Biodegradable agricultural pesticides

Abstract

The technology generally concerns antimicrobial materials and uses thereof.

Description

[0001] BIODEGRADABLE AGRICULTURAL PESTICIDES

[0002] TECHNOLOGICAL FIELD

[0003] The invention belongs to the field of agricultural pesticides, and specifically naturally derived and biodegradable pesticides showing potential for a broad range of antimicrobial activities and capability to activate plant defense mechanisms.

[0004] BACKGROUND

[0005] FAO’s (Food and Agriculture Organization of the UN) Scientific Review on the Impact of Climate Change on Plant Pests predicted that the rise in temperature and change in weather patterns due to climate change is going to increase the risk of pests spread in agricultural and forestry ecosystems, which in turn is going to pose a threat on the food security of the constantly increasing global population. Although many pesticides were found to be relatively efficient in preventing or reducing loss of crops by effectively controlling or destroying various pests’ populations, they were also shown to accumulate in the environment and to pose ecological threats to agricultural crops and to the food chain as a whole.

[0006] Salicylic acid (SA), a mono hydroxybenzoic acid with a hydroxy group at the ortho position, is a natural compound synthetized by plants. In plants, it fulfills the function of a plant hormone that binds and modulates the activity of various plant proteins partaking in growth and development and resistance to abiotic stress. It further serves as a secondary metabolite that confers antimicrobial resistance, with demonstrated anti- virulent activities against various types of plant pathogens, including bacteria and fungi [1-3], It has been further demonstrated that SA and its derivatives are effective antimicrobial and anti-virulent agents outside the plant kingdom with multiple prokaryotic and eukaryotic targets, which is why they are being extensively used as preservatives in the pharma, cosmetic and food industries. Specifically, they were shown to possess protective effects against a number of bacterial pathogens and to control the formation of bacterial biofilm via interference with quorum signaling (QS), a bacterial population density-dependent communication system. QS is a promising target for antibacterial drugs, and antimicrobial drugs in general, as it is highly selective for pathogen related growth mechanisms and leaves the growth and metabolism of host unaffected. In addition, unlike many antimicrobial drugs, it does not lead to the emergence of drug-resistant bacteria.

[0007] More recently, it has been shown that that SA interferes with QS in several Gramnegative bacteria, including several strains of pectobacteria [4]. P ectobacterium spp. have been related to a spectrum of disease symptoms such as wilt, soft rot, and blackleg in a wide range of monocot and dicot host plants, and together were found responsible for large economic losses in potato storage and production in the ornamental plants industry. P. brasiliense has gained increasing attention as a quickly spreading pathogen responsible for blackleg and soft rot infections in potato plants worldwide.

[0008] In P ectobacterium spp. QS signaling uses mainly N-acyl homoserine lactone (AHL), of which the most common are 3-oxo-hexanonyl homoserine lactone (3-oxo-C6- HSL) and 3-oxo-octanonyl homoserine lactone (3-oxo-C8-HSL). In P. brasiliense (Pb 1692), SA significantly downregulated the expression of QS related genes, the AHL synthase (expl) and the QS response regulator (expR), and also the expression of other virulence related genes such as pecS, pel, peh, and the transcription factor yheO which are dependent on the QS machinery [5].

[0009] With the positive example of SA anti-virulence in Pb 1692, SA has become an attractive target for the development of a new line of inhibitors to control virulence and infection of various plant pathogens such as bacteria and fungi [6-9]. As has been mentioned, the use of SA against P. brasiliense infections is particularly relevant for storage of potatoes and other tuberous crops. One problem with the use of SA, however, is that in moderate-to-high concentrations it can be irritating to the eyes, skin and respiratory tract and can cause moderate chemical bums, and if ingested, can lead to intoxication symptoms affecting the acid-base balance and the CNS leading to delirium and tremor. Another problem is that some SA derivatives, such as SA methyl ester (methyl salicylate) for example, are extremely volatile and are essentially ineffective.

[0010] REFERENCES

[0011] 1. Corina AV et al 2009. Salicylic acid, a multifaceted hormone to combat disease. Annu Rev Phytopathol 47 :177 -206.

[0012] 2. Kiessig DF et al 2018. Systemic acquired resistance and salicylic acid: Past, present, and future. Mol Plant-Microbe Interact 31(9):871-888.

[0013] 3. Joshi JR et al 2020. Direct binding of salicylic acid to pectobacterium N-Acyl- homoserine lactone synthase. ACS Chem Biol 15(7): 1883- 1891.

[0014] 4. Asfour H 2018. Anti-quorum sensing natural compounds. J Microsc Ultrastruct 6(l):l-10.

[0015] 5. Joshi JR et al 2016. Plant phenolic acids affect the virulence of P ectobacterium aroidearum and P . carotovorum ssp. brasiliense via quorum sensing regulation. Mol Plant Pathol 17 (4):487-500.

[0016] 6. Faure D and Dessaux Y 2007. Quorum sensing as a target for developing control strategies for the plant pathogen Pectobacterium. Eur J Plant Pathol 119(3):353-365.

[0017] 7. Da Rocha Neto AC et al 2015. Antifungal activity of salicylic acid against Penicillium expansum and its possible mechanisms of action. Int J Food Microbiol 215:64-70.

[0018] 8. Pedroso RDS et al 2019. In vitro and in vivo anti- Candida spp. activity of plant- derived products. Plants (Basel) 8(11): 494.

[0019] 9. Tuizer S et al 2022 Disalicylic Acid Provides Effective Control of Pectobacterium brasiliense. Microorganisms 10: 2516.

[0020] GENERAL DESCRIPTION

[0021] Numerous studies revealed that plant defense mechanisms that are activated by various types of microbial pathogens and herbivorous insects are regulated, in most cases, by a network of interconnecting signaling pathways dominated by certain types of plant signaling molecules, such as salicylic acid (SA), jasmonic acid (JA), and ethylene (ET). Further studies have shown that exogenous applications of these compounds often result in rapid activation of plant resistance or enhanced plant protectivity against a variety of microbial and other pathogens.

[0022] SA, and particularly the volatile secondary SA metabolite, has been known to play a central role in plant defense, as suggested by studies showing increased levels of SA in plant tissues after exposure to pathogens and enhanced resistance to a broad range of pathogens upon exogenous SA application. On the molecular level, SA has been implicated in the activation of several plant resistance genes, leading to the induction of a local defense that contains the growth of a virulent pathogen, and further, a longdistance signaling that provides broad spectrum and long-lasting resistance to secondary infections throughout the plant, i.e., systemic acquired resistance (SAR). JA is another interesting plant defense signaling molecule. JA is a volatile fatty- acid-derived molecule that has been implicated in some important aspects of plant life. Studies have shown that JA biosynthesis is activated during pollen and seed development, and further, in response to various biotic and abiotic stressors such as wounding, ozone, insect pests, and microbial pathogens. Further, mutants deficient in JA production exhibited enhanced susceptibility to a variety of plant pathogens, such as herbivore insects, fungi and bacteria of P ectobacterium spp. Owing to these properties, JA has been considered as a protective treatment for seeds to stimulate the natural plant defenses.

[0023] Moreover, it was suggested that SA and JA can act synergistically in improving and potentially broadening plant resistance. This thesis was supported by experiments showing that SA and JA can act synergistically in inducing the expression of PRlb, a pathogenesis-related gene. Gene analysis in Thaliana plants treated with combination of SA and JA revealed coordinated expression of more than 50 defense-related genes, suggesting a coordinated crosstalk between these two pathways and a regulatory potential to induce a broader scope of protection against various types of plant pathogens.

[0024] The sum of these properties supports the conclusion that the complementary mechanisms of SA and JA action on plant resistance genes that circumvent the risk of selection for pathogen resistant species, together with their natural origin, degradability and relative lack of toxicity, compared to other known and especially synthetic pesticides, all make them excellent candidates for protection of crops. The main problem, however, is that both SA and JA are highly volatile, and so are many of their known derivatives, and many of the known antimicrobial agents, which precludes them from being used as spreadable widely applied pesticides.

[0025] To overcome many of the difficulties associated with the use of volatile antimicrobial agents, such as SA and JA, the inventors have developed a methodology to reduce the volatility of such agents, and thus increase their induced effect over time. According to the novel methodology, covalent derivatives of antimicrobial agents, formulated as multimers, e.g., dimers of antimicrobial agents, or as amphiphilic derivatives thereof have been prepared. As demonstrated and discussed herein, homo or hetero-dimerization of antimicrobial agents or substitution of antimicrobial agents with lipophilic substituents, enabled not only an increase in the molecular weight and a decrease in volatility, but surprisingly also enabled a substantial increase in the effectivity of the new antimicrobial agents as compared to their respective building blocks, which may themselves be effective antimicrobials. Non-covalent combinations of antimicrobial building blocks have proven much less effective than their multimeric / dimeric form, an observation which suggests that the superior effect cannot be attributed to a dosedependent or a concentration-dependent effect, namely to the mere doubling in the amount of the antimicrobial materials.

[0026] Thus, in its broadest aspect, the invention provides an antimicrobial material comprising a water-soluble antimicrobial agent covalently associated to another same or different water-soluble antimicrobial agent or a lipophilic moiety, wherein the covalent association is hydrolyzed or labile.

[0027] In some embodiments, the antimicrobial material comprises one antimicrobial agent covalently associated to a lipophilic moiety that may or may not have antimicrobial properties. In some embodiment, the lipophilic moiety is selected to enable cellular penetration of the antimicrobial agent.

[0028] In some embodiments, the antimicrobial agent is covalently associated with a lipophilic agent selected amongst hydrocarbons, aromatic materials, and others. The lipophilic agent may be selected from oleanolic acid, ursolic acid, betulinic acid, boronic acid and others.

[0029] In some embodiments, the lipophilic agent is an antimicrobial agent having low solubility in water.

[0030] As used herein, the lipophilic agents are such which are more soluble in a lipid medium than in water. Typically, they may be defined as having a lipid solubility of at least 0.5 or 1 g / L, or at least 10 g / L, or at least 100 g / L, in an oil at room temperature (25°C) and atmospheric pressure. The water-soluble agents are those which exhibit a water solubility of at least 1 pg / L, or at least 10 pg / L, or at least 100 pg / L, or at least 0.5 or 1 g / L, in water at room temperature (25°C) and atmospheric pressure.

[0031] In some embodiments, the antimicrobial material comprises two antimicrobial agents covalently associated to each other. Such materials comprising two associated antimicrobial agents, being same or different, are regarded herein as ‘dimers’.

[0032] Thus, the invention further provides an antimicrobial material comprising two water-soluble antimicrobial agents, the antimicrobial agents being chemically associated to each other via a hydrolysable or a labile covalent bond or linker.

[0033] In some embodiments, each of the antimicrobial agents is selected amongst water soluble naturally derived antimicrobial agents. In another aspect, there is provided an antimicrobial material comprising two covalently associated same or different antimicrobial agents, wherein the covalent association being via or comprises a labile covalent bond or linker. In some embodiments, each of the antimicrobial agents is selected amongst water soluble naturally derived or naturally occurring antimicrobial agents.

[0034] Further provided is an antimicrobial material, as disclosed herein, for controlling a plant microbial source or a disease associated therewith, wherein the microbial source is bacteria, viruses, fungi and / or herbivorous insects.

[0035] The invention further provides an antimicrobial material comprising two covalently associated different antimicrobial agents, wherein the different antimicrobial agents are capable of inducing a synergistic antimicrobial effect as compared to the effect induced by each of the antimicrobial agents when used alone.

[0036] As used herein, materials of the invention are antimicrobial materials”, constructed to comprise one or two (or more) antimicrobial agents. The antimicrobial materials and each of the antimicrobial agents forming the material possess one or more inhibitory effects on at least one plant parasite selected from bacteria, viruses, fungi and herbivorous insects. The antimicrobial effect induced may be general or unspecific or may involve or cause reduced production of at least one mediator of Quorum Sensing (QS). Such mediators may be N-acyl-L-homoserine lactones (AHLs) or a product of a bacterial AHL synthetase or an ortholog thereof. Additionally, or alternatively, the inhibitory effect may involve or bring about reduced production of at least one plant cell wall degrading enzyme (PCWDE), such as pectate lyases (Pel), polygalacturonases (Peh), and proteases (Prt). Where bacteria (e.g., P ectobacterium spp., such as P. Brasiliense) are involved, the inhibitory effect may comprise or cause inhibition of at least one of bacterial motility, biofilm formation, virulence and bacterial growth.

[0037] The antimicrobial effect may be reflected in the ability of the antimicrobial material of the invention to prevent, inhibit, and / or control growth of microorganisms. The antimicrobial materials of the invention may thus be similarly regarded as bactericides, bacteriostats, fungicides, fungistats, algaecides and algistats. In some cases, the antimicrobial materials may be characterized based on their ability to inhibit or reduce or retard formation of load of a microorganism by inhibiting, reducing, or retarding growth of microorganisms and / or eradicating a portion or all of an existing population of the microorganisms. The microorganism may be, for example, unicellular microorganisms (prokaryotes, archaea, bacteria, eukaryotes, protists, fungi, algae, molds, yeast, euglena, protozoan, dinoflagellates, apicomplexa, trypanosomes, amoebae and others), or multicellular microorganisms.

[0038] The dimers or antimicrobial materials of the invention are structured of two antimicrobial agents which may be identical or different and which in their monomeric forms are optionally naturally occurring and water-soluble materials. In other words, in some embodiments, each of the antimicrobial agents associated to form the dimer may be characterized by water solubility, antimicrobial activity and natural occurrence. The selection of such materials for the construction of dimers of the invention, allows for a superiority that may be reflected in any one of:

[0039] 1. Increased activity;

[0040] 2. Prolonged activity;

[0041] 3. Reduced volatility ;

[0042] 4. Increased passive membrane permeability;

[0043] 5. Increased antimicrobial concentration as the breakdown components are themselves active antimicrobial agents;

[0044] 6. Increased bioavailability as the breakdown components are themselves water soluble and environmentally safe.

