Agricultural uses of plant biostimulants

Enhanced plant growth and yield by microbial inoculants has been linked in some cases to enhanced nutrient uptake and improved nutrient status of the plant. For example, (Wu et al. 2005) reported that plant growth promotion following inoculation of maize (Zea mays) with strains of Bacillus megaterium and Bacillus muciaraglaginous together with AMF was associated with improved nutritional assimilation of plant total N, P, and K. Application of PGPR resulted in a significant increase in N, P, and K uptake as well as root and shoot dry weight in cotton (Gossypium hirsutum) (Egamberdiyeva and Höflich 2004) and wheat (Triticum aestivum) (Shaharoona et al. 2008). (Adesemoye et al. 2010) reported that a three-strain mixture of Bacillus spp. PGPR promoted growth of tomato and increased plant uptake of ¹⁵ N-depleted fertilizer. In a three-year field study with maize, PGPR, AMF and a combination of the two increased yield and enhanced the total nutrient content of grain per plot (Adesemoye et al. 2008).

Several mechanisms have been reported for how specific microbial inoculants stimulate plant growth and nutrient uptake, including (i) asymbiotic nitrogen fixation (Boddey and Dobereiner 1995; Döbereiner 1997), (ii) solubilization of nutrients (de Freitas et al. 1997), (iii) sequestering of iron by production of siderophores, and (iv) production of volatile organic compounds (VOCs).

Bacteria with the capacity to fix atmospheric nitrogen (N₂) asymbiotically belong to many different genera, including Azoarcus spp. (Hurek et al., 1994), Beijerinckia spp. (Baldani et al., 1997), Klebsiella pneumoniae (Riggs et al., 2001), Pantoea agglomerans (Riggs et al., 2001), Azotobacter spp. (Mrkovacki and Milic 2001), Azospirillum spp. (Garcia de Salamone et al. 1996), Bacillus polymyxa (Omar et al. 1996), Burkholderia spp. (Baldani et al. 2001), Herbaspirillum spp. (Pimentel et al. 1991), and Gluconoacebacter diazotrophicus (Boddey et al. 2001). Among all these genera, Azospirillum is the most studied (Bashan et al. 2004; Bashan and de-Bashan 2010; Boddey and Dobereiner 1995; Hartmann and Bashan 2009). Azospirillum spp. can be found in close association with plant roots, including inside root tissues. The capacity of Azospirillum spp. to fix atmospheric nitrogen has been widely reported in different crops. (Malik et al. 2002), using ¹⁵ N tracer techniques, found that Azospirillum brasilense and Azospirillum lipoferum contributed between 7–12 % of the total nitrogen content of wheat. In contrast, with sugarcane (Saccharum officinarum), 60–80 % of total plant nitrogen came from nitrogen fixation by Azospirillum diazotrophicus (Boddey et al. 1991). Significant increases in nitrogen content by inoculation with Azospirillum spp. have been reported in many crops, including cotton, wheat, sugarcane, and corn (Garcia de Salamone et al. 1996; Fayez and Daw 1987; (Saubidet et al. 2000). It should be noted however that nitrogen fixation does not account for all of the observed growth promotion noted with the use of Azospirillum species.

Some microorganisms increase the availability of selected soil nutrients via enhanced solubilization of the nutrients, thereby allowing plants to take up nutrients in a more efficient way. Low availability of absorbable forms of P in soil represents an important problem for agricultural systems. Even when P fertilizers are added to soils, they may not be absorbed by plants because P is readily bound to soil particles, thereby being unavailable for plants (Gyaneshwar et al. 2002). Different bacterial genera have been identified as phosphorus solubilizers (de Freitas et al. 1997), including Pseudomonas spp. (Malboobi et al. 2009; Park et al. 2009), Bacillus spp. (Arkhipova et al. 2005; de Freitas et al. 1997; Sahin et al. 2004; Zaidi et al. 2006), Burkholderia spp. (Tao et al. 2008), Streptomyces spp. (Chang and Yang 2009), Achromobacter spp. (Ma et al. 2009), Microccocus spp. (Dastager et al. 2010), Flavobacterium spp. (Kannapiran and Ramkuma 2011), Erwinia spp. (Rodriguez et al. 2001), and Azospirillum spp. (Rodriguez et al. 2004). The two most reported mechanisms by which microorganisms solubilize P are production of organic acids (Goldstein 1995) and production of phosphatases (to release organic-P) (Rodriguez et al. 2006). Organic acids transform insoluble phosphate forms to soluble forms through their hydroxyl and carboxyl groups. These groups chelate the cations bound to phosphate, thereby converting it to soluble forms (Kpomblekou and Tabatabai 1994). Organic acids are also responsible for decreasing the pH of the surrounding soil, thereby releasing phosphate ions (Rodriguez and Fraga 1999). Many different types of organic acids have been linked to phosphate solubilization. The type of organic acid released by a microorganism depends on the species of the microorganism. For example, Bacillus licheniformis and Bacillus amyloliquefaciens species were found to produce mixtures of lactic, isovaleric, isobutyric, and acetic acids. In contrast, gluconic acid seems to be the most frequent organic acid produced by bacteria such as Azospirillum spp. (Rodríguez et al. 2004), Pseudomonas spp., Erwinia herbicola, Pseudomonas cepacia, and Burkholderia cepacia (Rodriguez and Fraga 1999). Organic phosphorus, which is about 30-80 % of soil phosphorus, plays an important role in the phosphorus cycle of agricultural soils (Tarafdar and Gharu 2006). The predominant forms of organic phosphorus are phytates (inositol hexa- and penta-phosphates), which constitute up to 60 % of soil organic phosphorus. However, phytate cannot be taken up by the plant. It must first be dephosphorylated by phosphatases (phytases and phosphatases), (Gyaneshwar et al. 2002; Singh and Satyanarayana 2011). Acid phosphatases and phytases produced by rhizosphere microorganisms are involved in organic- P solubilization. Gram negative bacteria such as Pseudomonas, Burkholderia, Enterobacter, Citrobacter, and Serratia have been reported to produce acid phosphatases (Gügi et al. 1991; Thaller et al. 1995). Organic phosphate in soils is predominantly present as phytate, and production of the enzyme phytase by inoculants including Pseudomonas spp. (Richardson et al. 2009) and Bacillus spp. (Idriss et al. 2002) has been shown to contribute to plant growth promotion. Arbuscular mycorrhizal fungi (AMF) are widespread in the plant kingdom and contribute significantly to plant P nutrition and growth in natural ecosystems (Smith et al. 2011). AMF colonize most agricultural species and have an important role in the P nutrition of many farming systems worldwide, especially in soils with low available P (Thompson 1987). The hyphae of AMF colonizing plants have some of the same functions as root hairs with respect to P acquisition (Jakobsen et al. 2005) and have an especially large influence on P uptake in varieties with short root hairs (Schweiger et al. 1995).

