Biosurfactants as Green Biostimulants for Seed Germination and Growth

The Pre-Sowing Seed Stimulation Is A Technique That Aims To Accelerating The Germination. It Covers The Seed Surfaces With Remarkable Molecules That Protect Them Against Microorganisms And/Or Enhance Their Metabolism, For Example Higher Plant Resistance Against Bacteria, Fungi, Yeasts And Virus. The Seed Stimulation Can Decrease The Initial Dose Of Fertilizers (Krawczyńska Et Al., 2012). In This Sense, Natural-Based Stimulation Methods Have Been Drawn Attention From The World Due To The Their Sustainable Approach. Biosurfactants Is One Of The Most Promissing Molecules As Biostimulants.


2.2.Glycolipids
Glycolipids are a group of chemical compounds formed by moieties of hydrophilic carbohydrate and hydrophobic lipid. They also present amphiphilic and interface-active characteristics. They are commonly hydroxylated fatty acids connected to a sugar residue by a glycosidic bond. This class could be produced by microbial metabolism, for example from the Pseudomonas aeruginosa bacteria which originates rhamnolipids; the Candida bombicola yeast that produces sophorolipids, and the consortium of yeasts and higher fungi (e.g., Candida antarctica and Pseudozyma aphidis) which generates mannosylerythritol lipids (Giessier-Blank et al., 2016).

Rhamnolipids
Rhamnolipids are glycolipids which chemical structures consist of a β-hydroxy fatty acid-based fraction and a rhamnose-based moiety which have one or two rhamnose residues; originating two classes of rhamnolipids: monorhamnolipids or dirhamnolipids. Likewise, the lipid portion can be represented by one or two ester-bonded β-hydroxy fatty acids, with different chain length and degree of saturation ( Figure 2). Pseudomonas aeruginosa are the best bacterium group for rhamnolipid production.

Sophorolipids
Sophorolipids (SLs) are synthesized by specific yeast species (Candida sp.) from renewable resources. Characteristically, they are formed by a mixture of lactonic and acidic varieties in the acetylation standards of the constituting glucose moieties, in the type of the hydrophobic portion, and in its bond to the hydrophilic sugar (sophorose) (Figure 3).

Mannosylerythritol lipids
Mannosylerythritol lipids (MELs) are nonionic biosurfactants, originated by plant-associated fungi of the genera Pseudozyma and Ustilago, also recognized by their low toxicity, excellent dispersion, emulsification, foaming, functional properties, production yields, solubilization, and wetting (Arutchelvi et al., 2008;Yu et al., 2015). MELs are amphiphilic molecules with an acetyl group and/or a fatty acid as the hydrophobic portion and a 4-O-β-D -mannopyranose-erythritol as a hydrophilic fraction (Arutchelvi and Doble, 2010). They can be classified according to the different location and number of the acetyl group at C-4 and C- 6

BIOSURFACTANTS AS BIOSTIMULANTS FOR SEED GERMINATION AND GROWTH
The production biosurfactants and their application as antimicrobials have been extensively studied. However, biosurfactants interact with bacterial, animal and vegetable cells in a wide range of ways. These interactions can act as, for instance as agricultural enhancers of plant immunity. In this sense, the applications of biosurfactants (groups) as biostimulants (agricultural) are briefly described below.