[0045] The reduced volatility observed for dimers of the invention, as compared with a volatility measured for each of the antimicrobial agents making up the dimers, is a property of the dimers reflected in their reduced boiling points and a reduced vapor pressure at ordinary temperatures and pressures. Thus, in another aspect, the invention provides a dimer according to the invention, wherein the dimer comprises two same or different covalently associated antimicrobial agents, wherein the dimer having a boiling point (or a volatility at ambient conditions) which is higher than the boiling point (or a lower volatility) of each of the two same or different agents when in a monomeric form.

[0046] Similarly, dimers of the invention exhibit a greater ability to cross or permeate through a pathogen barrier or cell membrane to induce their effect therein. This increase permeability ensures an increased antimicrobial effect, which may be maintained as long as the antimicrobial material is present and can permeate through the cell membrane. As such, the invention also provides a dimer according to the invention, wherein the dimer comprises two same or different covalently associated antimicrobial agents, wherein the dimer having a calculated passive membrane permeability which is higher than a calculated passive membrane permeabilities of each of the two same or different agents when in a monomeric form.

[0047] The two same or different antimicrobial agents are associated to each other directly or indirectly via a linker moiety, which may constitute a sacrificial material or by itself may have antimicrobial properties; or otherwise, may contribute to the stability or function of the antimicrobial material. Typically, to permit covalent association between the two agents, each of the agents may have or may be modified to include a functionality that permits association. The functionality is preferably inherent to the structure of each of the two antimicrobial agents; but in some cases, where needed, the antimicrobial agents may be synthetically modifiable to include such a functionality. Within the context of the present invention, the chemical modification of the antimicrobial agents does not reduce or diminish the antimicrobial properties of the agent.

[0048] The functionality permitting direct or indirect association between the two antimicrobial agents may be a heteroatom, such as N, O, or S, or a group such as an amine, an amide, a carboxylate, a carboxylic acid, an alcohol, a thiol or any other functionality permitting covalent functionalization.

[0049] The covalent association between the two antimicrobial agents may be direct, in which case a covalent bond may be formed between different functionalities, for example through an amine group on one agent and a carboxylic acid on another to form an amide bond. Where the association is via a linker moiety, the functionalities may be the same. The linker moiety may be an atom, such as a heteroatom (N, NH, O, S) or a group of atoms, such as a carbon-containing group that is bifunctional and capable of forming covalent bonds with a functionality present on each of the antimicrobial agents. The bifunctional material may have two identical bonding functionalities or two different bonding functionalities. The bifunctional material may have one or two carboxylic acid groups, one or two hydroxyl groups, one or two amine groups, etc. In some embodiments, the bifunctional material may be a diacid, a diol, a diamine or a mixed form thereof, such may be a linker having one hydroxyl functionality and a carboxylic acid functionality.

[0050] In some embodiments, the linker moiety is a heteroatom selected from -O-, -N=, -NH-, S and groups containing same.

[0051] In some embodiments, the linker moiety is derived from a diacid comprising two carboxylic acid moieties. In such embodiments, the linker moiety may be derived from HO(O=C)-X-(C=O)OH, wherein X is a group selected from -C1-C10alkylene, -C2- C10alkenylene, -C2-C10alkynylene, -C1-C5alkylene-O-C1-C5alkylene, -C1-C5alkylene-C6- C10arylene, -C6-C10arylene, -C2-C5alkenylene-C6-C10arylene and -C2-C5alkynylene-C6- C10arylene, -C3-C6hctcroarylcnc and others. In an antimicrobial material of the invention, the diacid may be presented as a reaction product (such as an ester or a diester, an amide or a diamide) resulting from a chemical interaction of the diacid groups with functionalities present on the antimicrobial agents.

[0052] In some embodiments, the linker moiety may be derived from a diol comprising two hydroxyl moieties. In such embodiments, the linker moiety may be derived from HO- X-OH, wherein X is a group as defined above. In some cases, X may be selected from - C1-C10alkylene, -C2-C10alkenylene, -C2-C10alkynylene, -C1-C5alkylene-O-C1-C5alkylene and others. In an antimicrobial material of the invention, the diol may be presented as a reaction product (such as an ester or a diester, an ether or a diether) resulting from a chemical interaction of the diol groups with functionalities present on the antimicrobial agents.

[0053] In some embodiments, the linker moiety is derived from a diamine comprising two amine groups. In such embodiments, the linker moiety may be derived from H2N-X- NH2, wherein X is a group as defined above. In some cases, X may be selected from -C1- C10alkylene, -C2-C10alkenylene, -C2-C10alkynylene, -C1-C5alkylene-O-C1-C5alkylene and others. In an antimicrobial material of the invention, the diamine may be presented as a reaction product (such as an amide or a diamide) resulting from a chemical interaction of the diamine groups with functionalities present on the antimicrobial agents.

[0054] In some embodiments, the linker moiety may be derived from HO(O=C)-X-OH, H0(0=C)-X-NH2, or H2N-X-OH, wherein X is a group selected as defined above. In some cases, X may be selected from -C1-C10alkylene, -C2-C10alkenylene, -C2- C10alkynylene, -C1-C5alkylene-O-C1-C5alkylene and others.

[0055] In some embodiments, the linker moiety comprises between 1 and 10 or between 1 and 5 carbon atoms and may thus be selected from -C1-C5alkylene, -C2-C5alkenylene, -C2-C5alkynylene, -C1-C5alkylene-O-C1-C5alkylene and others.

[0056] As used herein, the group ^-C1-Cioalkylene ' refers to a divalent moiety of alkyl, comprising between 1 and 10 carbon atoms, namely 1 to 10 -CH2- groups in a linear or branched sequence. In some embodiments, an alkylene group has 1 to 5 carbon atoms (C1-C5 alkylene). Examples of -C1-C10alkylene include methylene (Cl), ethylene (C2), propylene (C3) (e.g., n-propyl, isopropyl), butylene (C4) (e.g., n-butyl, tert-butyl, sec- butyl, isobutyl), pentylene (C5) (e.g., n-pentyl, 3-pentanyl, amyl, neopentyl, 3-methyl-2- butanyl, tert- amyl), and others. Unless otherwise specified, each instance of an alkylene group is independently unsubstituted or substituted with one or more substituents (e.g., not including Cl and F).

[0057] The term “-C2-Cioalkenylene” refers to a divalent alkenyl, comprising 2, 3, 4,

[0058] 5...0r 10 carbon atoms and 1 or 2 double bonds. In some embodiments, an alkenylene group has 2 to 5 carbon atoms (C2-C5alkenylene). Examples of C2-C5alkenylene groups include ethenylene (C2), 1-propenylene (C3), 2-propenylene (C3), 1-butenylene (C4), 2- butenylene (C4), butadienylene (C4), and the like.

[0059] The term “-C2-C10a.lkynyl.ene” refers to a divalent alkynylene, comprising 2, 3, 4,

[0060] 5. . . 10 carbon atoms and 1 or 2 triple bonds. In some embodiments, an alkynylene group has 2 to 5 carbon atoms (C2-C5alkynylene). Examples of C2-C5alkynylene groups include, without limitation, ethynylene (C2), 1-propynylene (C3), 2-propynylene (C3), 1- butynylene (C4), 2-butynylene (C4), and the like.

[0061] The term -C1-C5alkylene-O-C1-C5alkylene” refers to an interrupted alkylene having between 2 and 10 carbon atoms, as defined herein, wherein the alkylene is interrupted by an oxygen atom. Each of the “-Cl-C5alkylene” groups are selected independently.

[0062] The term “-C1-C5alkylene-Co-Cioarylene” refers to an alkylene comprising 1 to 5 carbon atoms, as defined herein, that is bonded to an arylene group comprising 6 to 10 carbon atoms. Arylene is a divalent moiety of aryl, a monocyclic or polycyclic aromatic ring or ring system. The arylene may be phenylene, 1 -naphthylene or 2-naphthylene. Examples of -C1-C5alkylene-C6-C10arylene include benzyl, phenylethylene, and others.

[0063] The term “-Co-Cioarylene” refers to an arylene, as defined herein, comprising 6 to 10 carbon atoms.

[0064] The terms “-C2-C5alkenylene-C6-Cioarylene” and “-C2-C5alkynylene-C6- Cioarylene” refer to alkenylene and alkynylene, respectively, that are bonded to an arylene moiety, each as defined herein.

[0065] The term -C3-Coheteroarylene” refers to a divalent heteroaryl comprising between 1 and 3 heteroatoms such as N, O and S, and between 4 and 7 carbon atoms. The heteroarylene is a 5-10 membered monocyclic or polycyclic aromatic ring system. Examples of heteroarylene groups include indolylene, quinolinylene, carbazolylene, and the like. In some embodiments, the linker moiety is derived from a dicarboxylic acid selected from ethanedioic acid, propanedioic acid, butanedioic acid, pentanedioic acid, hexanedioic acid, heptanedioic acid, octanedioic acid, nonanedioic acid, decanedioic acid and substituted forms thereof.

[0066] In some embodiments, the linker moiety is a succinyl linker, or is derived from succinic acid (butanedioic acid).

[0067] In some embodiments, a dimer of the invention comprises two same or different antimicrobial agents associated to each other via a 4-carbon chain, e.g., succinyl.

[0068] The bond or bonds associating the two antimicrobial agents is / are said to be hydrolysable, namely are dissociable in the presence of water to provide the two monomeric antimicrobial agents making up the dimer. Alternatively, the bond(s) is / are said to be labile, namely can dissociate under anhydrous conditions or by a mechanism other than hydrolysis, to provide the aforementioned monomeric agents.

[0069] In some embodiments, the bonds are labile bonds that upon dissociation generate the free antimicrobial agents that are water-soluble and water-stable in aqueous environments or under humid conditions. To stabilize or increase solubility in water, the dimers or antimicrobial materials of the invention may be provided in their free acid or free base forms or may be presented in a salt or acid forms.

[0070] The antimicrobial materials may be heterodimers in which the two antimicrobial agents are different, or homodimers in which the two agents are the same. In most general terms, the antimicrobial materials may thus have a structure A’-L-A, wherein each of A’ and A is an antimicrobial agent, L is a linker moiety which may be absent and each of “- “ constitutes a hydrolysable or labile covalent bond. In some cases, where L is missing, the dimer is of the form A’ -A.

[0071] The naturally occurring water-soluble antimicrobial agents forming materials of the invention , i.e., A and A’, may be selected amongst such agents that are water soluble and naturally occurring, which by themselves exhibit antimicrobial properties. These agents may have a water solubility that is at least 1 pg / L, or at least 10 pg / L, or at least 100 pg / L, or at least 0.5 or 1 g / L, in water at room temperature (25°C) and atmospheric pressure. Non-limiting examples of such antimicrobial agents include salicylic acid (SA), jasmonic acid (JA), oleanolic acid (OA), rosmarinic acid (RA), bonianic acid (BA), ursolic acid (UA), betulinic acid (TA) as well as various terpenes, alcohols, phenols and others. Each of the antimicrobial agents mentioned in the present application constitutes a separate and independent case for any of the aspects and embodiments disclosed herein.

[0072] Non-limiting examples of antimicrobial materials of the invention are dimers such as SA-SA, SA-JA, SA-OA, SA-RA, SA-BA, SA-UA, SA-TA, JA-JA, JA-OA, JA-RA, JA-BA, JA-UA, JA-TA, OA-OA, OA-RA, OA-BA, OA-UA, OA-TA, RA-RA, RA-BA, RA-UA, RA-TA, BA-BA, BA-UA, BA-TA, UA-UA, UA-TA, TA-TA, SA-L-SA, SA- L-JA, SA-L-OA, SA-L-RA, SA-L-BA, SA-L-UA, SA-L-TA, JA-L-JA, JA-L-OA, JA-L- RA, JA-L-BA, JA-L-UA, JA-L-TA, OA-L-OA, OA-L-RA, OA-L-BA, OA-L-UA, OA- L-TA, RA-L-RA, RA-L-BA, RA-L-UA, RA-L-TA, BA-L-BA, BA-L-UA, BA-L-TA, UA-L-UA, UA-L-TA, TA-L-TA, wherein each of SA, JA, OA, RA, BA, UA, TA is as defined above, L is a linker moiety and each of is a hydrolysable or labile covalent bond.

[0073] In some embodiments, L is absent.

[0074] In some embodiments, L comprises between 1 and 10 or between 1 and 5 carbon atoms.

[0075] In some embodiments, L is X as defined herein. In some embodiments, L is selected from -C1-C5alkylene, -C2-C5alkenylene, -C2-C5alkynylene, -C1-C5alkylene-O- C1-C5alkylene and others.

[0076] In some embodiments, L is derived from a dicarboxylic acid selected from ethanedioic acid, propanedioic acid, butanedioic acid, pentanedioic acid, hexanedioic acid, heptanedioic acid, octanedioic acid, nonanedioic acid, decanedioic acid and substituted forms thereof.

[0077] In some embodiments, L is a succinyl linker or is derived from succinic acid (butanedioic acid).

[0078] In some embodiments, L is succinyl.

[0079] In some embodiments, the antimicrobial material is a dimer comprising SA.

[0080] In some embodiments, the antimicrobial material is a dimer not comprising SA.

[0081] In some embodiments, the antimicrobial material is not SA-SA, or is not SA-L- SA, wherein L is any linker moiety. In some embodiments, the antimicrobial material is not bis(2-carboxyphenyl) succinate (or disalicylic acid; DSA), having a structure shown in Scheme 1.