In addition to P, Potassium (K) is another essential plant nutrient that can be solubilized by soil microorganisms and microbial inoculants. K-solubilizing microorganisms solubilize rock K minerals, such as micas, illite, and orthoclases, by excreting organic acids that directly dissolve rock K or chelate silicon ions to solubilize K (Friedrich et al. 1991; Parmar and Sindhu 2013). Application of Bacillus mucilaginosus and B. megaterium with rock K materials resulted in significant increases in available K in the soil and K uptake by eggplant roots and shoots (Han and Lee 2005). Sheng and He (2006) suggested that enhanced K uptake by inoculation with Bacillus edaphicus was due to the production of organic acids (citric, oxalic, tartaric, succinic, and α-ketogluconic) that directly dissolve rock K or chelate silicon ions.

Microbial inoculants have been reported to enhance uptake of other macro and micronutrients, and the mechanisms involved are still being elucidated. Increases in root biomass, root surface area, or root hairs could be indirect mechanisms that enhance the uptake of nutrients. A wide variety of microorganisms such as Pseudomonas spp. and Acinetobacter spp., Azospirillum ssp., Bacillus spp., and AMF have been reported to increase uptake of Zn (Kohler et al. 2008; Yazdani and Pirdashti 2011), Cu, Mn (Liu et al. 2000), Ca, Mg (Giri and Mukerji 2004; Khan 2005), and S (Banerjee et al. 2006). (Kohler et al. 2008) reported that inoculation with a combination of two different microorganisms, Pseudomonas mendocina and the AMF Glomus intraradices, significantly increased uptake of Fe, Ca, and manganese (Mn) in lettuce (Lactuca sativa).

Bacteria have evolved Fe (III) transport systems that enable them to grow in environments containing very low concentrations of iron. Fe-binding chelators, known as siderophores, bind or capture free Fe (III) and transport iron into the cell (Neilands and Nakamura 1991). Many different types of siderophores have been studied and identified in different bacterial genera including Bacillus (Park et al. 2005; Temirov et al. 2003; Wilson et al. 2006). The relation between siderophore production, plant growth promotion, and uptake of Fe has also been well documented for a variety of plants and microorganisms. Significant increases in tomato and rice growth parameters were reported using a siderophore-producing strain of Streptomyces (Rungin et al. 2012; Verma et al. 2011). (Sharma et al. 2013) reported that inoculation with a Pseudomonas putida strain increased iron uptake of rice due to siderophore production and increased the iron content in rice grains which enhanced the nutritional value of the rice.

Volatile organic compounds (VOCs) are low molecular-weight compounds, such as alcohols, aldehydes, ketones, and hydrocarbons, that have high enough vapor pressures under normal conditions to vaporize and enter the atmosphere (Ortíz-Castro et al. 2009). VOCs were initially described as contributing to some biocontrol activities of select rhizosphere microorganisms (Fernando et al. 2005; Vespermann et al. 2007). Later, the role of VOCs in plant growth promotion was reported. (Ryu et al. 2003) reported that the VOCs of some PGPR strains contained 2, 3 butanediol and acetoin that were related to growth promotion of Arabidopsis thaliana. In the same way, (Zhuang et al. 2007) found that VOCs produced by one of the same PGPR strains used by (Ryu et al. 2003) regulated genes involved in cell wall modifications and auxin production in A. thaliana.

The production of plant growth-regulators by many bacterial species and their effect on plant growth was reported more than 30 years ago (Barea et al. 1976). Microbial inoculants, such as PGPR, can alter root architecture and promote plant development via the production or degradation of the major groups of plant hormones (Bhattacharyya and Jha 2012; Dodd et al. 2010; Idris et al. 2007). Microbial inoculants can also modify plant hormone status (Belimov et al. 2009). Phytohormones, like auxins, cytokinins (CKs), gibberellins (GAs), and ethylene (ET), can be synthesized by beneficial microorganisms. These plant hormones regulate multiple physiological processes, including root initiation, root elongation, and root hair formation. They typically operate in complex networks involving cross-talk and feedback (Dodd et al. 2010; Zahir et al. 2003).

Microbial production of the auxin indole-3-acetic acid (IAA) has been extensively reported (Ali et al. 2009). IAA plays a critical role in plant growth and development by influencing many plant functions, including promotion of cell elongation and cell division, apical dominance, root initiation, differentiation of vascular tissue, ethylene biosynthesis, mediation of tropic responses, and the expression of specific plant genes Most studies using microorganisms that produce IAA have reported a link between IAA production and root development and morphology (Aloni et al. 2006; Döbbelaere et al. 1999a). Many different bacterial species can produce IAA through various mechanisms. For example, (Spaepen et al. 2008) found that IAA production by A. brasilense was regulated by root exudates. When root exudates decrease and become a limiting factor for bacterial growth, bacterial production of IAA increases, thereby triggering lateral root and root hair formation. A. brasilense Sp245, which produces IAA, increased leaf length and shoot dry weight of spring wheat compared to a non-inoculated control (Spaepen et al. 2008). Aeromonas spp., A. brasilense, and Comamonas acidovorans are among the many IAA species that promote plant growth in rice (Oryza sativa) (Mehnaz et al. 2001), wheat (Kaushik et al. 2000), and lettuce (Lactuca sativa) (Barazani and Friedman 1999).

Cytokinins are a broad group of plant growth regulators that share the capacity to induce plant cell division in bioassays. After biosynthesis in root tips and developing seeds, cytokinins are transported to the shoot via the xylem where they regulate several processes, such as cell division, leaf expansion, and senescence (Spaepen et al. 2009). The capacity of microorganisms to produce cytokinins as one mechanism of plant growth promotion was confirmed using bacterial mutants (García de Salamone et al. 2001). Inoculation with Bacillus subtilis caused increased cytokinin content in shoots and roots of lettuce plants and an increase in plant shoot and root weight of approximately 30 % (Arkhipova et al. 2005). Application of the cytokinin-producing bacterium B. licheniformis resulted in enhanced cell division, higher chlorophyll content, and increased fresh weight and cotyledon size of cucumber (Cucumis sativus) plants (Hussain and Hasnain 2009). The importance of cytokinin-producing bacteria under drought conditions is a topic of renewed interest (Arkhipova et al. 2007).

Gibberellins (GA) are mainly involved in regulating plant cell division and elongation and hence, they influence almost all stages of plant growth, including seed germination, stem and leaf growth, floral induction, and fruit growth (Spaepen et al. 2009). As with auxins and cytokinins, GAs mainly act in combination with other phytohormones. Frankenberger and Arshad (1995) reported that bacteria are able to release GA into the rhizosphere. Several Azospirillum species produce different GAs that are responsible for plant growth promotion that occurs upon inoculation onto plants (Piccoli et al. 1997). Apart from Azospirillum spp., production of gibberellin-like substances has also been claimed in numerous bacterial genera, including Acetobacter diazotrophicus, Herbaspirillum seropedicae (Bastián et al. 1998), and Bacillus spp. (Gutiérrez-Mañero et al. 2001). The exact mechanism of plant growth promotion by GAs remains unclear, although GA-producing PGPR can act by promoting root growth, more particularly by increasing root hair density in root zones involved in nutrient and water uptake, as shown for A. lipoferum inoculation of maize seedlings (Fulchieri et al. 1993). Gibberellin production by Azospirillum spp. and Bacillus spp. has been implicated in the increased ¹⁵ N uptake observed after inoculation of wheat roots (Kucey 1988).

