil microbial-based biostimulant on soil ecosystem and particularly on qualitative and quantitative microbial populations in rhizosphere (Mar Vázquez et al., 2000; Lace et al., 2015).There is an increasing interest for the use of mycorrhiza due to their positive effects on sustainability in agricultural and horticultural field. Therefore, caused widely accepted benefits through increased nutrition efficiency for (macronutrients, especially P, and micronutrients), water uptake, tolerance to biotic and abiotic stress, plant photosynthesis and production (Gianinazzi et al., 2010; Hamel et al., 2007; Harrier et al., 2004; Siddiqui et al., 2008; van der Heijden et al., 2004). Recent resaerch also demonstrate the existence of hyphal networks which interconnect not only with fungal-plant partners but also, individual plant-plant community. This can have significant ecological and agricultural implications because there are multiple evidences which show fungal conduits allowing for interplant signaling (Johnson and Gilbert, 2015; Simard et al., 2012). another research show AMF form tripartite, associations with plants and rhizobacteria.wich are relevant in practical field situations (Siddiqui et al., 2008). In order to increase the benefits from the plant mycorrhizal associations, both crop management practices and plant cultivars should be adapted within interaction of plant and microorganisms (Plenchette et al., 2005; Hamel and Plenchette, 2007; Gianinazzi et al., 2010; Sheng et al., 2011). as metagenomics are an interesting tool to monitor and study microbial associations in the rhizosphere should be more focused in future research in order to have a highly effective symbiosis within plant and beneficial microorganisms.

Conclusion
Plant biostimulants include diverse substances and microorganisms that enhance the plant growth. The definition and concepts od plant biostimulants is still evolving. Among different definitions one of the most common definition has been reported by EBIC as ” substances or/and microorganisms whose function when applied to the plant or to the rhizosphere, is to stimulate natural process to enhance benefitial nutrient uptake. all the represented examples in this review show that many scientific studies have demonstrated the potential of various categories of biostimulants to improve the crop production and resistance to biotic and abiotic stresses such as drought and soil salinity. Biostimulants such as humic substances has positive effects on macro and micro nutrients uptake and obviously stimulant root growth and plant nutrition uptake which it enhances the plant growth and yield hydrolyzed amino acids and protein also stimulate root growth and plant metabolism which all of them positively increase crops productivity. Brown algae extracton increase the AM fungi growth and also is used as fertilizer to promotes plant growth. Biostimulants based on inorganic salt increase the crop resistance to biotic and abiotic a stress. But still further research is required to explain and clarify the mood of action of such biostimulants. Microbial biostimulants have a great effect on beneficial microorganism which are responsible for plant growth through increasing root nutrition uptake mechanism. effects of humic substances will help to clarify how specific humic substances elicit plant growth, nutrient uptake, and abiotic/biotic stress-tolerance responses. an obvious area for future research and development of biostimulants is the combination of some of the various categories presented in this review. For example, combinations of microbial inoculants with seaweed extracts or humic substances could theoretically deliver more reproducible benefits to crop production. effective regulation as well as clear definition of biostimulants could stimulate both the market trade and application of biostimulant products

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