[0082] Dimers or antimicrobial materials of the invention may be carried in various liquid or solid formulations which may be in a ready-for-use form or in a form of a concentrate or a dilutable form. Generally speaking, formulations of the invention comprise one or more of the dimers or materials disclosed herein, optionally in a carrier liquid or solid and further optionally in combination with one or more other active or inert ingredient.

[0083] Formulations of the invention may comprise at least one active ingredient capable of preventing, killing, or repelling a pest or an active ingredient that acts as a plant regulator, desiccant, defoliant, synergist, or nitrogen stabilizer. Inert or inactive ingredients may be selected amongst solvents, carriers, adjuvants, or any other material for dissolving any of the ingredients, for aiding in the delivery of the active ingredient, for inducing adhesivity or stickiness of the formulation to or spread out on the surface of the target, e.g., leaves, for dilution or reconstitution purposes, etc.

[0084] The medium used for making formulations of the invention is typically water or water-based.

[0085] Thus, the invention further provides compositions and formulations configured as antimicrobial or pesticidal formulations for use as plant-protection formulations, adapted for application by any method known in the art, including spraying, brushing, powdering, by irrigation, etc.

[0086] Further provided is an antimicrobial formulation comprising at least one material, as disclosed herein, for controlling a plant microbial source or a resultant disease caused thereby, wherein the microbial source is bacteria, viruses, fungi and / or herbivorous insects.

[0087] Formulations of the invention are generally purposed for crop protection and thus may be applied by any known means onto a surface region of the crop or in its growing vicinity. Additionally, formulations of the invention may be used pre-harvest or postharvest on agricultural products such as seeds, grains, flowers, fruits, vegetables, bulbs, and tubers, or may be embedded in functional packaging of foods and agricultural products for preventing the decomposition or rancidification of the food or product overtime. Formulations of the invention may further find use in the storage of crops, including grains, fruits, or vegetables, and cultivated flowers.

[0088] The invention further provides a method of treating or preventing or reducing or diminishing or ridding of growth of plant pathogens or herbivorous insects (including for examples bacteria, viruses, fungi), the method comprising applying to surface infested or prone to infestation by the pathogens or insects, e.g., the surface optionally being a plant, plant part, or to a region in a vicinity of the plant or plant part, a formulation comprising at least one antimicrobial material of the invention and an environmentally acceptable carrier.

[0089] As noted herein, formulations of the invention may be applied by a variety of ways, including for example spraying, brushing, powdering, by irrigation, etc. In some embodiments, the formulation may be applied by foliar application. In some cases, for special applications, trunk injection or laser ablation may be used for application.

[0090] In some embodiments, the formulation may be applied onto a surface of a seed, or a bulb, or a tuber, or an agricultural product by forming a coating thereon which comprises the material of the invention. Coating may be achievable by dipping, dust coating, blowing or smearing.

[0091] In some embodiments, methods of the invention may further include combining at least one dimer or agent of the invention with another plant active agent of a miticide, nematicide, fungicide, herbicide, a plant growth regulator, microbial agricultural chemical, soil conditioner and / or fertilizer.

[0092] The invention further provides a method of treating a soil region to reduce microbial infections in plant populations therein, the method comprising treating or delivering, e.g., by irrigation, to said soil region a formulation according to the invention.

[0093] The invention further provides an add-on plant protection kit comprising a solid or a liquid composition of at least one material of the invention, wherein the solid or liquid composition is configured for addition into a commercial or premade pesticide or fertilizer formulation.

[0094] The invention further provides:

[0095] An antimicrobial material being a dimer of two different antimicrobial agents associated to each other, directly or indirectly, via hydrolysable or labile covalent bond(s).

[0096] In some embodiments of a material according to the invention, each of the antimicrobial agents is selected amongst water soluble naturally derived or naturally occurring antimicrobial agents.

[0097] In some embodiments of a material according to the invention, it is for controlling a plant microbial source or a plant disease caused thereby, wherein the microbial source is bacteria, viruses, fungi and / or herbivorous insects.

[0098] In some embodiments of a material according to the invention, the two different antimicrobial agents are directly associated via a hydrolysable or labile covalent bond. In some embodiments of a material according to the invention, the two different antimicrobial agents are associated through a hydrolysable or labile linker moiety.

[0099] In some embodiments of a material according to the invention, the linker moiety is derived from a diacid, a diol, a diamine or a mixed form thereof.

[0100] In some embodiments of a material according to the invention, the linker moiety is derived from a diacid comprising two carboxylic acid moieties.

[0101] In some embodiments of a material according to the invention, the linker moiety is derived from a diacid of the formula HO(O=C)-X-(C=O)OH, wherein X is a group selected from -C1-C10alkylene, -C2-C10alkenylene, -C2-C10alkynylene, -C1-C5alkylene-O- C1-C5alkylene, -C1-C5alkylene-C6-C10arylene, -C6-C10arylene, -C2-C5alkenylene-C6- C10arylene and -C2-C5alkynylene-C6-C10arylene, and -C3-C6hctcroarylcnc.

[0102] In some embodiments of a material according to the invention, the linker moiety is derived from a diol comprising two hydroxyl moieties.

[0103] In some embodiments of a material according to the invention, the linker moiety is derived from a diol of the formula HO-X-OH, wherein X is a group selected from -C1- C10alkylene, -C2-C10alkenylene, -C2-C10alkynylene, -C1-C5alkylene-O-C1-C5alkylene, - C1-C5alkylene-C6-C10arylene, -C6-C10arylene, -C2-C5alkenylene-C6-C10arylene and -C2- C5alkynylene-C6-C10arylene, and -C3-C6hctcroarylcnc.

[0104] In some embodiments of a material according to the invention, the linker moiety is derived from a diamine comprising two amine groups.

[0105] In some embodiments of a material according to the invention, the linker moiety is derived from a diamine of the formula H2N-X-NH2, wherein X is a group selected from -C1-C10alkylene, -C2-C10alkenylene, -C2-C10alkynylene, -C1-C5alkylene-O-C1- C5alkylene, -C1-C5alkylene-C6-C10arylene, -C6-C10arylene, -C2-C5alkenylene-C6- C10arylene and -C2-C5alkynylene-C6-C10arylene, and -C3-C6hctcroarylcnc.

[0106] In some embodiments of a material according to the invention, the linker moiety comprises between 1 and 5 carbon atoms.

[0107] In some embodiments of a material according to the invention, the linker moiety comprises a group selected from -C1-C5alkylene, -C2-C5alkenylene, -C2-C5alkynylene, - C1-C5alkylene-O-C1-C5alkylene and others.

[0108] In some embodiments of a material according to the invention, the linker moiety is derived from a dicarboxylic acid selected from ethanedioic acid, propanedioic acid, butanedioic acid, pentanedioic acid, hexanedioic acid, heptanedioic acid, octanedioic acid, nonanedioic acid, decanedioic acid and substituted forms thereof.

[0109] In some embodiments of a material according to the invention, the linker moiety is a succinyl linker, or is derived from succinic acid (butanedioic acid).

[0110] In some embodiments of a material according to the invention, each of the naturally occurring water soluble antimicrobial agents is selected from salicylic acid (SA), jasmonic acid (J A), oleanolic acid (OA), rosmarinic acid (RA), bonianic acid (BA), ursolic acid (UA), and betulinic acid (TA).

[0111] In some embodiments of a material according to the invention, the material comprising SA.

[0112] In some embodiments of a material according to the invention, formed of an antimicrobial agent different from SA.

[0113] In some embodiments of a material according to the invention, the material comprising JA.

[0114] In some embodiments of a material according to the invention, the material comprising OA.

[0115] In some embodiments of a material according to the invention, being selected from SA-JA, SA-OA, SA-RA, SA-BA, SA-UA, SA-TA, JA-OA, JA-RA, JA-BA, JA- UA, JA-TA, OA-RA, OA-BA, OA-UA, OA-TA, RA-RA, RA-BA, RA-UA, RA-TA, BA- UA, BA-TA, UA-UA, UA-TA, SA-L-JA, SA-L-OA, SA-L-RA, SA-L-BA, SA-L-UA, SA-L-TA, JA-L-OA, JA-L-RA, JA-L-BA, JA-L-UA, JA-L-TA, OA-L-RA, OA-L-BA, OA-L-UA, OA-L-TA, RA-L-BA, RA-L-UA, RA-L-TA, BA-L-UA, BA-L-TA, and UA- L-TA, wherein each of SA, JA, OA, RA, BA, UA, TA is as defined herein, L is a linker moiety as defined herein and each of is a hydrolysable or labile covalent bond.

[0116] An antimicrobial material being a dimer of two antimicrobial agents, associated to each other, directly or indirectly, via hydrolysable or labile covalent bond(s), wherein each of the two antimicrobial agents is selected amongst water soluble naturally derived antimicrobial agents.

[0117] In some embodiments of a material according to the invention, being a homodimer or a heterodimer.

[0118] In some embodiments of a material according to the invention, for controlling a plant microbial source or a plant disease caused thereby, wherein the microbial source is bacteria, viruses, fungi and / or herbivorous insects. In some embodiments of a material according to the invention, the two antimicrobial agents are directly associated via a hydrolysable or labile covalent bond.

[0119] In some embodiments of a material according to the invention, the two antimicrobial agents are associated through a hydrolysable or labile linker moiety.

[0120] In some embodiments of a material according to the invention, the linker moiety is derived from a diacid, a diol, a diamine or a mixed form thereof.

[0121] In some embodiments of a material according to the invention, the linker moiety is derived from a diacid comprising two carboxylic acid moieties.

[0122] In some embodiments of a material according to the invention, the linker moiety is derived from a diacid of the formula HO(O=C)-X-(C=O)OH, wherein X is a group selected from -C1-C10alkylene, -C2-C10alkenylene, -C2-C10alkynylene, -C1-C5alkylene-O- C1-C5alkylene, -C1-C5alkylene-C6-C10arylene, -C6-C10arylene, -C2-C5alkenylene-C6- C10arylene and -C2-C5alkynylene-C6-C10arylene, and -C3-C6hctcroarylcnc.

[0123] In some embodiments of a material according to the invention, the linker moiety is derived from a diol comprising two hydroxyl moieties.

[0124] In some embodiments of a material according to the invention, the linker moiety is derived from a diol of the formula HO-X-OH, wherein X is a group selected from — C1-C10alkylene, -C2-C10alkenylene, -C2-C10alkynylene, -C1-C5alkylene-O-C1- C5alkylene, -C1-C5alkylene-C6-C10arylene, -C6-C10arylene, -C2-C5alkenylene-C6- C10arylene and -C2-C5alkynylene-C6-C10arylene, and -C3-C6hctcroarylcnc.

[0125] In some embodiments of a material according to the invention, the linker moiety is derived from a diamine comprising two amine groups.

[0126] In some embodiments of a material according to the invention, the linker moiety is derived from a diamine of the formula H2N-X-NH2, wherein X is a group selected from -C1-C10alkylene, -C2-C10alkenylene, -C2-C10alkynylene, -C1-C5alkylene-O-C1- C5alkylene, -C1-C5alkylene-C6-C10arylene, -C6-C10arylene, -C2-C5alkenylene-C6- C10arylene and -C2-C5alkynylene-C6-C10arylene, and -C3-C6hctcroarylcnc.

[0127] In some embodiments of a material according to the invention, the linker moiety comprises between 1 and 5 carbon atoms.

[0128] In some embodiments of a material according to the invention, the linker moiety comprises a group selected from -C1-C5alkylene, -C2-C5alkenylene, -C2-C5alkynylene, - C1-C5alkylene-O-C1-C5alkylene and others. In some embodiments of a material according to the invention, the linker moiety is derived from a dicarboxylic acid selected from ethanedioic acid, propanedioic acid, butanedioic acid, pentanedioic acid, hexanedioic acid, heptanedioic acid, octanedioic acid, nonanedioic acid, decanedioic acid and substituted forms thereof.

[0129] In some embodiments of a material according to the invention, the linker moiety is a succinyl linker, or is derived from succinic acid (butanedioic acid).

[0130] In some embodiments of a material according to the invention, each of the naturally occurring water soluble antimicrobial agents is selected from salicylic acid (SA), jasmonic acid (J A), oleanolic acid (OA), rosmarinic acid (RA), bonianic acid (BA), ursolic acid (UA), and betulinic acid (TA).

[0131] In some embodiments of a material according to the invention, the material comprising SA. In some embodiments of a material according to the invention, the material is formed of an antimicrobial agent different from SA. In some embodiments of a material according to the invention, the material comprising JA. In some embodiments of a material according to the invention, the material comprising OA.

[0132] In some embodiments of a material according to the invention, being selected from SA-SA, SA-JA, SA-OA, SA-RA, SA-BA, SA-UA, SA-TA, JA-JA, JA-OA, JA-RA, JA-BA, JA-UA, JA-TA, OA-OA, OA-RA, OA-BA, OA-UA, OA-TA, RA-RA, RA-BA, RA-UA, RA-TA, BA-BA, BA-UA, BA-TA, UA-UA, UA-TA, TA-TA, SA-L-SA, SA- L-JA, SA-L-OA, SA-L-RA, SA-L-BA, SA-L-UA, SA-L-TA, JA-L-JA, JA-L-OA, JA-L- RA, JA-L-BA, JA-L-UA, JA-L-TA, OA-L-OA, OA-L-RA, OA-L-BA, OA-L-UA, OA- L-TA, RA-L-RA, RA-L-BA, RA-L-UA, RA-L-TA, BA-L-BA, BA-L-UA, BA-L-TA, UA-L-UA, UA-L-TA, and TA-L-TA, wherein each of SA, JA, OA, RA, BA, UA, TA is as defined herein, L is a linker moiety as defined herein and each of is a hydrolysable or labile covalent bond.