The phytohormone ethylene (ET) is well known as a ripening hormone, but it also has a role in other processes, such as seed germination, cell expansion, and leaf and flower senescence. In addition, ET is produced under conditions of both abiotic and biotic stress and is therefore known as the stress hormone (Spaepen et al. 2009). High ET concentrations have an inhibitory effect on root growth, resulting in reduced overall plant growth (Abeles and Wydoski 1987). ET biosynthesis is highly regulated mostly at the key step catalyzed by the enzyme ACC synthase, and hence, regulation of ACC synthase can lead to regulation of ET (Chae and Kieber 2005). Some PGPR express the enzyme l-aminocyclopropane-1-carboxylatedeaminase (ACC deaminase), which can degrade ACC to α-ketobutyrate and ammonia. A wide range of soil microorganisms, including the fungus Penicillium citrinum (Honma 1993) produce the enzyme ACC deaminase that degrades ACC to α-ketobutyrate and ammonia (Blaha et al. 2006; Chinnadurai et al. 2009). Inoculation of diverse plant species with bacteria expressing ACC deaminase activity stimulates plant growth (Glick et al. 2007; Saleem et al. 2007). (Glick et al. 1998) proposed a model to explain the role of bacterial ACC deaminase in plant growth promotion. As ACC is exuded by plant roots, it is hydrolyzed by bacteria that produce ACC deaminase. ACC concentration outside the roots decreases and more ACC is exuded by the plant. As a result, ACC levels in the plant are lowered and the ET content is reduced (Glick et al. 1998). Once ET is reduced there is an increase in plant biomass (Contesto et al. 2008; Saleem et al. 2007). Bacteria with ACC deaminase activity have been extensively used for alleviating diverse stresses in plants. By reducing the stress hormone ET, these bacteria are able to protect plants from the growth inhibition caused by ET under numerous stress conditions, such as flooding, toxic compounds (both organic compounds and heavy metals), high salt concentrations, drought, and pathogenic attack (Glick et al. 2007; Saleem et al. 2007).

Yield losses due to drought and salinity stress are increasing mainly due to climate change and intensive agriculture that leads to soil degradation. It has recently been discovered that some microbial inoculants known to have a positive effect on plant development can also help plants overcome or tolerate abiotic stress conditions, thereby reducing potential yield losses. Increases in soil salinity have become a serious problem for agriculture crops in the last years. Application of some bacteria, such as Rhizobium spp. and Azospirillum spp., increased plant tolerance to salinity conditions (Cordovilla et al. 1999; Hamaoui et al. 2001). Applications of a strain of A. lipoferum to wheat plants reduced the negative effect of saline conditions (Bacilio et al. 2004). Drought stress limits growth and production of crop plants, particularly in arid and semiarid areas (Kramer and Boyer 1997). Greenhouse studies revealed that inoculation of maize seedlings with A. brasilense resulted in the mitigation of many negative effects of drought stress (Casanovas et al. 2002). Among the specific effects were increased water content, reduction in the decrease of water potential, increased foliar area and total plant biomass, and increased accumulation of proline. Proline is an osmoprotectant that regulates and helps protect the plant from drought stress. In other experiments, inoculation of wheat with A. brasilense in non-irrigated soil resulted in a yield increase of 12 %. In addition, grains harvested from Azospirillum- inoculated plants under drought conditions had increased Mg, K, and Ca contents compared to non-inoculated plants (Creus et al. 2004). Other bacterial species, such as Pseudomonas spp. and Bacillus spp., have also been reported to stimulate plant growth under dry conditions. Inoculation under drought conditions increased shoot and root biomass and water content (Marulanda et al. 2009). Increased root elongation and root biomass are common in drought-tolerant species (Sharp et al. 2004). Modifications to root development allow better water and nutrient uptake due to greater root exploration of the soil (Padilla and Pugnaire 2007).

With tolerance to drought, bacterial production of hormone-like compounds has been shown to play a key role in ameliorating effects of drought (Döbbelaere et al. 1999). For example, inoculation of white clover (Trifolium repens) with the IAA-producing strains of P. putida and B. megaterium, resulted in increased shoot and root biomass and water content under drought conditions (Marulanda et al. 2009). In addition to IAA, abscisic acid (ABA) is another plant hormone that plays an important role in the whole plant response to drought stress. (Cohen et al. 2008) reported that plants inoculated with the PGPB A. brasilense Sp245 showed more ABA content than non-inoculated plants, and this resulted in enhanced plant drought tolerance. Degradation of the stress hormone ethylene by ACC-deaminase also resulted in greater drought tolerance. Inoculation with Pseudomonas spp. containing ACC-deaminase partially eliminated the effects of drought stress on growth, yield, and ripening of pea (Pisum sativum) (Arshad et al. 2008). One strain of Achromobacter piechaudii ARV8, isolated from an arid desert environment, significantly increased the fresh and dry weights of both tomato and pepper seedlings exposed to water stress. In addition, the bacterium reduced the production of ethylene by tomato seedlings, following water stress (Mayak et al. 2004).

AMF are known to enhance plant establishment and drought tolerance (Querejeta et al. 2003) by various mechanisms including (a) improved water uptake, by which AMF effectively extend plant roots making the uptake of water much more efficient; (b) better mineral nutrition, especially phosphorus, as a consequence of effectively extending roots; (c) alterations in root architecture; (d) modification of some physiological and enzymatic activities, especially those involved in plant antioxidative responses; and (e) induction of the plant hormone ABA, which can play an important role in mediating some plant responses to different stresses including drought (Gamalero et al. 2009). Many plant species, including corn, soybean (Glycine max), wheat, onion (Allium cepa), and lettuce, have shown enhanced drought tolerance as a consequence of mycorrhizal symbioses (Augé 2001; Brundrett 1991a, 1991b). Under these conditions, AMF enhanced root surface area and promoted dense root growth, resulting in improved drought tolerance. Moreover, plants colonized by AMF were able to maintain higher water use efficiency, and growth was increased at a faster rate when irrigation was restored. Such adjustment of osmotic potential is one of the most important factors for plant survival under drought conditions. In addition, AMF may affect plant water potential by modification of soil structure. Hyphae of AMF can increase soil structure by binding soil particles and producing glomalin, an insoluble glue-like substance (Augé 2001). AMF may also play a role in the protection of roots from heavy metal toxicity by mediating interactions between metals and plant roots (Leyval et al. 1997).