Lipopeptides
Lipopeptides are the main class of antimicrobial peptides produced by Bacillus sp. with a wide range of biotechnological applications (Shafi et Ongena et al. (2007), where surfactin and pure fengycin were applied directly to the plant, providing a significant ISR response as a protective effect, similar to that induced by a strain of Bacillus subtilis cells inoculated at the root of bean and tomato plants. The results show a significant decrease of the disease (28%) using surfactin, but not with fengycin, which showed a disease reduction of 14%, which was equivalent to the control plants. Despite the induction of resistance to pathogens in plants by the use of surfactin being efficient, this lipopeptide exerts no direct antagonism to the growth of pathogens. Surfactin by itself is not fungitoxic, but when combined with iturin it exerts a synergistic antifungal effect (Maget-Dana et al., 1992). In contrast, fengycins and iturins exhibit high fungitoxicity (Vanittanakom et al., 1986). Few papers report the use of lipopeptides directly associated to plant growth. However, some studies indicate the passive use of lipopeptides as potential biostimulants, increasing the bioavailability of nutrients to plant-associated beneficial microorganisms (Santos et al., 2016). To improve the immune system and serve as a support to plant metabolism, a biostimulator should be able to penetrate plant tissue. However, penetration time is limited since the biostimulator should remain in liquid form. This must be taken into account, since plants undergoing treatment are exposed to different climatic conditions and other extrinsic factors (Kolomazník et al., 2012). However, the permeability of the pores can be influenced by reducing the surface tension of the solution (Gaskin et al., 1996), by means of, for example, using surfactants or biosurfactants. Another very important factor for plant growth is the microbial consortium in the soil. Some microbiological tests are useful in assessing the quality of the soil before planting. To determine the effects of contaminants or even of soil management and the dynamics of microbial communities, one of the tests most commonly used as an indicator is the release of CO 2 by microbial respiration (De Paula et al., 2010; Marchiori Júnior and Melo, 1999; Zhong et al., 2010). According to Machado (2018), who used respirometry techniques to test the influence of adding surfactin to the soil, surfactin acted as a biostimulant, increasing the release of CO 2 from 9.78 to 216.29 mg C-CO 2 / kg of soil with the addition of 4,000 mg / kg of surfactin (Machado, 2018). Therefore, lipopeptides play a crucial role in agriculture, and could be better exploited for plant growth and disease management. The biodegradability of biosurfactants is a positive factor because they do not persist in the environment, are less toxic and more biocompatible (Ławniczak et al., 2013). Recent research investigates the applicability of lipopeptide biosurfactants as an alternative to pesticides and chemical fertilizers in order to improve the sustainability of current agricultural practices. The relevance of the topic and the few reports in the literature justify a more thorough research regarding the application of biosurfactants as biostimulants in plants.

Rhamnolipids
Rhamnolipids are well-known biosurfactants that can be used in a wide variety of applications including detergent formulation, food and pharmaceutical industry, cosmetics and bioremediation. More recently, some studies have shown that rhamnolipids can also be used as agriculture additives since they can act as direct antimicrobial agents as well as a stimulant to enhance plant's immunity (Vatsa et al., 2010). Rhamnolipids can be applied as biocontrol agents in crops, controlling the growth and spread of several phytopathogenic fungi, such as  2009) studied the use of rhamnolipids from Pseudomonas aeruginosa to trigger grapevine defense responses against B. cinerea. The authors detected Ca 2+ influx, reactive oxygen species production and mitogen-activated protein kinase activation in the first two minutes after the application. These signaling events can induce plant defense mechanisms, including the expression of a wide variety of genes and hypersensitive response. The cytosolic concentration of Ca 2+ reached 6 µM with the addition of 0.05 mg/mL of rhamnolipids. The calcium influx generally leads to the activation of mitogen-activated protein kinase and the production of reactive oxygen species, which is frequently involved in plant cell signaling succeeding pathogen perception. The authors also demonstrated that 0.01 mg/mL of rhamnolipids was enough to induce early signaling events and expression of defense genes such as chitinase genes. The authors propose that rhamnolipids are acting as microbe-associated molecular patterns (MAMPs) in grapevine. MAMPs, also known as general elicitors, are involved in non-specific immunity in plants, being effective against a wide variety of pathogens (Bent and Mackey, 2007;Varnier et al., 2009). Sanchez et al. (2012) reported that rhamnolipids were able to stimulate an innate immune response in thale cress including accumulation of signaling molecules and activation of defense genes. The use of rhamnolipids increased the plant resistance against the bacterium Pseudomonassyringae pv tomato, the oomycete Hyaloperonospora arabidopsidis, and the fungus B. cinerea. The authors demonstrated that the increased resistance induced by rhamnolipids can involve different signaling pathways depending on the nature of the pathogen. For example, ethylene is involved in the resistance to H. arabidopsidis and P. syringae pv tomato, jasmonic acid is crucial for the resistance to B.cinerea, while salicylic acid takes part of the defense against all pathogens tested. Sanchez et al. presented evidence that plant defense mechanisms involving salicylic acid are potentiated by rhamnolipids when the plant is exposed to B. cinerea or P. syringae pv tomato. As well as Varnier et al. (2009), the authors concluded that rhamnolipids can act as MAMPs to trigger non-specific immunity in plant cells. According to Monnier et al. (2018), rhamnolipids from P. aeruginosa stimulate an effective defense of rapeseed foliar tissues toward B. cinerea. The defense triggered by rhamnolipids includes, besides chemical mechanisms as the production of reactive oxygen species and the expression of defense genes, physical protections as callose deposits and stomatal closure. Table 1 summarizes the researchers found on literature about the use of rhamnolipids as plant immunity stimulants against phytopathogenic microorganisms. The analysis of Table 1 indicates that there are a reduced number of publications on the application of rhamnolipids as agricultural enhancers of plant immunity. Thus, it should be deeper investigates, since rhamnolipids can replace the current agrochemicals, leading to more sustainable agriculture productions.