[0133] An antimicrobial formulation comprising a material according to the invention.

[0134] In some embodiments of a formulation according to the invention, it is an aqueous formulation.

[0135] In some embodiments of a formulation according to the invention, it is a plantprotection formulation.

[0136] A method of treating or preventing growth of plant pathogens or herbivorous insects, the method comprising applying to a surface infested or prone to infestation by the pathogens or insects a formulation comprising at least one material or an antimicrobial formulation according to the invention.

[0137] In some embodiments of a method according to the invention, the surface is a plant, plant part, or to a region in a vicinity of the plant or plant part.

[0138] In some embodiments of a method according to the invention, the formulation is applied onto a surface of a seed, or a bulb, or a tuber, or an agricultural product by forming a coating thereon.

[0139] A method of treating a soil region to reduce microbial infections in plant populations therein, the method comprising treating or delivering to said soil region a formulation comprising a material according to the invention.

[0140] In some embodiments of a method according to the invention, the formulation is delivered via irrigation.

[0141] A plant protection kit comprising a solid or a liquid formulation of at least one material according to the invention, wherein the solid or liquid formulation is configured for addition into a commercial or premade pesticide or fertilizer formulation; and instructions of use.

[0142] An antimicrobial material according to the invention, the antimicrobial material being in a form suitable for preventing or eradicating or diminishing growth of a plant microbial source selected from bacteria, viruses, fungi and / or herbivorous insects.

[0143] In some embodiments of a material according to the invention, for use pre-harvest or post-harvest.

[0144] In some embodiments of a material according to the invention, for direct application onto a plant or plant part.

[0145] In some embodiments of a material according to the invention, the dimer is a homodimer or a heterodimer of SA.

[0146] In some embodiments of a material according to the invention, the dimer is a homodimer or a heterodimer of OA.

[0147] In some embodiments of a material according to the invention, the dimer is a homodimer or a heterodimer of J A.

[0148] In some embodiments of a material according to the invention, the dimer is a heterodimer. BRIEF DESCRIPTION OF THE DRAWINGS

[0149] In order to better understand the subject matter that is disclosed herein and to exemplify how it may be carried out in practice, embodiments will now be described, by way of non-limiting example only, with reference to the accompanying drawings, in which:

[0150] Figs. 1A-1B illustrate Minimum Inhibitory Concentration (MIC) of DSA (1A) and SA (IB) in P. brasiliense Pcbl692. Inoculum density was 1 x 106per mL CFU, bacteria were exposed to 0.3-1.5 mM DSA or 1.5-12 mM SA and incubated for 18 h at 28°C. Final bacterial concentrations were assessed by measuring absorbance at 600 nm. Dots represent average of three experiments with 8 replicates each (bar = SE; n = 3)

[0151] Fig. 2 illustrates the effect of sub-MIC concentrations DSA or SA on biofilm formation of P. brasiliense Pcbl692. Biofilm formation was measured after 72 h of in LB medium (with the compounds or water) at 28°C. Bars represent the absorbance of crystal violet dye measured at 590 nm. Data points represent results of 3 experiments with 6 replicates per treatment (means± SE), using one-way ANOVA with post hoc Tukey HSD test to analyze differences (GraphPad Prism 8.0). Bars labeled with different letters indicate significant differences (p < 0.05).

[0152] Figs. 3A-3B illustrate the effect of sub-MIC concentrations DSA or SA on the motility of P. brasiliense Pcbl692. Pb 1692 was applied to the center of soft agar plates following exposure to SA or DSA at specified concentrations, or DDW treated control. Motility diameter was measured after incubation (24 h at 28°C). Bars represent results of 4 experiments with 3 replicates each (means± SE), using one-way ANOVA with post hoc Tukey HSD test to analyze differences. Bars labeled with different letters indicate significant differences (p < 0.05).

[0153] Figs. 4A-4B illustrate the effect of sub-MIC concentrations DSA or SA on the production AHL, quorum sensing (QS) signaling molecules, in P. brasiliense Pbl692, using C. violaceum CV026 reporter strain and the AHL induced violacein pigment. Results were estimated by visual analysis of the violacein production (outer circle) in the DSA, SA and DDW-control treated plates (4A) and quantitative analysis of violacein at 550 nm calculated as % inhibition rate relative to control (4B). Bars represent results of 8 replicates per treatment (means± SE), using one-way ANOVA and post hoc Tukey HSD test to analyze differences. Bars labeled with different letters indicate significant differences (p < 0.05). Figs. 5A-5B illustrate the effect of sub-MIC concentrations DSA or SA on AHL production in P. brasiliense Pbl692, using the E. coli pSB401 bioluminescence reporter. Luminescence (250 nm) and absorbance (600 nm) measured every 30 min for 18 h. Relative luminescence (RLU = LU / OD600nm) was calculated using E. coli pSB401 grown in the absence of Pbl692 supernatants as blank (5A). Growth curves of E. coli pSB401 (OD) in the presence of different DSA and SA concentrations is also shown (5B). Data points represent results of 8 replicates per treatment (OD-optical density, LU-light units, RLU-relative light units).

[0154] Figs. 6A-6C illustrate the effect of sub-MIC concentrations DSA or SA on the activities of plant cell wall degrading enzymes (PCWDEs), Pectate lyase Pel, (6A), polygalacturonase Peh, (6B) and proteolytic Prt, (6C) in P. brasiliense Pbl692. Enzymatic activity was determined following overnight exposure of bacterial suspensions the specified concentrations of the tested compounds and DDW-control, measuring the size of haloes caused by substrate degradation. Results (% activity relative to control) represent 4 replicates (means± SE), using one-way ANOVA and post hoc Tukey HSD tests. Bars labeled with different letters indicate significant differences (p < 0.05).

[0155] Figs. 7A-7B illustrate the effect of sub-MIC concentration DSA or SA on the pathogenicity (virulence) of P. brasiliense Pcbl692 in potatoes (7A), potatoes and calla lily (7B). Pbl692 was exposed to DSA, SA or DDW-control, bacterial inoculum was normalized to 5 x 106 CFU per mL for potato and 1 x 107 CFU per mL for calla lily. Virulence was determined as the percentage of decayed or infected tissue, 24 h post inoculation for calla lily and 48 h for potato tubers, relative to plant tissues treated with control bacteria (DDW treated). Bars represent two experiments with 10 replicates for calla lily and 4 replicates for potato each (means± SE), using one-way ANOVA and post hoc Tukey HSD tests. Bars labeled with different letters indicate significant differences (p < 0.05). Representative pictures are also shown (bottom left and right).

[0156] Figs. 8A-8B illustrate the effects of SA-JA composite (10 mM) on P. brasiliense Pcbl692 in the potato. Virulence was determined as % decayed tissue 24 h post inoculation of potato tubers with bacterial suspension (106CFU) relative to the decay induced by untreated bacterial cultures (control). Data represents two experiments with three replicates each (means± SE). Bars labeled with different letters indicate significant differences (p < 0.05). Representative pictures are also shown. Figs. 9A-B provides sublimation rates for composite 1, OA-SA, SA and OA using TGA. (9A) gradual heating at 30 - 550 °C; (9B) isothermal heating at 80 °C, 2 h.

[0157] Figs. 10A-C provide (10A) Illustration of the proposed physical model of passive membrane permeability. (10B) Illustrations of the SA and OA 3D models. (10C) Two conformer structures with minimum energy.

[0158] Fig. 11 provides physical membrane values calculated for OA-SA, SA, and OA compounds.

[0159] Figs. 12A-C provides photos of potato tubers treated with OA-SA composite, salicylic acid and oleanolic acid, (12A), (12B) and (12C), respectively.

[0160] Figs. 13A-B illustrates the effects of the OA-SA composite, salicylic acid and oleanolic acid (10 mM concentration each), on PC ssp. brasiliense Pcbl692 on potato. (13A) Percentage of potato decay. (13B) bacteria count CFU / ml of the potato with the treatment of the OA-SA composite, salicylic acid, and oleanolic acid Potato tubers were injected with 10 μL of bacterial suspension [IO*5colony -forming units (CFU)] and incubated at 30 °C. Virulence was determined as the percentage of decayed tissue 24 h after the inoculation of potato tubers, relative to the decay induced by untreated bacterial cultures (control). Data represents the means ± standard errors (SE) of two independent experiments with three replicates for potatoes in each experiment. Treatments that are not labeled with the same letter in each panel are significantly different (P < 0.05). Representative pictures of infected potato tuber discs are shown for each treatment.

[0161] Figs. 14A-B provides an illustration of a molecular networking of crude extracts in a positive ionization mode of PC ssp. brasiliense Pcbl692 on potato after treatment of the OA-SA composite, salicylic acid, and oleanolic acid (14A) Molecular networking connected to tuberonic acid glucoside; (14B) Molecular networking connected to jasmonic acid.

[0162] DETAILED DESCRIPTION

[0163] To demonstrate the uniqueness and generic characteristics of materials of the invention, the inventors have developed two new methodologies for forming the dimers, exemplified herein by the formation of a homodimer of SA and a heterodimer of SA-JA. Both methodologies, apart from being natural-based and eco-friendly in terms of amounts of solvents to avoid environmental damage, yielded products that proved to be highly efficient and advantageous in terms of efficiency and span of antimicrobial and other activities impacting of plant resistance and plant defense mechanisms in general. More specifically, a homodimer of SA, bis(2-carboxyphenyl) succinate (disalicylic acid; DSA), was synthetized from two salicylic acids associated via a succinyl linker. The chemical structures of SA and DSA are shown in Scheme 1 below.

[0164] Superior antimicrobial activity and anti- virulence properties of DSA were initially revealed in experiments showing that the inhibitory effect of DSA on P. brasiliense was apparent at significantly lower Minimal Inhibitory Concentrations (MIC) than SA. These results were consistent with a parallel in silico analysis by computational docking tools, predicting that DSA may have a greater inhibitory potential on the bacterial QS (quorum sensing) related genes, such AHL synthase (expl) in P. brasilense, than SA. This is while implication of DSA in QS machinery could suggest a broader long-lasting mechanism of action on multiple levels of plant defense. This prediction proved to be true in further experiments showing that DSA acts on specific virulence determinants related to QS machinery, inhibiting biofilm formation, secretion of plant cell wall-degrading enzymes, motility, and production of AHL (acyl-homoserine lactone), eventually manifested in an impaired virulence of P. brasiliense. Inhibited AHL production upon DSA application on P. brasiliense was further reproduced with biosensors such as Chromobacterium violaceaum CV026 and E. coli pSB401. (EXAMPLE 1)

[0165] These results suggest that inhibition of the QS machinery is only one of the mechanisms by which DSA inhibits specific virulence determinants. Nonetheless, they propose a new method for producing DSA and call for repurposing thereof for new application as an effective and a multipotent plant protecting agent.

[0166] The inventors have further demonstrated that the same effects of a broad antimicrobial activity and plant protectivity were not specific to DSA per se but were rather characteristic of the dimeric structures and could therefore encompass other SA dimers. Another example that was studied and exemplified in this application is an SA- JA dimer produced by a novel method from SA and JA associated by a 5-carbon linker via two ester bonds. The chemical structure of the SA-JA conjugate is shown in Scheme 2 below.

[0167] Scheme 2

[0168] One of the main incentives for the synthesis of dimers such as SA-JA has been to reduce volatility and to allow spray-ability and a wider applicability of the resulting product. This assumption has proved to be true in the subsequent volatility, showing a significantly lower volatility of the dimer compared to the monomeric building blocks. By calculations of membrane permeability, accounting for the predicted 3D structure of the dimer and the monomers and the respective internal resistance of the membrane and at the water-membrane interface, suggested that the physical values of the dimer had the potential of a better membrane permeability than the monomers. A preliminary study of the effect of SA-JA on the soft-rot phenotype in potatoes induced by P. brasiliense showed that the dimer dramatically reduced the degree of necrosis in the treated potato tubers compared to control. (EXAMPLE 2)

[0169] Overall, the studies in this application demonstrate the attractiveness of the new SA dimers in the prospect of replacing the commonly used pesticides that produce continuing risks of emerging resistance and environmental population. Being natural, biodegradable, eco-friendly, effective and cheap, these new chemical entities can lead to new lines of products with wide applicability in agriculture, food preservation and related industries.

[0170] Any method and material similar or equivalent to those described herein can be used in the practice or testing of the present invention. Some embodiments of the invention will be now described by way of examples with reference to respective figures.

[0171] EXAMPLE 1: DS A - an efficient antimicrobial acting on Quorum Sensing (DS) Materials and Methods

[0172] Chemicals, bacteria, and growth conditions

[0173] Commercial Bis(2-carboxyphenyl) succinate (Santa Cruz) and salicylic acid (Sigma Aldrich), 98% and 99% pure, respectively, were used for preparing stock solutions in 1% DMSO, control treatments were using 1% DMSO. Bacterial strains, P. brasiliense Pcbl692 and Chromobacterim violaceum CV026 were cultivated at 28°C; E. coli strains at 37°C, using LB medium (Difco Laboratories, USA) under continuous shaking (150 rpm).

[0174] Synthesis

[0175] Bis(2-carboxyphenyl) succinate 1 eq. of succinyl chloride (154 mg, 1 mmol) was added dropwise to a flask containing 2 eq. salicylic acid (276, 2 mmol). The reaction mixture was stirred at 40°C overnight. The solvent evaporated and DSA was purified on a silica gel column using DCM: MeOH (98:2, Yield 76%).