Co-inoculation of AMF and PGPR is also a promising strategy to increase plant drought tolerance. It was reported that co-inoculation of the AMF Glomus mosseae and G. intraradices and PGPR Bacillus spp. on lettuce increased plant growth, photosynthetic rate, water use efficiency, and stomatal conductance after drought stress. The effect of the co-inoculation was better than inoculation with only AMF or Bacillus spp. Furthermore, Bacillus spp. inoculation also improved AMF colonization and growth (Vivas et al. 2003).

Fungal-based microbial inoculants have also been reported to induce plant drought tolerance. Previously reported fungi include many genera, such as Neotyphodium spp., Cuvularia spp., Colletotrichum spp., Fusarium spp., Alternaria spp., and Trichoderma spp. which were reported to elicit drought tolerance in tomato (Solanum lycopersicum), pepper (Capsicum annuum), ryegrass, (Lolium perenne) tall fescue (Festuca arundinacea), wheat, and barley (Hordeum vulgare) (Bae et al. 2009; Singh et al. 2011). Interestingly, in addition to exhibiting increased drought tolerance, the inoculated plants expressed a range of adaptations to biotic stress and other abiotic stresses including mineral imbalance and salinity (Kim et al. 2012).

Application of microbial inoculants to mitigate salinity stress has been reported. Inoculation with some PGPR, including strains of B. subtilis, Bacillus atrophaeus, Bacillus spharicus, and Staphylococcus kloosii, increased chlorophyll content, nutrient content, and yield of strawberry (Fragaria ananassa) plants growing in high saline soils (Karlidag et al. 2013). In a similar way, under salinity stress inoculation of canola with A. lipoferum resulted in significant increases in shoot and root weights. Azospirillum lipoferum also increased antioxidant levels in the plants. It was postulated that positive effects were due to bacterial production of ACC-deaminase, which reduced ethylene levels caused by the salinity condition (Baniaghil et al. 2013).

AMF have also been shown to increase tolerance to salt stress. Recent reviews have identified numerous physiological mechanisms by which AMF improve plant salinity tolerance (Evelin et al., 2009; Porcel et al. 2012). Improvement in salt tolerance of maize plants inoculated with AMF (G. mosseae) was related to higher accumulation of soluble sugars in the roots, independent of plant nutritional (P) status (Feng et al. 2002). This effect could be responsible for improved plant water status, chlorophyll concentration, and photosynthetic capacity by increasing photochemical efficiency (Sheng et al. 2008). In a manner similar to drought conditions, plant growth under saline stress may be regulated via changes in phytohormone concentrations (Pérez-Alfocea et al. 2010). Hence the capacity of microbial inoculants to induce changes in phytohormones could be one of the mechanisms involved in enhancing plant tolerance to salinity (Dodd and Pérez-Alfocea 2012; Dodd et al. 2010).

In recent years, research has shown that microbial inoculants can also play an indirect role on soil remediation and soil fertility. Bioremediation is recognized as an important tool to restore contaminated sites, reforest eroded areas, and restore degraded ecosystems. The plant is the major component of the phytoremediation processes, and application of microbial inoculants in bioremediation of contaminated soil and reforestation of degraded lands is a promising research area (de-Bashan et al. 2010a; 2010b; 2012; Grandlic et al. 2008; Kuiper et al. 2004; Ma et al. 2011). Application of Kluyvera ascorbata increased several plant parameters and promoted plant growth of tomato, canola (Brassica napus), and Indian mustard (Brassica juncea) grown in soils containing high concentrations of zinc and nickel (Burd et al. 2000). In another study, Pseudomonas spp. increased growth of canola and common reed (Phragmites australis) in the presence of copper or polycyclicaromatic hydrocarbons (Reed and Glick 2005; Reed et al. 2005). The properties of plants used for phytoremediation could be improved by microbial inoculants, but it is important to choose microorganisms that can survive and succeed when used in phytoremediation practices (De-Bashan et al. 2012).

The examples of microbial inoculants discussed above consist of one or a small number of microbial strains which are fermented or grown separately and are then removed from the fermentation medium, concentrated, and then formulated with some carrier into the final product form. An additional category of inoculants consists of mixed culture fermentation to produce a complex microbial mixture together with fermentation metabolites. On example of this category of inoculant-based biostimulants is the product referred to as EM, for “effective microorganisms.” As described by Hu and Qi (2013), the inoculant called EM was first described in non-refereed presentations in the early 1900s. The contents of EM were subsequently summarized (Hu and Qi 2013) as containing “about 80 species of microorganisms, which included photosynthetic bacteria, lactic acid bacteria, yeasts, actinomycetes, and fermenting fungi like Aspergillus and Penicillium.” The microbes are fermented together in the presence of organic wastes and molasses (Khaliq et al. 2006). Field experiments with cotton showed that EM increased the efficiency of mineral and organic fertilizers, and the combination of EM with organic matter increased yields 23 % compared to treatment with organic matter alone (Khaliq et al. 2006). In contrast, (Mayer et al. 2010) found that EM did not increase yield or soil quality in trials conducted over 4 years in Central Europe. Results of a 13 years trial on wheat in China (Hu and Qi, 2013) found that long-term applications of EM significantly increased yield of wheat, grain nutrition, and biomass of straw. Another example of a biostimulant based on a mixed culture fermentation resulting in microorganisms and their metabolites are variations of the product SoilBuilder. Treatment of plants with SoilBuilder and a different formulation of the same mixed culture fermentation product significantly increased weight of squash (Cucurbita pepo) seedlings with 0, 50, and 100 mM added NaCl (Yildrim 2007). In the same study, treatment with the biostimulants significantly increased weights of transplanted squash plants at the same NaCl concentrations, and at 50 mM NaCl, there was significantly less Na⁺ in leaves of treated plants. In a study aimed at determining effects of inoculants on emissions of nitrous oxide (N₂O) in agriculture, application of SoilBuilder significantly decreased the cumulative emissions of nitrous oxide (N₂O) by 80 % in soils fertilized with urea-ammonium nitrate (Calvo et al. 2013). Hence, specific inoculants based on mixed culture fermentation have been shown to have diverse beneficial effects on plants.

Overall, microbial inoculants are gaining more attention due to their potential not only in the agriculture industry but also as tools to solve some environmental problems. In the last years there has been more focus on this potential application as a tool to enhance nutrient uptake in integrated crop management practices. Additional work is needed in order to provide consistent results between laboratory and greenhouse studies and appropriate field applications.