Sophorolipids
The DE102014209346A1 German patent presents the use of sophorolipids to increase a crop yield, achieved even if the plant pathogen is not combated or the clinical picture is not changed (Sieverding, 2017). Experiments at field, greenhouse and laboratory were projected to treat each crop plants (barley, wheat, soybeans, and tomato) with distinguish sophorolipid samples in variated growth stages. The application was done to plant seeds or leaves considering sophorolipid samples alone or in mixture with commercial plant protection products (insecticides, fungicides, and nutrients). Such procedures can defend plants from insect infestation and illness at a primary stage of growth. At field tests, sophorolipids provided an increase in yield for agricultural crops. Sophorolipid seed treatment hurries and improves initial plant growth in sprouting and rooting (Sieverding, 2017). An additional report about the advantage of the sophorolipids use is an increase in yield, when used as adjuvants as shown at the US 2012/0220464 patent (Giessier-Blank et al., 2016). A landfill with contaminated sludge derived originated in copper mineral production represent a threat to the nearby ecosystem due to the dust emission with heavy metals and the complete absence of vegetation. With the view to enrich the quality of the landfill and promote plants development for future cultivation, a researcher's group of Wroclaw University of Technology analyzed the performance, regarding germination and plant growth, of biopreparations with biosurfactants and microorganisms in these plant species: Avena sativa, Lupinus luteus, Pisum sativum, Sinapsis alba, and Zea mays. The results made it possible to conclude that biosurfactants application before germination promotes a stimulation of all studied seeds. Nevertheless, the direct efficiency of biosurfactants for plant growth development was not certified (Krawczyńska et al., 2012). Numerous surfactants or wetting agents used in crop protection exhibit an elevated spreading behavior. One example is the trisiloxanes, that are harmful to health and could be either included directly in the crop protection product formulations or as an increment to the aqueous spray solutions, as a tank mix additive, at any moment before the application. In this context, Giessler-Blank et al. . The best results were obtained by two successive washings with 20 g/L of sophorolipid solution plus ultrasonication (35 kHz) that effectively extracted 71.2% Cd, 100% roxithromycin, 96.6% sulfadiazine, and 88.2% tetracycline. Concomitantly, the rates of ARGs decreased to 10 -7 and 10 -8 . Supplementary, lettuce cultivation after the second washed soil exposed a reduction in ARG abundance in lettuce tissues and separate amounts of antibiotic-resistant bacterial endophytes, besides that a relevant enhancement in vegetable growth indices was confirmed by the measurement of chlorophyll content dry/fresh weight, root surface area and soluble protein content (Ye et al., 2016). These results evidenced that lettuce farming in the washed soil was an appropriate and effective tactic for vegetable safety control, and also provided a more appropriate environment for the establishment and subsistence of microorganisms to reinstate microbial metabolism in the washed soil. Extras researches about the utilization of sophorolipids in agriculture are presented on Table 2. In front of the reduced number of published works, this class of biosurfactant is opened for more studies related to improve plant immunity in the agricultural environment.
Table2. Extras reports about sophorolipid application in agricultural environment. Improvement of soil quality by solubilizing and breaking down hydrocarbons (e.g., phenanthrene), crude oils and heavy metal removal (e.g., zinc/copper/arsenic). (Sun et al., 2018) Stimulation of dissipation of antibiotics and antibiotic resistance genes in soil

Mannosylerythritol Lipids
MELs have been used in several industrial processes due to their specific properties, such as low toxicity, biodegradability, and excellent surface activity (such as emulsification, dispersion, solubilization, foaming, and wetting) Beck et al., 2019;Coelho et al., 2020). However, besides these characteristics, no practical use in agriculture has been developed. According to Yoshida et al. (2015), one potential agronomic application of MELs is as pesticides against phytopathogenic microorganisms. Some studies had shown that MELs, as well as the other biosurfactants, can act as antimicrobial agents . Kitamoto 2015) demonstrated that the application of MEL-A in wheat leaves was able to suppress the development of powdery mildew fungus Blumeria graminis f. sp. tritici strain T-10, possibly due to the inhibition of conidial germination. On the other hand, when applied on rice leaves, no effect was noticed, and on strawberry leaves, the application of MEL enhanced significantly the disease symptoms. Thus, the ability of MELs to act as pesticides by decreasing the hydrophobicity of plant surfaces seems to depend on the plant and on the target pathogen . Up to date, MELs have been poorly explored as agricultural additives and few studies can be found on literature. The mechanisms behind their action as antimicrobial agents and their interaction with plant surfaces must still be elucidated, so more research is needed in this area, using different plant species and pathogenic microorganisms. Others approaches of MELs utilization are exhibited on Table 3.