[0176] Homology modeling and docking

[0177] The Expl homology model (UniProtKB identifier P33882) was generated based on the crystal structures of homologous proteins, Esal (PDB code: 1KZF) and TofI (PDB code: 3P2H). Prior to docking, the model was processed with the Protein Preparation tool in Maestro (Schrodinger, USA) to assign correct protonation states for all residues at physiological pH. The grid box was centered on the merged site that consists of the binding sites for the acyl chain carrier protein and S-adenosyl-L-methionine (SAM). The structures of the ligands were minimized with the OPLS3e force field and processed by the Ligprep procedure to assign correct protonation states at physiological pH. Docking of three ligands was performed, Sadenosyl-L-methionine (SAM), salicylic acid (SA), and bis(2-carboxyphenyl) succinate (DSA). In Induced Fit Flexible docking (IFD) analysis, residues within 5 A° of any of the resulting top 20 ligand positions (from initial rigid docking) were subjected to conformational search and minimization while residues outside this region were kept fixed. The final 20 new receptor conformations were taken forward for redocking. Glide SP (standard precisions) was used for all docking calculations. The binding affinity of each complex was evaluated by the IFD Score.

[0178] MIC assay

[0179] MIC assays (by CLSI guidelines) were used for determining antimicrobial activity of DSA and SA. Briefly, bacterial cultures were grown in LB overnight and normalized to 1 x 106 colony forming units (CFU) per mL with fresh LB. Aliquots (20 pl) were then used to inoculate 180 mL LB containing 2-fold serial dilutions of each of the tested compounds. Overnight cultures of Pcbl692 were grown in LB. Cultures were diluted to a final inoculum of 106 CFU / mL in 96-well microtiter plates, compounds were added at appropriate concentrations and MIC was determined after 18 h incubation at 28°C. QS assays were performed at sub-inhibitory concentrations, i.e., lower than MIC values.

[0180] Biofilm inhibition assay

[0181] The assay was performed using microtiter dish assay with crystal violet (CV) for biofilm staining and quantified by measuring the absorbance at 550 nm in a microplate reader (the absorbance of CV dye bound to biofilm). Mean from eight replicates was calculated after subtraction of blank.

[0182] Qualitative assays for the detection of AHL in Pcbl692

[0183] The assay used CV026 strain of C. violaceum (reporter strain) in which the violet pigment violacein is induced in the presence of AHL with N-acyl C4 to C8 side chains, and P. brasiliense. Pcbl692 (test strain) to assess the effect of DSA and SA on the production of AHL. Pcbl692 was grown as described above, centrifuged (7,000 x g, 5 min, at 28°C) and bacterial pellets were re-suspended in LB. CV026 was grown in fresh LB medium supplemented with kanamycin (10 pg / mL). DSA or SA were applied in the center on a paper disc on LB plates and Pc 1692 and CV026 were plated in circles in distances of few millimeters. Plates were incubated overnight at 28°C and subjected to visualization assessment.

[0184] Quantitative assay for the detection of AHL in Pcbl692

[0185] The assay was based on occulting of Pcbl692 (test strain) and CV026 (reporter strain) with quantitation of the violet color. Pcbl692 was grown in LB (48 h, 28°C) with or without DSA or SA at sub-MIC concentrations (0.11 mM - 1 mM), centrifuged (5,223 g, 10 min, 4°C), and the supernatant was filtered. Supernatant (500 pl) was mixed with LB (500 pl) inoculated with 5xl06CV026 (with kanamycin) and incubated (24 h, 28°C). The overnight culture (1 mL) was centrifuged (13,793 g, 10 min) to precipitate the insoluble violacein, the pellet was re-suspended DMSO (ImL), centrifuged (13,793 g, 10 min) to remove the cells and the violacein was quantified at 585 nm using UV-Vis spectrophotometer (SPARK, TEKAN machine). Percentage of violacein inhibition was calculated by the following equation:

[0186] Quantitative assay for the detection of AHL using bioluminescence The assay used E. coli JM109 strain carrying the pSB401 plasmid with the fused luxRI’luxCDABE bioluminescent reporter gene, enabling to detect AHL with acyl chains ranging C6 to C8. This QS biosensor was used to quantitatively assess the secretion of AHL by Pcbl692 in the presence of DS A or SA. Pellets of overnight grown bacteria were resuspended in LB with or without DSA and SA at sub-MIC concentrations and incubated (8 h). Control and treated samples were centrifuged and supernatant (10 pl) was mixed with 190 μL of 5 x 106 CFU / mL E. coli pSB401 in LB in 96-well plates. The supernatant (10 μL) containing DSA and SA was diluted 20-folds in LB (final volume 200 pl), the reporter strain (200 pl) was used as a blank and 1% DMSO as control. Plates were incubated (37°C, 17 h) and bioluminescence and OD were determined simultaneously by UV-Vis spectrophotometer every 30 min at 250 nm and 600 nm, respectively. Bioluminescence was calculated as relative light units (RLU) per unit OD at 600 nm.

[0187] Activities of hydrolytic enzymes

[0188] Activities of Pcbl692 pectate lyase (Pel), polygalacturonase (Peh) and proteolytic enzymes (Prt) were determined after overnight exposure of bacteria (in LB at 28°C) to non-lethal concentrations (50% inhibition) of DSA and SA, using previously described semiquantitative assays (Chatterjee et al. Appl Environ Microbiol. 1995;61(5)). Plates were poked to form 4 mm holes that were filled with the supernatants from overnight grown cultures and incubated (at 28°C, 24 h). Enzymes’ activities were assessed as the size of the observed haloes. Two independent experiments were carried out, each with four replicates of each compound concentration.

[0189] Virulence assays

[0190] Virulence was evaluated by assessing symptom severity in two plants- Zantedeschia aethiopica (calla lily) and Solanum tuberosum “Lady Rosetta”. Fully expanded young leaves of calla lily potato tubers were externally disinfected by soaking in 0.7% sodium hypochlorite (20 min) and washed with sterilized DW. Whole disinfected potato tubers were used for inoculation. Discs (20 mm in diameter) were excised from disinfected leaves of calla lily and transferred to petri dishes containing Murashige and Skoog (MS) medium. Bacterial strains were grown overnight (in LB at 28°C) with continuous shaking and diluted to 1 x 107 cfu mL-1 (OD 600 *4 0.1) in sterile DDW containing non-lethal concentrations of DSA and SA. Bacterial suspensions were shaken (150 rpm for 2 h at 28°C) before inoculation. Leaf discs and potato tubers were pierced at the center with a sterile tip and inoculated with 10 mL bacterial suspensions. Inoculated plant material was incubated (at 28°C). For potato tubers, disease severity was determined as the percentage of rotten tissue determined by weighing the whole tuber and subtracting the tuber weight after the decayed portion was scraped away 48 h after inoculation. For calla lily disease severity was determined as the percentage of decayed tissue relative to the total area of the disc 24 h after inoculation. Two independent experiments were carried out, each with four replicates of each compound concentration.

[0191] Results

[0192] 1.1 A novel method for the synthesis ofDSA

[0193] Bis(2-carboxyphenyl) succinate (DSA) was synthetized by a novel method using one eq. SA acid and 2 eq. succinyl chloride mixed at 50°C overnight under reflux conditions. Due to the use of succinyl chloride to dissolve SA and the exclusion of other solvents, this method has reduced environmental damage and can be considered eco- friendly and advantageous for producing plant protection agents.

[0194] 1.2 Determination of Minimum Inhibitory Concentration (MIC)

[0195] MICs were determined as the lowest concentration of DSA or SA with no measurable growth (increase in absorbance). MIC values for DSA and SA are shown in Figs. 1A and IB, respectively. MIC of DSA (1.5 mM) was 4-fold lower than of SA (6 mM). The following experiments, biofilm formation, motility, signal molecules accumulation etc., used non-lethal or sub-MIC concentrations of DSA and SA (<1 mM) that did not exceed 50% growth inhibition relative to the water treated control.

[0196] 1.3 Molecular docking of SAM, DSA and SA to Expl

[0197] The QS signaling molecules in P. brasilense, 3-oxohexanoyl homoserine lactone (3-oxoC6HSL) or 3-oxooctanoyl homoserine lactone (3-oxo-C8HSL; AHL), are biosynthesized from Sadenosyl methionine (SAM) and acylated carrier protein by AHL synthase Expl, a LuxI ortholog. Analysis of docking results showed that SAM binds to the SAM cavity of the enzyme, forming five hydrogen bonds: two with the backbone Phe44 and ArglOl and three with the side chains Glu43, Asp45, and Glul70. The adenine moiety of SAM participates in p-p stacking interactions with the side chain Trp34 and Phe82. SAM further makes two salt bridges with Glu43 and Asp45. SA also binds to the SAM cavity, forming one hydrogen bond with the backbone ArglOl, two with the side chains Arg24 and Asp45, and one p-p interaction with Trp34, and salt bridges with Arg24. DSA also binds to the SAM cavity although part of the structure is oriented in a different direction. DSA makes one hydrogen bond with the backbone ArglOl, two hydrogen bonds with the side chains Arg24 and Serl44, and one salt bridge with Arg24. Superimposition of SAM, SA, and DSA enzyme binding sites suggested that DSA and SA can compete with the natural precursor for binding site interactions, thereby interfering with AHL production. According to the prediction, the best binder to the merged site in Exp I is SAM (-432.36 kcal mol-1), followed by DSA (-427.6 kcal mol-1) and SA (-421.6kcal mol-1), which would lead to impaired virulence and virulence-related activities as was subsequently shown in the experiments below.

[0198] 1.4 Biofilm formation

[0199] The effect of DSA on the biofilm formation ability of P. brasiliense Pcbl692 was studied using a microtiter dish assay under non-inhibitory concentrations of DSA and SA. The results are shown in Fig. 2 (DSA grey, SA black). Significant inhibition of biofilm formation was observed with DSA at concentrations as low as 0.3 and 0.4 mM compared to only partial inhibition with SA at concentrations of 0.6 mM 1.0 mM and 1.5 mM. Both compounds acted in dose dependent manner.

[0200] 1.5 Effect of DSA and SA on motility

[0201] Motility is an important virulence trait of many plant-pathogenic bacteria. The effects of DSA and SA on the motility of P. brasiliense Pbl692. The results are shown in Figs. 3A-3B. While the highest motility rate was observed in the DDW control treatment, DSA significantly inhibited Pbl692 motility at the concentrations as low as 0.6 mM compared to SA that remained ineffective at 0.6 mM and ImM and slightly inhibited the motility at 1.5 mM. Overall, DSA had a stronger inhibitory effect on Pb 1692 motility.

[0202] 1.6 DSA suppress the production of QS signaling molecules in Pbl692

[0203] AHL are the most common signaling molecules of QS in Pectobacteria, and the main signals that regulate the synthesis of plant cell wall degrading enzymes (PCWDEs), the primary virulence determinants of soft rotting bacteria such as P. brasiliense. The effect of DSA on the production of AHL in P. brasiliense Pbl692 was studied using C. violaceum CV026 as a reported strain and qualitative and quantitative analysis of the AHL induced violacein pigment (purple). The results are shown in Figs. 4A-4B. DSA, SA (sub-MIC concentrations) or DDW-control applied to the paper disc at the center diffused to Pbl692in the inner circle, affecting the production of violacein by the reporter in the outer circle (Fig. 4A). While control and SA permitted the visualization of the violacein signal in the outer circle, DSA significantly inhibited the production of violacein at the concentration of ImM. Subsequent quantitative analysis showed that DSA significantly inhibited the production of violacein at the concentration as low as 0.1 mM compared to the same extent of inhibition with 0.8 mM SA (Fig. 4B), suggesting that DSA is about 8-fold more potent in inhibiting AHL production in P. brasiliense Pbl692.

[0204] An additional experiment used the E. coli pSB401 biosensor produced on the background of E. coli JM109, with the fusion luxRI'luxCDABE bioluminescence reporter enabling to quantify the AHL production in response to QS signaling molecules. Supernatants of bacterial cultures were pre-exposed to DSA, SA (sub-MIC concentrations) or DDW-control and bioluminescence levels were correlated with the amount of AHL during 8 h. The results are shown in Figs 5A-5B. DSA strongly inhibited bioluminescence at the concentration as low as 0.4 mM and achieved 25% inhibition at the concentration of 0.6mM, while the same extent of inhibition could be observed in none of the SA concentrations (Fig. 5A). The effect of DSA on AHL production was 3- to 4-fold higher than the effect of SA. Parallel analysis of bacterial growth by OD showed that the reporter strain growth was not affected by any of the above treatments (Fig. 5B). The results were consistent with the experiment using the C. violaceum CV026 reporter.

[0205] Overall, the results suggest that DSA is a significantly more effective inhibitor of AHL production than SA at sub-MIC concentrations, meaning without apparent effect on bacterial growth, which is entirely consistent with its putative role in QS.

[0206] 1.7 Effect of DSA compound on exoenzyme activity

[0207] Exoenzyme activity is a virulence determinant that is crucial for necrotrophic pathogens, including Pectobacteria, which is mediated by the secretion and activity of plant cell wall degrading enzymes (PCWDEs) that promote soft rot decay. DSA and SA effects on the activities of exoenzymes such as pectate lyases (Pel), polygalacturonases (Peh), and proteases (Prt) were studied in P. brasiliense Pbl692, comparing DSA, SA (sub-MIC concentrations) or DDW-control. The results are shown in Figs. 6A-6C. DSA at the concentration of 1.2 mM achieved about 50% reduction in Pel activity, and about 40-50% reduction in Peh and Prt activities (Figs. 6A, B, C, respectively) compared to up to 20-30% reduction with the same co concentration of SA. At all concentrations, DSA proved to be a better inhibitor of these three different PCWDEs activities than SA.