The stimulatory effects of humic acids have been shown in some studies to result in enhanced tolerance to salinity stress (Table 1). In studies with bean plants (Aydin et al. 2012), all control plants grown with eight salt sources were killed at the highest dose of 120 mM, while no plant dies following treatment with various humic acids at 0.05 and 0.1 % w/w, with seven of the salt sources including NaCl.. In the same study, treatments with humic acids in saline soils were associated with reduced soil electrical conductivity and reduced leakage of proline from plants, effects which were related to plant tolerance to the saline conditions. Similar results were reported in a study on maize (Mohamed 2012) where humic acids protected against plant death resulting from three forms of calcium salts. Co-applications of humic acids and phosphorus reduced saline stress and increased yields of pepper growing in a moderate salt dose of 8 mM NaCl (Cimrin et al. 2010), and the Na concentration in shoots and roots decreased with increasing doses of humic acid. In a greenhouse test on chrysanthemum (Chrysanthemum indicum) grown two seasons in field soils, applications of humic acids ameliorated the negative effects of salinity alone on vegetative growth and flowering (Mazhar et al. 2012). In a field study with pistachio (Pistacia vera), humic acid ameliorated negative effects on plant growth resulting from irrigation with low to moderate rates of NaCl, and this effect was related to a reduction in proline accumulation and decreased levels of abscisic acid (ABA) in plants treated with humic acids compared to controls (Moghaddam and Soleimani 2012).

Investigating the potential of humic acids to aid plants in drought tolerance has begun. Humic acid extracted from vermicompost was evaluated for effects on growth and physiology of rice seedlings grown under water deficient conditions (García et al. 2012). Seedling growth promotion induced by humic acid began at 10 days after germination, which coincided with onset of water stress symptoms. Seedlings treated with humic acid had increased shoot and root dry weights compared to controls under the water-stress conditions. At 25 days after germination under water stress, the levels of chlorophyll, carotenoids, protein, and carbohydrates were greater in treated plants than in controls, indicating that the photosynthetic capacity of water-stressed plants was increased by humic acid. In the same study, effects on plant physiology were evaluated, and these are discussed in the following section.

Humic substances exert beneficial effects on plant physiology by improving soil structure and fertility and by affecting nutrient uptake and root architecture (Trevisan et al. 2010). Fractions of humic acids interact directly with root structures. Studies of humic substances marked with 14 C isotopes have shown that these humic fractions are associated in greater quantities with the cell wall within the first few hours of humic substances–root interaction (3 h) and subsequently (18 h) become part of the soluble component of the cells (Berbara and García 2014). Most humic substances bind tightly to plant cell walls and can be absorbed by roots where some of them can transfer to the shoots (Nardi et al. 2009). This direct plant uptake allows the humic substances to exert direct effects on plant metabolism (reviewed in Nardi et al. 2009). The specific physiological effects of humic substances on plants depend on the source, the concentration, and the molecular weight of the humic fractions applied (Nardi et al. 2002). (Trevisan et al. 2010) reviewed the signaling events that affect the physiological effects of humic substances on plant metabolism. As described above, humic acids have auxin-like effects on plants, and this primary effect was cited as the main biological factor accounting for the diverse beneficial effects on plants. For example, enhanced lateral root development by humic substances is related to mechanisms of cell division that are under the control of auxin. In a study on maize, (Canellas et al. 2009) showed that humic acids increased ATPase activity in root cells and caused increases in root area, while others increased root density. These results were interpreted as suggesting that hydrophobic interactions of humic acids in the rhizosphere may release auxin-like compounds that promote root growth. Support for this suggestion was reported in two independent studies using the auxin-synthesis promoter DR5 fused to a GUS reporter gene in transgenic tomato (Canellas et al. 2011; Dobbss et al. 2010).

The role of H⁺-ATPase activity, also referred to as proton pump activity, in the beneficial effects of humic substances has been reported in several studies. The main function of the plasma membrane H⁺-ATPase is to create a driving force for uptake and efflux of metabolites and ions across the membrane by generating an electrochemical gradient. (Jindo et al. 2012) reported that humic acid enhanced root growth of maize seedlings by release of auxin-like compounds that induce H⁺-ATPase activity in the plasma membrane of root cells, resulting in acidification of the apoplast and leading to loosening of cell walls and elongation of root cells. Increased H⁺-ATPase activity has been associated with humic acid-induced enhanced root elongation and lateral root formation of maize (Canellas et al. 2002; Zandonadi et al. 2007; Canellas et al. 2009). The functionality of H⁺ATPase and the proton pump has also been linked to nutrient uptake. Treatment of oat (Avena sativa) seedlings with humic substances enhanced proton extrusion from roots, which related to increased nutrient uptake (Pinton et al. 1997). In another study with maize roots, (Pinton et al. 1999) found enhanced nitrate uptake induced by humic substances was related to enhanced plasma membrane H⁺ATPase activity. However, increased nitrate uptake by plants following treatment with humic substances can also occur by transcriptional activation of specific genes in roots and shoots. In a study on maize, (Quaggiotti et al. 2004) reported that the expression of two putative maize nitrate transporters and a gene encoding an H + −ATPase isoform was enhanced by humic substances.

Another area of study for understanding how humic substances affect plant growth and development is the role of reactive oxygen species (ROS), which include ¹O₂, O₂⁻, OH, and H₂O₂. ROS can regulate plant growth through different pathways than those regulated by auxins. ROS signaling is involved in many plant metabolic processes, including regulation and development of plant growth, responses to biotic and abiotic stresses, and cell death (Suzuki et al. 2012). In a study with Arabidopsis roots, disruption of the transcriptional factor UPB1, which regulates cell proliferation and differentiation, resulted in changes in the balance of ROS and a delay in root differentiation (Tsukagoshi et al. 2010). Berbara and García (2014) demonstrated with histochemical staining that ROS was produced in rice plant roots following application of humic acid. In this study, ROS production, especially production of H₂O₂ was dependent on the concentration of humic substances. Following treatment with moderate levels of humic substances, the resulting production of ROS did not cause lipid peroxidation. As a result, growth and lateral root formation were favored. In contrast, treatment with high levels of humic substances elevated levels of ROS, which can lead to lipid peroxidation and negative effects on root growth and development.

Effects of humic substances on plant genes and their metabolites have been further characterized in reports using protemotics, trancriptomics, and microarrays. Proteomic analyses of maize roots following applications of humic substances revealed that a total of 42 proteins were differentially expressed by HS, including proteins related to energy, metabolism, and cellular transport (Carletti et al. 2008). This study concluded that the major pathways in the roots affected by humic substances were sucrose metabolism, malate dehydrogenase, ATPases, and cytoskeletal proteins. Transcriptomic analyses with microarrays of metabolic targets of humic acids were reported on winter oilseed rape (Brassica napus), a crop which has low nitrogen use efficiency (Jannin et al. 2012). In plants treated with a humic acid fraction that elicited significant increases in plant dry weight and chlorophyll content 30 days after treatment, genes that were up- or down-regulated were identified. At 1 day after treatment (DAT) with humic acid, no genes were differentially regulated; at 3 DAT, 720 genes in shoots and 366 in roots were significantly affected; and at 30 DAT, no genes in roots and 102 genes in shoots were affected. All the affected genes were involved in plant metabolic pathways, demonstrating that humic acid treatments can result in many potential changes in plant physiology. A transcriptomic approach based on detection of cDNA-AFLP markers was used to study the regulation of Arabidopsis plant genes in response to treatment with humic substances (Trevisan et al. 2011). In total, 133 genes were found to respond to humic substances, and real-time PCR analyses confirmed transcription of 32 of these genes. In a study with humic substances on the woody ornamental plant Lantana camara, genetic analyses of MADS-box AGAMOUS-like (AGL) genes were conducted (Costa et al. 2008). MADS box transcription factors are thought to have played an important role in the evolutionary development of angiosperms, and one sublineage of MADS box genes, AGL genes, is known to have a diverse role in flower and fruit development in many plant species (Pan et al. 2010). Application of humic substances to lantana enhanced activity of AGL genes.