[0208] 1.8 Infection assays following treatment with DSA or SA The effect of DSA or SA was further studied with respect to bacterial pathogenicity using two unrelated hosts, potato tubers and calla lily leaf and plant tissues. P. brasiliense Pcbl692 was exposed to DSA or SA (sub-MIC concentrations) and DDW- control for 2h and inoculated on the tested plants. The results are shown in Figs. 7A-7B for potatoes and calla lily, respectively. In both host, DSA inhibited the manifestation of pathogenic symptoms at much lower concentrations than SA, with complete inhibition of symptoms in potatoes with 0.1-0.2 mM DSA compared to 0.4 mM SA (Fig. 7A) and 50% inhibition of symptoms in calla lily with 0.2 mM DSA compared to no inhibition with the same SA concentration (Fig. 7B). Overall, the effect of DSA was about 4-fold more significant than SA, with no effect on bacterial growth at all concentrations.

[0209] Conclusions

[0210] Effective and safe pesticides that can be synthesized in sufficient and cost- effective amounts is one of the leading incentives of the current agriculture R&D. Even more challenging is finding compounds that may act by multifunctional or multi-hurdle mechanisms to lower effective concentrations and overcome the emerging resistance. Owing to the important role of QS in the pathogenesis of many plant and animal bacteria, several anti-QS compounds have become subjects to intense basic and translational research, including in agriculture.

[0211] SA is a well-known Gram-positive and Gram-negative antibacterial compound, including Pectobacteria. Here it was shown that DSA, a new double SA conjugate, can act as as an effective antimicrobial agent that was superior to SA by the ability to impair virulence and inhibit manifestation of pathogenic symptoms. The protective effects of DSA and SA were evaluated in a series of physiological assessments in plant pathogenic bacterial model and molecular docking calculations. DSA proved to have significantly better ability than SA in suppressing virulence related activities, such as biofilm formation, motility, PCWDEs production and production of AHL signaling molecules. The MIC value of DSA for P. brasiliense Pbl692 growth inhibition was 4-times lower than SA, which could not be explained by a simple stoichiometry of the two compounds (expected to be half of that of SA). Subsequent computing docking analysis suggested that DSA may have a higher affinity and binding rate to the active site of the AHL synthase Expl compared to SA, which can explain the unexpectedly high MIC values. Further, DSA significantly impaired the biofilm formation and motility of P. brasilense at a rate that exceeded the actual concentration of 2-hydroxybenzoic acid (SA) in the suspension (if DSA had been degraded). Inhibition of biofilm formation was 7-fold more effective with 0.4 mM DSA than SA, with no effect on growth. Motility was reduced by 3-fold more with 0.6 mM DSA than SA and 5-fold more with 1.5 mM DSA than SA.

[0212] Still further, as was suggested by two biosensor strains, DSA significantly reduced AHL production in P. brasilense. In both experiments, DSA inhibited AHL synthesis by Expl in a concentration-dependent manner and significantly more efficiently than SA. A stronger effect of DSA on QS inhibition was predicted using computational docking tools. These findings, however, cannot rule out the involvement of other mechanisms by which DSA can exert its anti-virulent activity.

[0213] To conclude, present findings provide direct evidence for the inhibitory effect of DSA on bacterial virulence, which significantly exceeds the effect of SA by several virulence determinants and in concentrations that do not impair growth. The differences between DSA and SA could not be explained by simple stoichiometry but rather by a higher affinity of DSA to the Expl active site, or another mechanism such as a better membrane permeability or accumulation in bacterial cells. In any of these scenarios, DSA holds great potential as a novel advantageous anti-virulence agent.

[0214] EXAMPLE 2: SA- J A conjugate — a novel effective antimicrobial agent

[0215] Materials and Methods

[0216] Chemicals and bacteria

[0217] Methyl jasmonate (J A), salicylic acid (SA), and 1,5-dibromopropane were purchased from Sigma Aldrich. P. brasiliense Pcbl692 was cultivated at 30°C in LB (Difco Laboratories, Detroit, MI, USA) under continuous shaking (150 rpm).

[0218] Reaction progress

[0219] The progress of synthesis reactions was monitored using TLC on pre-coated silica plates (Merck 60F-254, 250pm). Spots were visualized using UV light. Merck silica gel (100-200 mesh) served for general purification using column chromatography. Analytical HPLC-MS on Agilent 1260 series Liquid Chromatograph / Mass Selective Detector (MSD) (Single Quadrupole) equipped with an electrospray interface and a UV diode array detector served to verify products identity. Analytical HPLC served for quantity determination, using an Agilent 1100 series Liquid Chromatograph equipped with a UV diode array detector: XBridge C18 50x3.0mm 3.5pm column, ImL / min flow, and a gradient of 10-90% MeCN in water with 10 mM ammonium carbonate over 9 minutes, 215-395 nm, 254 nm. ESI MS with positive ionization. 1 H-NMR and 13C- NMR spectra were recorded on a Bruker 400 / 700 spectrometer. Chemical shifts were referenced to solvent peaks: 6H 3.30 and 6C 49.0 for CD3OD.

[0220] Synthetic procedures

[0221] 5-bromopentyl 2-hydroxybenzoate - Compound 1: Salicylic acid (500 mg, 3.62 mmol) was added to K2CO3 solution (552 mg, 4 mmol) in 7 ml dry DMF at 40°C. 1,5- dibromopentane (0.55 ml, 3.9 mmol) was added dropwise. The reaction mixture was stirred overnight. The solvent was evaporated, and the product was purified on a silica gel column using DCM: MeOH, 98:2. Yield - 44% (2 ml).

[0222] (Z)-2-((5-(2-(3-oxo-2-(pent-2-en-l-yl)cvclopentyl)acetoxy)pentyl)oxy)benzoic acid: Jasmonic acid (0.2 ml, 0.95 mmol) was added to K2CO3 solution (94 mg, 0.68 mmol) in 1 ml dry DMF at 40°C. Compound 1 (0.26 ml, 0.95 mmol), was added dropwise. The reaction mixture was stirred at 40°C overnight. The solvent was evaporated, and the composite was purified on silica gel column using DCM: MeOH, 97:3. MS ES+ 417. 1H NMR (400 MHz, CDC13): 8 10.8 (s, 1H), 7.8 (m, 1H), 7.4 (m, 1H, J = 8.0 Hz), 6.9 (m, 1H), 6.8 (m, 1H, J = 8.0 Hz), 5.4 (m, 1H), 5.2 (m, 1H), 4.3 (m, 3H), 4.1 (m, 1H), 3.6 (s, 1H), 2.6 (m, 1H), 2.3 (m, 5H), 2.0 (m, 3H), 1.8 (m, 4H), 1.5 (m, 4H), 0.9 (m, 3H). 13CNMR (CDC13, 400MHz): 218.4, 172.4, 170.1, 161.7, 135.6, 133.8, 129.8, 125.1, 119.0, 117.5, 65.2, 64.3, 51.5, 38.7, 37.6, 28.8, 27.7, 25.4, 22.6, 20.5, 14.1.

[0223] (Z)-5-bromopentyl 2-(3-oxo-2-(pent-2-en-l-yl)cvclopentyl)acetate: Jasmonic acid (2 g, 15 mmol) was dissolved in 20 ml DMF, K2CO3 (1.98 g, 15 mmol) was added. The reaction was stirred at room temperature for Ih. 1,5- dibromopentane (9.5 ml, 75 mmol) was added dropwise. The reaction was stirred at room temperature overnight and was monitored using TLC (5% EtOAc in hexane). The reaction was gravitationally filtered, and the filtrate was concentrated under reduced pressure. The residue was portioned between water and EtOAc (60 mL each). The organic phase was washed with water (3 x 60 ml), dried over sodium sulfate, filtered, and evaporated. The residue was purified on a PuriFlash system using hexane as eluent. This resulted in 2.8 g, 7.82 mmol of the desired product, 5% Yield. Membrane permeability predictor model by Schrodinger

[0224] Computational model for predicting Membrane dG Insert and RRCK permeability via size dependent partition approach was used as the RRCK permeability predictor in the physics-based permeability prediction module within the Schrodinger’s Small- Molecule Drug Discovery Suite. Macrocycle sampling for macrocycles was used in conjunction with OPLS2005 force field and an implicit solvent model of chloroform (PRIME).

[0225] In situ virulence assay

[0226] Virulence was measured by assessing severity of symptoms in Solanum tuberosum (potato). 10 mM of SA-JA in peptone was used for all treatments (peptone, SA, and JA). The experiment was performed on small (about 25-50 g) potato tubers that were surface sterilized by soaking in 0.4% sodium hypochlorite for 20 min and washed twice with sterilized DW. Whole disinfected potato tubers were used for infection assays. Potato tubers were treated by dripping 40 uL respective treatments and incubated at 30°C for 18 h. Bacterial strains were grown overnight in LB at 30°C with continuous shaking and diluted to 108 CFU / mL (OD600 = 0.1). Potato tubers were pierced at the center with a sterile tip and inoculated with 10 pl of bacterial suspension (106 CFU). The inoculated plant material was incubated at 30°C. The disease severity was expressed as the percentage of rotten tissue, determined by weighing decayed tissues 24 h post inoculation. Two independent experiments were carried out with three replicates each.

[0227] Results

[0228] 2.1 Synthesis

[0229] The JA-SA composite was prepared using a linker composed of a 5 carbons chain to allow flexibility. The three compounds were conjugated through the two carboxylic acids in both substances to form 2 ester bonds (see also Scheme 2). The high number of functional groups in SA and JA permitted low reaction yield.

[0230] Two synthetic routes were tested: to produce Composite 1:

[0231] JA was prepared through hydrolysis of methyl jasmonate in 1 M methanolic NaOH solution (Scheme 3). Composite 2 was obtained by adding SA to K2CO3 solution in dry DMF at 40°C. 1,5- dibromopentane was added dropwise for overnight. JA was added to K2CO3 solution in dry DMF at 40°C. Composite 2 then added dropwise overnight to obtain Composite 1. Steps in the reaction are shown in Scheme 3.

[0232]

[0233] Scheme 3 a.l,5-dibromopentane, K2CO3, DMF, 40 0 C. b.(±)methyl jasmonate,! M methanolic NaOH solution c. K2CO3, DMF, 40 0 C.

[0234] To increase yield, SA was converted to silver salicylate thereby activating the carboxylic acid position. 5-bromopentyl jasmonate was prepared from 1,5- dibromopentane and JA through hydrolysis (LiOH in THF: water 1:1). Composite 3 was obtained by reacting JA with 1,5-dibromopentane. Silver salicylate was added to the reaction solution containing Composite 2 in DMF, the obtained mixture was stirred overnight at room temperature. The synthesis procedure and identity of the products were monitored using LC-MS. Steps in the reaction are shown in Scheme 4.

[0235]

[0236] Scheme 4 a. 1) LiOH, THF: H2O 1:1, 1.5h RT, 2) IN HCL. b. 1,5- dibromopentane, K2CO3, DMF, 40 0 C. c. NaOH, AgNO3, H2O, RT. d. DMF, RT.

[0237] 2.2 Passive membrane permeability

[0238] Physical models of passive membrane permeability were based on Solubility Diffusion Theory (Leung et al. 2012 J Chem Inf Model', Diamond and Katz 1974, J Membrane Biol). A key concept of the theory is that membrane permeability is inversely related to the resistances presented in the permeation process, including the internal resistance of the membrane and the resistance at the water-membrane interface. If the aqueous unstirred layers and interfacial resistance are assumed to be negligible, then conceptually, the rate-limiting step involves traversing a barrier region, the region of the membrane where the free energy of the molecule reaches a maximum. Here we used Schrodinger Prime Structure-Based Membrane Permeability Predictor model to predict passive membrane diffusion of Composite 1. This model is based on two main parameters: a) Membrane AG insert - the total free energy penalty for the ligand state change due to insertion into the membrane. This is the sum of membrane HDLD and membrane state penalty (low value required); and b) log perm RRCK, a value that takes into account the volume of the molecule in membrane penetration (a high value is required).

[0239] The free energy penalty for the neutral form of the ligand in its conformation inside the membrane to enter the membrane (i.e., move from the high dielectric region to the low dielectric region, hence HDLD). Membrane HDLD is the free energy penalty for the neutral form of the ligand in its conformation inside the membrane to enter the membrane (i.e., move from the high dielectric region to the low dielectric region, hence HDLD) and membrane state penalty is the free energy penalty for neutralizing (or tautomerizing) the ligand. The results of the calculations are structures of ligands in a conformation that is predicted to be the most likely conformation in the membrane.

[0240] Both physical membrane values for Composite 1 reflected better membrane permeability than the buildings blocks. Model prime energy was 0.69. Membrane AG insert value for composite 1 was -0.7 compared to 15.5 and 8.2 for SA and JA, respectively, while log perm RRCK value was -4.6 compared to -5.3 and -5.

[0241] 2.3 Volatility determinations

[0242] Volatilities of Composite 1 were determined gravimetrically, using the rate of weight loss from a 0.0145 mmol loading dose dispersed over aluminum surfaces, at RT in a laboratory hood and at a wind speed of about 45 m per min (see Hammack and Petroski 2004 J Chem Ecol). Volatility is dominated by intermolecular force, which is determined by molecular weight, therefore lower volatilities of the composite should be ascribed to its larger molecular weight. Weight loss determinations were replicated three times for each dispenser type and dosage. As expected, due to the greater molecular weight and the molecular bonds that resulting from it, the weight loss of composite 1 was lower than its building blocks, 42 % compared to 58 % and 65 % of SA and JA, respectively.

[0243] 2.4 In situ antimicrobial effect of SA- J A composite on bacteria To determine the effect of Composite 1 on bacterial growth in potatoes, potato tubers were incubated with 1.5 mM solutions of Composite 1 for 18 hrs, and SA and JA in concentration of 1.5 mM prior to inoculation with the bacteria. The treated potato tubers were inoculated with P. brasiliense PC 1692 bacterial cultures and compared to pepton treated tubers as controls. The results are shown in Figs 8a-8b. Treatment with the SA-JA composite caused marked decrease of decayed tissue compared to Sa or JA treated tubers or pepton treated controls (Fig. 8a). Bacteria count showed significant decrease with the DS A treatment compared SA or JA treatments or control (Fig. 8b).

[0244] Conclusions

[0245] The use of SA and JA is limited due to their high volatility. Here, a novel potent antimicrobial agent was synthetized using two alternate routes to provide a conjugated SA-JA composite. This product proved to be surprisingly less volatile and was suggested to have a higher membrane penetration compared to SA or JA alone. In addition, it demonstrated significant antimicrobial activity in reducing the potato tissue decay induced by P. brasiliense, suggesting activation of plant defense system. All these properties, in addition to its inherent biodegradability, make the new composite an attractive natural candidate for replacing the commonly used pesticides.

[0246] EXAMPLE 3: OA-SA composite- an efficient antimicrobial material

[0247] General information and methods

[0248] All chemicals and solvents used for synthesis were purchased from commercial suppliers and applied directly in the experiment without further purification.

[0249] Chromatography procedure and analysis

[0250] The progress of the reaction was monitored by TLC on pre-coated silica plates (Merck 60F-254, 250 pm in thickness), and spots were visualized by UV light. Merck silica gel (100-200 mesh) was used for general column chromatography purification. Analytical HPLC-MS was performed using an Agilent 1260 series Liquid Chromatograph / Mass Selective Detector (MSD) (Single Quadrupole) equipped with an electrospray interface and a UV diode array detector. Analytical HPLC was performed using an Agilent 1100 series Liquid Chromatograph equipped with a UV diode array detector. HPLC analyses were performed by the method: XBridge C18 50x3.0mm 3.5pm column, flow of ImL / min, gradient of 10-90% MeCN in water with 10 mM ammonium carbonate over 9 minutes, 215-395 nm, 254 nm. ESI MS with positive ionization.

[0251] NMR

[0252] 1H NMR and 13C NMR spectra were recorded on a Bruker 400 / 700 spectrometer. Chemical shifts were reported in parts per million relatives to internal standard tetramethylsilane (Si(CH3)4 = 0.00 ppm). 1H NMR coupling constants (J) were reported in Hertz (Hz), and multiplicity is indicated as the following: s (singlet), d (doublet), t (triplet), dd (doublet doublet), m (multiple).

[0253] Chemicals

[0254] 1,5-dibromopropane, oleanolic acid and salicylic acid were purchased from Alfa. Aesar Company.

[0255] Synthetic procedures: 5-bromopentyl 10-hydroxy-2,2,6a,6b,9,9,12a- heptamethyl-l,3,4,5,6,6a,6b,7,8,8a,9,10,l l,12,12a,12b,13,14b-octadecahydropicene- 4a(2H)-carboxylate (2)

[0256] Oleanolic acid (500 mg, 1.095 mmol) was added to a solution containing K2CO3 (304 mg, 2.2 mmol) and in 1 ml dry DMF at 400 C. 1,5 -dibromopentane (0.165ml, 1.21 mmol) was added dropwise. The reaction mixture was stirred at overnight for overnight. The solvent evaporated and the product was purified on a silica gel column using DCM: MeOH, 98:2. Yield - 44% (283 mg). 1H NMR (700 MHz, CDC13): 8 5.2 (s, 1H), 4.0 (m, 2H), 3.4 (m, 2H), 3.2 (s, 1H), 2.9 (s, 1H), 1.9 (m, 1H), 1.8 (m, 4H), 1.6 (m, 8H), 1.5 (m, 7H), 1.4 (m, 1H), 1.3 (s, 3H), 1.1 (m, 5H), 1 (d, 4H), 0.9 (s, m, 10H) 0.7 (m, 5H) ppm. 13CNMR (CDC13, 700MHz): 177.7, 143.8, 122.9, 78.9, 63.7, 55.2, 47.5, 46.6, 45.8, 41.3, 39.3, 38.7, 38.4, 33.8, 33.6, 33.5, 33.2, 33.1, 32.7, 32.5, 32.2, 31.8, 28.1, 27.8, 27.6, 27.1, 25.8, 24.7,23.6, 23.4, 23.0, 18.3, 17.0, 15.5, 15.3. MS APCI+ 589 (MH20+).

[0257] 5-((2-hydroxybenzoyl)oxy)pentyl 10-hydroxy-2,2,6a,6b,9,9,12a- heptamethyl-l,3,4,5,6,6a,6b,7,8,8a,9,10,ll,12,12a,12b,13,14b-octadecahydropicene- 4a(2H)-carboxylate (1)

[0258] Compound 2 (400 mg, 0.68 mmol) was added to a solution containing K2CO3 (94 mg, 0.68 mmol) and in 1 ml dry DMF at 40 0 C. salicylic acid (94 mg, 0.68 mmol) was added dropwise. The reaction mixture was stirred at 400C for overnight. The solvent evaporated and compound 1 was purified on a silica gel column using DCM: MeOH, 97:3. Yield - 22% (100 mg). 1H NMR (700 MHz, CDC13): 8 10.8 (s, 1H), 7.8 (m, 1H, J = 8.0 Hz), 7.4 (m, 1H, J = 6.0 Hz), 6.9 (m, 1H, J = 8 Hz), 6.8 (m, 1H, J = 6 Hz), 5.2 (s, 1H), 4.3 (m, 1H), 4.0 (m, 2H), 3.2 (s, 1H), 2.9 (s, 1H), 1.9 (m, 1H), 1.8 (m, 4H),1.7 (m, 1H), 1.6 (m, 6H), 1.5 (m, 7H), 1.4 (m, 1H), 1.3 (s, 3H), 1.1 (m, 5H), 1 (d, 4H), 0.9 (s, m, 10H) 0.7 (m, 5H) ppm. 13CNMR (CDC13, 700MHz): 177.7, 170.2, 161.7, 143.8, 135.6, 129.8, 122.9, 119.1, 117.6, 112.5, 78.9, 65.2, 63.7, 55.2, 47.5, 46.6, 45.8, 41.3, 39.3, 38.7, 38.4, 33.8, 33.6, 33.2, 33.1, 32.7, 32.5, 31.8, 28.3, 28.2 28.1, 27.6, 27.1, 25.8, 23.6, 23.4, 23.0, 22.7, 18.3, 17.0, 15.5, 15.3.

[0259] Membrane Permeability Predictor model by Schrodinger

[0260] The computational model for predicting Membrane dG Insert and RRCK permeability via the size-dependent partition approach was implemented as the RRCK permeability predictor in the physics-based permeability prediction module within the Schrodinger’s Small-Molecule Drug Discovery Suite. macrocycle sampling for macrocycles in conjunction with the use of OPLS2005 force field and an implicit solvent model of chloroform (PRIME).

[0261] Conf gen calculation

[0262] The core technology for rapidly generating diverse con-formers in ConfGen was originally developed for the dockingprogram Glide, and has been modified for the task ofreproducing bioactive conformations in a relatively small setof total conformations. ConfGen uses the infrastructure fromthe general molecular modeling program MacroModel, which allows for access to multiple all-atom force fields, redundant conformer elimination, and multiple processorcomputing. Technology development is an ongoing process, and the description presented here is for ConfGen version 2.1211. we used dihedral function by packing 4 atoms for each molecule, OA-S A and for OA, 3-OH, 13-C,O-succicyl linker, C- carboxy atom and 3-OH, 13-C, 17-C, C- carboxy atom, respectivly.

[0263] Bacterial strains and growth conditions

[0264] The bacterial strains used in this study, Pcbl692 was cultivated at 30 °C. it was grown in LB medium (Difco Laboratories, Detroit, MI, USA) under continuous shaking (150 rpm) in a TU-400 incubator shaker (MRC, Holon, Israel).

[0265] In situ assay

[0266] Virulence was measured by the assessment of symptom severity in Solanum tuberosum (potato). To explore the effect of the composite, 10 mM of OA-SA in peptone were used for all treatments (pepteone, SA and OA). The experiment was performed on small (about 2.5-50 g) potato tubers that were surface sterilized by soaking in 0.4% sodium hypochlorite for 20 min and then washed twice with sterilized distilled water. Whole disinfected potato tubers were used for infection assays. Potato tubers were treated by appropriate treatment, by dripping 40 uL followed by incubation at 30 °C for 18 h. Bacterial strains were grown overnight in LB liquid medium at 30 °C with continuous shaking and diluted to 10sCFU / mL (OD600 = 0.1). Potato tubers were then pierced at the center with a sterile tip and inoculated with 10 μL of bacterial suspension (106CFU). The inoculated plant material was incubated at 30 °C. The disease severity was expressed as the percentage of rotten tissue, which was determined by weighing decayed tissues after 24 h of inoculation. Two independent experiments were carried out, with three replicates for each treatment.

[0267] Minimal inhibitory concentration (MIC) assay

[0268] MIC was assessed using the standard broth microdilution protocol as described by Weigand et al with a change in incubation time from 20 h to 24 h. Briefly, overnight cultures were grown in LB for p. carotovorum and p. aeruginosa or BIH for v aureus at 30 °C for p. carotovorum and 37 °C for p .aeruginosa and ,y. aureus and diluted to yield an inoculum of approximately 1 x 107CFU / ml. Cultures were further diluted 1 / 10000 and inoculated with different treatments. Growth was scored after 24 h incubation at 30 °C and 37 °C, the MIC value taken as the concentration that inhibited macroscopic growth.

[0269] The MICs of crude extract, fractions, compounds were determined as follows: the test sample was first dissolved in Dimethylsulfoxide (DMSO) and for oleanolic acid, 10% (v / v) Tween 20 was added. The solution obtained was added to LB to a final concentration of 12mM for salicylic acid and 1,5-pentadiol (the compound that will obtain after enzymatic breakdown) and diluted twofold to obtain concentration ranges of 0.75-12mM and final concentration of 3mM for oleanolic acid and diluted twofold to obtain concentration ranges of 0.19-3mM.

[0270] Due to dissolving problems (Oleanolic acid residue), the UV absorbance test could not be performed. 24h after application, the original crude solution was diluted 1 / 10, 10 times and all the samples were re-inoculated and counted, following an additional 24 h incubation at 30 °C.

[0271] Molecular Networking by GNPS (Global Natural Products Social Molecular Networking) FTP client, WinSCP, was used to upload the converted MS / MS data to the MzXML format using the host ost ccms-ftp01.ucsd.edu; these data to system were then transferred automatically to the GNPS system. The uploaded data were available in GNPS website readily for uploading data to create the molecular networking on the GNPS website (http: / / gnps.ucsd.edu). In the basic option setting, precursor ion and fragment ion mass tolerance were set to 0.5 and 0.02, respectively. The advanced network setting systems were set to minimum pairs cos of 0.7, network TopK of 10, maximum connected component size of 100, minimum matched fragment ions of 4, the minimum cluster size of 2. For further analysis, the spectra were searched and matched toward GNP spectral library'. They were set to the library search minimum matched of 4, search analog of “do search”, score threshold of 0.7, maximum analog search mass difference of 100. Cosine similarity score that shows closer score to 1 indicates higher similarity matched with the library spectra or representing identical spectra, whereas the score closer to 0 indicates no similarity. The calculation of cosine similarity was considered based on fragment ions, precursor ions and peak intensities.

[0272] Visualization of Molecular Networking Using Cytoscape

[0273] The molecular networking data obtained from the GNPS system were imported to Cytoscape 3.7.2 to visualize and simplify molecular networking in one display. Cytoscape was used for analyzing the whole profile of metabolites in all crude extracts and correlation between standard compounds and their analogs.

[0274] 3.1 Results and discussion

[0275] Synthetic pathways are limited and exclude reagents such as acidity agents, so there cannot be protections on the functional sites that we do not want them to react, for example, on the C-3 hydroxyl on OA for producing the composite, any deportations of this group will cause brake of the compound. Moreover, our first criterion was the design of a synthesis that would avoid any protecting group to limit the overall number of steps. This should be taken into consideration for possible byproducts, purification of the product, and low yields throughout the synthesis stages.

[0276]

[0277] The linker includes di-ester on both sides of the pentyl at the composition of C- 28 carboxylic acid of the oleanolic acid on one side, and on the hydroxyl or carboxylic acid of the salicylic acid on the other side. The four carbons chain without branching allows flexibly of the composite. Following our previous experience in syntheses of this kind of compound, 1,5-dibromopentane was initially selected as a coupling reaction for the proof of concept. Indeed, compound 2 was readily obtained in one step from OA using slightly modified conditions from the literature, and it was coupled with the SA in the same conditions to synthesize the desired conjugate 1 with a yield of 22% (Scheme 5). a.1,5-dibromopentane, K2CO3, DMF, 40 °C. b. salicylic acid, K2CO3, DMF, 40 °C.

[0278] 3.2 Volatility Determinations The relative sublimation rates for solids of composite 1, OA and SA, were measured by thermogravimetric analysis (TGA). The samples (about 2 mg for each) were subjected to TGA with gradual heating at 30 - 550 and isothermal heating at 80 °C for 2 h under a constant flow of nitrogen (Cojocaru et al., 2013), Fig. 9. Volatility was defined as the weight loss of the compound under isothermal conditions. There is a large range of about 200 degree in sublimation temperatures of the new composite and SA, there is a significant difference between the sublimation temperatures. Moreover, on isothermal heating no weight loss was observed during 2h, while for SA weight loss was observed within as early as 20 minutes.