Investigations into the effects of humic substances on plant physiology have also been directed to studies of abiotic stress. In a study on rice grown under water stress, (García et al. 2012) reported that humic acids induced peroxidase activity in leaves and roots, which led to reduced hydrogen peroxide content, maintenance of membrane permeability, and increased proline content. Increased proline content was also reported to be associated with humic acid-induced mitigations of salinity stress in bean (Aydin et al. 2012). Proline is an antioxidant amino acid that responds to stress events, functions as an osmolyte in root membrane permeability (Berbara and García 2014), stabilizes proteins, and inhibits lipid peroxidation. Hence, proline can be considered an important indicator of abiotic stress (García et al. 2012). In another study on rice under water-stress conditions, García et al. (2013) reported that humic acid maintained peroxidase activity below levels in plants without humic acid and that lipid peroxidation was lower in water-stressed plants with humic acid than in stressed control plants. However, abscisic acid (ABA) levels were similar in stressed plants with and without humic acid. These results suggested that the protection from water stress resulted from ABA-independent mechanisms. Further, regulation of tonoplast aquaporin genes (OsTIPs) by humic acid was indicated as a possible mechanism.

In comparison to the literature on humic acids, there are fewer reports documenting a role of fulvic acids in the amelioration of environmental stresses. However, plant stresses induced by harmful elements in soil and by drought have been shown to be affected by fulvic acids. Studies on effects of fulvic acids on yield of wheat under drought-stress conditions were discussed above in the section on yield increases. Fulvic acid effects on selenium stress were also reported. Selenium (Se) is harmful to plants and exists in soils in parts of China in high levels, resulting in reduced plant growth (Peng et al. 2001). Treatment of wheat with fulvic acids in soils with low Se concentration promoted root growth, and in soils with high Se content, fulvic acids reduced plant symptoms of stunting, leaf chlorosis, and wilt (Peng et al. 2001). Aluminum (Al) is another element that reaches levels in soil that restricts plant growth. Asp and Berggren (1990) studied the effects of Al interference on uptake of Ca²⁺ in beech in the presence and absence of fulvic acids. In the presence of fulvic acids and Al, uptake of Ca²⁺ was increased, and the fulvic acid-complexed Al was not available for uptake by roots. In a similar study with maize, (Harper et al. 1995) reported that FA reduced the negative effects of Al on root elongation. In China, FAs have been reported to play a role in reduction of stress from the buildup in soil of three rare earth elements (REEs) (La³⁺, Gd³⁺, and Y³). (Gu et al. 2001) reported that these REEs are widely used as micronutrients in fertilizers of wheat in China and that the resulting increased soil concentrations pose an environmental problem. Foliar sprays of wheat with FAs increased the bioaccumulation of the REEs which could reduce their buildup in soil. As part of a long-term assessment of fast-growing trees for phytoremediation in Spain, treatments of fulvic acid-rich amendment were applied to wild olive trees growing in eroded soils that had been polluted during a chemical spill related to mining (Murillo et al. 2005). The concentrations of trace elements in leaves of plants treated and not treated with fulvic acids were assessed to determine if fulvic acid would increase uptake of potentially toxic trace elements. The overall results indicated that treatments with fulvic acid stimulated growth and chlorophyll content without excessively enhancing uptake of the trace elements. Effects of fulvic acids on Pb toxicity of plants was investigated using broad bean in growth chamber tests (Shahid et al. 2012). Results showed that fulvic acids at low concentrations complexed toxic free Pb²⁺ and increased Pb uptake without resulting in Pb toxicity. At high concentrations of fulvic acids, Pb uptake and toxicity were reduced, presumably due to the high binding constant of fulvic acid for Pb. Effects of fulvic acid on maize under drought conditions were reported in a net-house trial (Anjum et al. 2011b). In this test, drought was applied at the tassel stage which is also when fulvic acid was applied as a foliar spray. Plants treated with fulvic acid under drought conditions exhibited increased plant growth (plant height, leaf area, shoot dry weight) and yield (grain yield, kernel rows per cob, and 100-kernel weight).

Several studies have been conducted to help elucidate how fulvic acids affect plant physiology in an effort to explain the observed plant growth enhancement and stress tolerance. The capacity of fulvic acid to enhance development of roots in a mini-hydroponic system with micro-Tom tomatoes was shown to be related to auxin by using the auxin-insensitive mutant dgt. This mutant does not respond to exogenous IAA, and it did not respond to treatment with fulvic acid, while the wild-type did (Dobbss et al. 2007). Treatment of maize with fulvic acid increased net photosynthesis, transpiration rate, and the intercellular concentration of CO₂, effects that were related to plant growth promotion (Anjum et al. 2011b). In the same study, proline accumulation was enhanced by fulvic acid treatment in both water-stressed and well-watered plants. Enhanced proline following treatments with fulvic acid leading to amelioration of abiotic stress was also noted by (Peng et al. 2001) in the studies on selenium stress discussed above. On soybean and ryegrass, increased concentration of chlorophyll was noted following application of fulvic acid (Chen et al. 2004). Fulvic acid applied to cell cultures of Greek fir interacted with the signaling pathway for plant hormones and increased intercellular levels of ATP and glucose-6-phosphate, physiological effects that were related to growth promotion (Zancani et al. 2011).

The examples cited above indicate that there is much evidence that humic and fulvic acids can interact with soil nutrients and elicit physiological responses in plants that lead to increased plant growth and in some cases to amelioration of abiotic stresses. The large number of studies demonstrating the specific physiological responses of plants to humic substances lends credence to their use. Berbara and García (2014) stated that the application of humic substances is becoming routine in agriculture. The increasing number of publications showing that humic and fulvic acids deliver economically important benefits to plants through enhanced yield, quality, or stress tolerance supports the potential for humic substances to become routine inputs in some agriculture. However, to reach this goal, more replicated field studies are needed to understand the specific ways that humic substances can add value to crop production systems.

Protein hydrolysates have been shown to stimulate carbon and nitrogen metabolism and to increase nitrogen assimilation. Maini (2006) summarized the early literature showing enhanced activity of NAD-dependent glutamate dehydrogenase, nitrate reductase and malate dehydrogenase in maize following application of Siapton. These results were expanded upon by (Schiavon et al. 2008) who showed that an alfalfa protein hydrolysate applied to hydroponically-grown maize increased the activity of three enzymes in the tricarboxylic acid cycle (malate dehydrogenase, isocitrate dehydrogenase and citrate synthase) and five enzymes involved in N reduction and assimilation (nitrate reductase, nitrite reductase, glutamine synthetase, glutamate synthase and aspartate aminotransferase). Increased gene expression of the three TCA cycle enzymes, nitrate reductase and asparagine synthetase was confirmed by RT-PCR in the roots following application of alfalfa hydrolysate. (Ertani et al. 2013b) reported similar effects of a meat hydrolysate. These data suggest that protein hydrolysates may promote nitrogen assimilation in plants via coordinated regulation of C and N metabolism. (Ertani et al. 2009) compared the effects of hydrolysates from alfalfa (Medicago sativa) and meat flour on maize seedling growth and showed increased activities of glutamine synthase (GS) as well as nitrate reductase in leaves and roots compared to the control. Up-regulation of isoforms GS1 and especially of GS2, which is responsible for assimilation of ammonia produced by nitrate reduction, was observed, consistent with a stimulatory effect of the hydrolysates on nitrogen assimilation.

Exogenous application of glutamate slowed primary root growth and enhanced root branching behind the root tip of Arabidopsis, suggesting a signaling role for glutamate and potentially more precise placement of roots within nutrient-rich patches in soil (Walch-Liu et al. 2006a, 2006b; Forde and Lea 2007).

There is considerable evidence that protein hydrolysates and specific amino acids including proline, betaine, their derivatives and precursors can induce plant defense responses and increase plant tolerance to a variety of abiotic stresses, including salinity, drought, temperature and oxidative conditions (Ashraf and Foolad 2007; Chen and Murata 2008; Kauffman et al. 2007; Apone et al. 2010; Ertani et al. 2013a). (Kauffman et al. 2007) showed that application of an animal membrane hydrolysate, Macro-Sorb Foliar to perennial ryegrass prior to exposure to prolonged high air temperature stress increased photochemical efficiency and cell membrane integrity compared to control plants. (Apone et al. 2010) reported that an amino acid/peptide/sugar mixture derived from plant cell walls induced the expression of three stress marker genes and two genes involved in the oxidative stress response in Arabidopsis plants and enhanced the tolerance of cucumber plants to oxidative stress. (Ertani et al. 2013a) showed that an alfalfa hydrolysate applied to maize grown hydroponically under increasing salt stress increased plant biomass, reduced the activity of antioxidant enzymes and the synthesis of phenolics, but increased leaf proline and flavonoid content, phenylalanine ammonia-lyase activity and gene expression relative to salt-stressed controls.

Glycine betaine, the N-methyl-substituted derivative of glycine, and proline act as osmoprotectants or osmolytes, stabilizing proteins, enzymes and membranes from the denaturing effects of high salt concentrations and non-physiological temperatures (reviewed by Ashraf and Foolad 2007; Chen and Murata 2008; 2011; dos Reis et al. 2012; Ahmad et al. 2013). Accumulation of glycine betaine and proline is generally correlated with increased stress tolerance and exogenous application of these compounds has been shown to enhance tolerance to abiotic stresses in a variety of higher plants including maize, barley, soybean, alfalfa and rice (Chen and Murata 2008; dos Reis et al. 2012; Ahmad et al. 2013). In addition to their roles in stabilizing proteins and membranes, glycine betaine and proline have been shown to scavenge reactive oxygen species and induce expression of salt stress responsive genes, and genes involved in transcription factors, membrane trafficking and reactive oxygen species (Kinnersley and Turano 2000; Ashraf and Foolad 2007, Einset et al. 2007; 2008; Anjum et al. 2011a; dos Reis et al. 2012; Liang et al. 2013).

Other amino acids have an effect on tolerance to abiotic stresses. Exogenous application of glutamate and/or ornithine, precursors of proline, can also enhance tolerance to salt stress (Chang et al. 2010; da Rocha et al. 2012). Arginine, which plays an important role in nitrogen storage and transport in plants, has been shown to accumulate under abiotic and biotic stress (Lea et al. 2006). The non-protein amino acids beta-aminobutyric acid (BABA) and gamma-aminobutyric acid (GABA) increase plant resistance to abiotic and biotic stresses and are thought to act as endogenous signal molecules (Shelp et al. 1999; Kinnersley and Turano 2000; Bouché and Fromm 2004; Jakab et al. 2005; Zimmerli et al. 2008). (Shang et al. 2011) showed that exogenous application of GABA reduced postharvest chilling injury in peache (Prunus persica) and led to enhanced endogenous accumulation of GABA and proline. L-glutamine inhibited the BABA-induced resistance to heat shock and a bacterial pathogen in Arabidopsis (Wu et al. 2010).

Amino acids and peptides play a role in the tolerance of plants to a range of heavy metals. Proline accumulation is induced in many plants subjected to heavy metal stress and some metal-tolerant plants exhibit high constitutive proline content even in the absence of excess metal ions (reviewed by Sharma and Dietz 2006). Proline may function in osmoregulation, by offsetting the water deficit that can arise with heavy metal exposure; it may chelate metal ions within plant cells and in the xylem sap; it may act as an antioxidant, scavenging free radicals formed as a result of heavy metal uptake; and it may function as a regulator (reviewed by Sharma and Dietz 2006). Nickel-hyperaccumulating plants also exhibit higher histidine concentrations and histidine appears to be involved in transport of Ni from root to shoot (Krämer et al. 1996; Kerkeb and Krämer 2003). There is evidence that other amino acids, including asparagine, glutamine and cysteine and peptides such as glutathione and the phytochelatins are important in chelation of Zn, Ni, Cu, As and Cd (Sharma and Dietz, 2006 Sytar et al. 2013).

Seaweed extracts have a multitude of effects on plant metabolism (reviewed by Khan et al. 2009), and recent gene expression analyses have provided further insight into the pathways involved. (Fan et al. 2013) observed increases in total soluble protein content, antioxidant capacity, phenolics, and flavonoid content in spinach treated with brown algal extracts. These effects were correlated with increases in transcript abundance of key enzymes involved in nitrogen metabolism (cytosolic glutamine synthetase), antioxidative capacity (glutathione reductase), and glycine betaine synthesis (betaine aldehyde dehydrogenase and choline monooxygenase). Chalcone isomerase activity, a key enzyme in the biosynthesis of flavanone precursors and phenylpropanoid plant defense compounds, also increased following treatment with seaweed extract (Fan et al. 2013).

(Jannin et al. 2013) used microarray analysis to assess the effects of a brown algal extract on the expression of 31,561 genes in Brassica napus. Sixty percent of these genes were not identified, but, of the remainder, about 1,000 known genes were differentially expressed and grouped into nine clusters representing the major metabolic functions of plants. Of these the most affected by algal extract application were those involved in carbon and photosynthesis, cell metabolism, nitrogen and sulfur metabolism, and responses to stress. Genes involved in carbon fixation, including Rubisco and carbonic anhydrase were upregulated by algal extract, which should lead to enhanced starch synthesis. This hypothesis was supported by the microscopic observation of increased number and size of starch granules following extract treatment. With respect to N and S metabolism, genes coding for proteins involved in uptake and assimilation of N were strongly upregulated, and qPCR analysis showed the induction of nitrate transporters in roots of treated plants. One of the genes coding for nitrate transporters, NRT1.1, has been suggested to also have a role in N sensing and in auxin transport (Krouk et al. 2010; Castaings et al. 2011), leading to enhanced lateral root growth. This hypothesis was supported by the increase in root dry weight of extract-treated plants observed in this study (Jannin et al. 2013). Similarly, genes encoding proteins involved in S uptake, assimilation, and storage were upregulated, which was correlated with the enhanced sulfate uptake and accumulation observed in shoots and roots of extract-treated plants (Jannin et al. 2013).

It is well established that purified seaweed cell wall polysaccharides and derived oligosaccharides can enhance plant growth (reviewed by González et al. 2013). Oligo-alginates derived from brown seaweeds enhanced nitrogen assimilation and basal metabolism in plants (Khan et al. 2011a; Sarfaraz et al. 2011), and oligo-carrageenans derived from red algae enhanced photosynthesis, nitrogen assimilation, basal metabolism, cell division, and protection against viral, fungal, and bacterial infections in tobacco (Nicotiana tabacum) and chickpea (Cicer arietinum) (Bi et al. 2011; Castro et al. 2012; Vera et al. 2012). (González et al. 2013) speculate that oligo-alginates and oligo-carrageenans may interact with plasma membrane receptors that use a co-receptor involved in signal transduction leading to simultaneous activation of plant growth and defense against pathogens as has been observed with brassinosteroid-dependent and microbial-associated molecular patterns (MAMPs)-dependent signaling pathways, which have a common co-receptor (Kemmerling et al. 2011).

Seaweed extracts have been shown to alleviate a variety of abiotic stresses including drought, salinity, and temperature extremes (Nabati et al. 1994; Zhang and Ervin 2004; Mancuso et al. 2006; Khan et al. 2009; Craigie 2011). Our current understanding of how plants respond to environmental stresses has been informed by recent advances in genomics and transcriptomics. Response is mediated via an intricate network of signals that perceive the stress and set in motion molecular, biochemical, and physiological processes that may be unique to each stress (Hirayama and Shinozaki 2010; Krasensky and Jonak 2012; dos Reis et al. 2012). At the molecular level stress-inducible genes are expressed that code for proteins that directly protect against stress, including osmoprotectants, detoxifying enzymes, and transporters and genes that code for proteins that are regulatory in nature such as transcription factors, protein kinases, and phosphatases. Metabolism may be altered by the synthesis of endogenous regulatory molecules, such as salicylic and abscisic acids, and by compatible solutes, such as proline and glycine-betaine that stabilize proteins and cell structures, maintain cell turgor, and scavenge reactive oxygen species. At the cellular level there may be changes in the plant membrane, cell wall architecture, cell cycle, and cell division. (Krasensky and Jonak 2012; dos Reis et al. 2012).

The mode of action of seaweed extracts in enhancing stress tolerance in plants is not well understood, but the presence of bioactive molecules in the extracts, such as betaines (Blunden et al. 1997) and cytokinins (Zhang and Ervin 2004), may play a role. Seaweed extracts also increase the endogenous concentrations of stress-related molecules, such as cytokinins, proline, antioxidants, and antioxidant enzymes in treated plants (Zhang and Ervin 2004; 2008; Zhang et al. 2010; Aziz et al. 2011; Lola-Luz et al. 2013; Fan et al. 2013). (Rayirath et al. 2009) showed that extracts of A. nodosum and its lipophilic fraction increased tolerance of Arabidopsis to freezing temperatures and that this was associated with protection of membrane integrity, reduced expression of chlorophyllase genes, and increased expression of three cold tolerance genes. (Nair et al. 2012) determined that the lipophilic components (LPC) of the seaweed extract increased proline content in Arabidopsis plants undergoing freezing stress and that this increase was associated with increased expression of proline synthesis genes. In addition, the concentration of total soluble sugars in the cytosol and the proportion of unsaturated fatty acids increased in LPC-treated plants under freezing stress. Using transcriptomic and metabolomic approaches they demonstrated that 1,113 genes were differentially expressed in the LPC-treated Arabidopsis undergoing freezing stress and of these 463 were up-regulated, including those associated with stress responses, sugar accumulation, lipid metabolism and response to abscisic acid. In a similar study, (Jithesh et al. 2012) showed that treatment of Arabidopsis undergoing salt stress with A. nodosum extracts induced many positive regulators of salt tolerance and down-regulated other genes.

Seaweed extracts contain a variety of plant hormones including cytokinins, auxins, abscisic acid, gibberellic acid and salicylic acid determined indirectly by bioassays (reviewed by Khan et al. 2009 and Craigie 2011; Khan et al. 2011b) and directly by methods such as high pressure liquid chromatography (HPLC), gas chromatography–mass spectrometry (GC-MS), and liquid chromatography-mass spectrometry (LC-MS) (Stirk et al. 2003; Gupta et al. 2011). Cytokinins and cytokinin-like compounds are the most widely reported in seaweed extracts, followed by auxins and auxin-like compounds (Stirk et al. 2003; Khan et al. 2009; Craigie 2011), and these have often been speculated to be responsible for the plant growth enhancing effects of the extracts (Khan et al. 2009; Craigie 2011). (Wally et al. 2013) determined the phytohormone concentrations in 12 seaweed extracts from different sources by ultraperformance liquid chromatography-electrospray tandem mass spectrometry (UPLC-ESI-MS/MS) and concluded that the phytohormone levels present in seaweed extracts were insufficient to cause significant effects in plants when applied at the recommended rates. However, using phytohormone biosynthetic and insensitive mutants of Arabidopsis mutants coupled with transcript analysis they showed that “alteration in plant phenotype following seaweed extract application results from a modulation of biosynthesis, quantity, and ratios of the endogenously produced cytokinins, auxins and abscisic acid metabolites, rather than from the exogenous phyohormones present within the extracts themselves” (Wally et al. 2013). They observed increases in concentrations of cytokinins and abscisic acid and their metabolites in leaf tissue and decreases in auxin concentrations following treatment with seaweed extract. They suggest that this stimulation of endogenous cytokinin and abscisic acid biosynthetic pathways and repression of auxin biosynthesis likely explains the increased vegetative plant growth and enhanced resistance to drought and salinity stress observed by others following seaweed extract application. The bioactive compounds within the seaweed extracts that lead to activation of the plant phytohormone biosynthetic pathways remain to be determined.