[0279] 3.3 Passive membrane permeability

[0280] Molecular permeation through membranes is a fundamental biological process that is important for small neutral molecules. The predictor model is based on solubilitydiffusion theory. Here we used Schrodinger Prime Structure-Based Membrane Permeability Predictor model to predict passive membrane diffusion of composite 1. This model is based on two main parameters; Membrane AG inserts - The total free energy penalty for the ligand state change due to insertion into the membrane. This is the sum of membrane HDLD and membrane state penalty (low value required); and log perm RRCK, a value that takes into account the volume of the molecule in membrane penetration (a high value is required). Moreover, we took into consideration the option that two molecules will enter the membrane together thanks to electrostatic force, like H-bonds. For this prediction, we use Confogen, a module that uses potential energy surface to find low energy conformations of a small molecule. The potential energy surface of a compound help with understanding key aspects of a molecule's behavior using dihedral as coordinate type (describe on methods section). This gives us an indication of how adaptable the molecule is and how much strain is needed for it to adopt a particular conformation. Calculations of two conformers of both composite 1 and OA were made (SA does not have enough conformers for these calculations). Membrane permeability predictor calculation was made for the two conformations with the minimum energy.

[0281] The main results of the calculations are the structures of the ligands in a conformation that predicted to be the most likely conformation in the membrane. Both physical membrane values for the new composite, 1, SA-OA, reflect better membrane permeability than the building blocks. The model dprime energy is 3.8. Membrane AG inserts value for composite 1 is -6.77 compared to 15.5 and 2.02 for SA and OA, respectively, while log perm RRCK value is -4.9 compare to -5.3 and -5.19. Furthermore, comparing the one molecule with the two conformers, the calculation results of membrane AG insert value for the new composite is much higher than the 2*0 A, -11.45 and 5.7, respectively.

[0282] 3.4 In Situ Antimicrobial effect of OA-SA composite on Bacteria

[0283] Pectobacterium carotovorum ssp. brasiliense 1692 (Pcbl692) is an important emerging pathogen of potatoes causing blackleg in the field and soft rot during postharvest storage. PC 1692 has emerged as a pathogen of major economic importance in countries such as Canada, USA, South Africa, and New Zealand. In all of the countries in which it has been isolated, PC 1692 is more aggressive than other Pectobacterium spp.

[0284] MIC experiments, using up to 12 mM, showed no effect on the bacteria. To examine if the new composite activates the plant's defense system, thereby prevents the culture of bacteria, and reduces the disease, we performed a simple test on potatoes. Lagonenko et al. (2013) showed that SA inhibits the biofilm formation and motility of P. carotovorum. Moreover, SA has been found to reduce infection in tissue-cultured potatoes infected by Dickeya solani. To determine the effect on bacterial growth in treated potatoes, we treated the potatoes with peptone (as a control), our newly synthesized OA-SA composite, as well as with salicylic acid and oleanolic acid separately in a concentration of 10 mM. After 18 h of incubation at 30° C, the potato tubers were inoculated with PC 1692 bacterial cultures were used in this assay on potato tubers as described in the experimental section).

[0285] Treatment of the potato tubes with OA-SA composite showed a two-thirds decrease of the decay compared to the peptone treated potato tubers (controls), that produced characteristic tissue necrosis in the host (Fig. 10), typical of the development of soft rot. The potato tubes that were treated with SA showed a decline in the rot, compared to those that were treated with OA, which the observed decrease was not significant.

[0286] Moreover, bacteria count showed a 3-fold decrease compared to the control, and surprisingly, even to SA and OA treatments. It can be clearly seen that the treatment with the new composite prevents the growth of bacteria, unlike the other treatments. The potato tubes that were treated with SA alone showed a decline in the rot, compared to those that were treated with OA alone, in which the observed decrease was negligible (Fig. 11). 3.5 Molecular Networking by GNPS (Global Natural Products Social Molecular Networking) of potatoes extract after treatment with OA-SA

[0287] MS / MS spectra of the potatoes extract with all the treatments were performed and further analysis using the GNPS website; all acquired MS / MS data were converted into MzXML as an open file format by Proteo Wizard . Then, the converted data were uploaded to create molecular networking on the GNPS website (http: / / gnps.ucsd.edu). All molecular networking data obtained from the GNPS system were imported to Cytoscape 3.7.2 version, to visualize and simplify molecular networking in one display. The node colors were set and they represented MS / MS data of compounds present in crude extracts or standard compounds. Cytoscape was used for rapid analysis of the whole profile of metabolites in ah crude extracts, as well as for the correlation between standard compounds and their analogs. The result of the molecular networking of crude extracts in a positive mode without blank is shown in Fig. 13. Colors for extracts of control as well as OA-SA, SA, and OA treatments are depicted in Fig. 9. The results showed several exclusive compounds group for the OA-SA treatment extract (orange), particularly 2 main molecular networking; one compound group containing 2[(lR,2R)3oxo2[(Z)5[3,4,5trihydroxy6(hydroxymethyl)oxan2yl]oxypent2enyl]cyclope ntyl]acetic acid (m / z [M+NH4]+) 406.21, two stereoisomers that one of them apparently tuberonic acid glucoside, TGA. The tuber-inducing factor in potato, tuberonic acid, TA, or its glucopyranosyl derivative is derived from jasmonic acid. In previous studies, TA and were increased by mechanical wounding in rice and tobacco, supporting similar results found in tomato plants. These results indicate a relationship between the wounding response in plants and TA and TAG accumulation. The second molecular networking of jasmonic acid, JA, (m / z [M+H]+21 1.13) a well-known volatile fatty-acid-derived signaling molecule, is involved in several aspects of plant biology including pollen and seed development, and defense against wounding, ozone, insect pests, and microbial pathogens. The molecular networking of JA showed the node of MS / MS spectra related to TA through the ion at m / z 209.11 [M+H]+ with a cosine similarity score of 0.84 and 0.95, respectively. Fig. 13B.

[0288] Conclusions

[0289] In conclusion, our data demonstrate that OA-SA composite; a novel synthetic compound designed from the natural compounds, oleanolic acid, and salicylic acid has properties distinct from its building blocks and is efficacious in reducing decay caused by Pectobacterium carotovorum ssp. brasiliense 1692 (PC 1692). Furthermore, it exhibits a significant decrease in bacteria count compared to the control, and surprisingly, even to SA and OA treatments.

[0290] Mass spectrometry-based molecular networking is a powerful dereplication strategy; it not only identifies known metabolites in complex mixtures but also suggests the presence of related analogues. In this work we examine the chemical different in potatoes extract after treatment with new compound and shown exclusive networks, particularly, jasmonic acid network that approve the activation of our compound.

[0291] These findings indicate OA-SA composite is a promising new preservation compound for potatoes with the potential for use in different types of fruits and vegetables. The findings that the new composite that based on the natural compound is particularly important concerning the development new approach of effective composite utilization of the activity of natural materials while preserving the environment.

Claims

CLAIMS:

1. An antimicrobial material being a dimer of two different antimicrobial agents associated to each other, directly or indirectly, via hydrolysable or labile covalent bond(s).

2. The material according to claim 1, wherein each of the antimicrobial agents is selected amongst water soluble naturally derived or naturally occurring antimicrobial agents.

3. The material according to claim 1, for controlling a plant microbial source or a plant disease caused thereby, wherein the microbial source is bacteria, viruses, fungi and / or herbivorous insects.

4. The material according to any one of claims 1 to 3, wherein the two different antimicrobial agents are directly associated via a hydrolysable or labile covalent bond.

5. The material according to any one of claims 1 to 3, wherein the two different antimicrobial agents are associated through a hydrolysable or labile linker moiety.

6. The material according to claim 5, wherein the linker moiety is derived from a diacid, a diol, a diamine or a mixed form thereof.

7. The material according to claim 6, wherein the linker moiety is derived from a diacid comprising two carboxylic acid moieties.

8. The material according to claim 7, wherein the linker moiety is derived from a diacid of the formula HO(O=C)-X-(C=O)OH, wherein X is a group selected from -C1- C10alkylene, -C2-C10alkenylene, -C2-C10alkynylene, -C1-C5alkylene-O-C1-C5alkylene, - C1-C5alkylene-C6-C10arylene, -C6-C10arylene, -C2-C5alkenylene-C6-C10arylene and -C2- C5alkynylene-C6-C10arylene, and -C3-C6hctcroarylcnc.

9. The material according to claim 6, wherein the linker moiety is derived from a diol comprising two hydroxyl moieties.

10. The material according to claim 9, wherein the linker moiety is derived from a diol of the formula HO-X-OH, wherein X is a group selected from -C1-C10alkylene, -C2- C10alkenylene, -C2-C10alkynylene, -C1-C5alkylene-O-C1-C5alkylene, -C1-C5alkylene-C6- C10arylene, -C6-C10arylene, -C2-C5alkenylene-C6-C10arylene and -C2-C5alkynylene-C6- C10arylene, and -C3-C6hctcroarylcnc.

11. The material according to claim 6, wherein the linker moiety is derived from a diamine comprising two amine groups.

12. The material according to claim 11, wherein the linker moiety is derived from a diamine of the formula H2N-X-NH2, wherein X is a group selected from -C1-C10alkylene,-C2-C10alkenylene, -C2-C10alkynylene, -C1-C5alkylene-O-C1-C5alkylene, -C1- C5alkylene-C6-C10arylene, -C6-C10arylene, -C2-C5alkenylene-C6-C10arylene and -C2- C5alkynylene-C6-C10arylene, and -C3-C6hctcroarylcnc.

13. The material according to any one of claims 5 to 12, wherein the linker moiety comprises between 1 and 5 carbon atoms.

14. The material according to any one of claims 5 to 13, wherein the linker moiety comprises a group selected from -C1-C5alkylene, -C2-C5alkenylene, -C2-C5alkynylene, - C1-C5alkylene-O-C1-C5alkylene.

15. The material according to claim 6, wherein the linker moiety is derived from a dicarboxylic acid selected from ethanedioic acid, propanedioic acid, butanedioic acid, pentanedioic acid, hexanedioic acid, heptanedioic acid, octanedioic acid, nonanedioic acid, decanedioic acid and substituted forms thereof.

16. The material according to claim 15, wherein the linker moiety is a succinyl linker, or is derived from succinic acid (butanedioic acid).

17. The material according to any one of claims 1 to 16, wherein each of the naturally occurring water soluble antimicrobial agents is selected from salicylic acid (SA), jasmonic acid (JA), oleanolic acid (OA), rosmarinic acid (RA), bonianic acid (BA), ursolic acid (UA), and betulinic acid (TA).

18. The material according to any one of the preceding claims, comprising SA.

19. The material according to any one of claims 1 to 17, formed of an antimicrobial agent different from SA.

20. The material according to any one of the preceding claims, comprising JA.

21. The material according to any one of the preceding claims, comprising OA.

22. The material according to any one of the preceding claims , being selected from SA-JA, SA-OA, SA-RA, SA-BA, SA-UA, SA-TA, JA-OA, JA-RA, JA-BA, JA-UA, JA- TA, OA-RA, OA-BA, OA-UA, OA-TA, RA-RA, RA-BA, RA-UA, RA-TA, BA-UA, BA-TA, UA-UA, UA-TA, SA-L-JA, SA-L-OA, SA-L-RA, SA-L-BA, SA-L-UA, SA-L- TA, JA-L-OA, JA-L-RA, JA-L-BA, JA-L-UA, JA-L-TA, OA-L-RA, OA-L-BA, OA-L- UA, OA-L-TA, RA-L-BA, RA-L-UA, RA-L-TA, BA-L-UA, BA-L-TA, and UA-L-TA, wherein each of SA, JA, OA, RA, BA, UA, TA is as defined in claim 16, L is a linker moiety as defined in any one of claims 6 to 16 and each ofis a hydrolysable or labile covalent bond.

23. An antimicrobial material being a dimer of two antimicrobial agents, associated to each other, directly or indirectly, via hydrolysable or labile covalent bond(s), wherein each of the two antimicrobial agents is selected amongst water soluble naturally derived antimicrobial agents.

24. The material according to claim 23, being a homodimer or a heterodimer.

25. The material according to claim 23 or 24, for controlling a plant microbial source or a plant disease caused thereby, wherein the microbial source is bacteria, viruses, fungi and / or herbivorous insects.

26. The material according to any one of claims 23 to 25, wherein the two antimicrobial agents are directly associated via a hydrolysable or labile covalent bond.

27. The material according to any one of claims 23 to 25, wherein the two antimicrobial agents are associated through a hydrolysable or labile linker moiety.

28. The material according to claim 27, wherein the linker moiety is derived from a diacid, a diol, a diamine or a mixed form thereof.

29. The material according to claim 28, wherein the linker moiety is derived from a diacid comprising two carboxylic acid moieties.

30. The material according to claim 29, wherein the linker moiety is derived from a diacid of the formula HO(O=C)-X-(C=O)OH, wherein X is a group selected from -C1- C10alkylene, -C2-C10alkenylene, -C2-C10alkynylene, -C1-C5alkylene-O-C1-C5alkylene, - C1-C5alkylene-C6-C10arylene, -C6-C10arylene, -C2-C5alkenylene-C6-C10arylene and -C2- C5alkynylene-C6-C10arylene, and -C3-C6hctcroarylcnc.

31. The material according to claim 28, wherein the linker moiety is derived from a diol comprising two hydroxyl moieties.

32. The material according to claim 31, wherein the linker moiety is derived from a diol of the formula HO-X-OH, wherein X is a group selected from — C1-C10alkylene, -C2- C10alkenylene, -C2-C10alkynylene, -C1-C5alkylene-O-C1-C5alkylene, -C1-C5alkylene-C6- C10arylene, -C6-C10arylene, -C2-C5alkenylene-C6-C10arylene and -C2-C5alkynylene-C6- C10arylene, and -C3-C6hctcroarylcnc.

33. The material according to claim 28, wherein the linker moiety is derived from a diamine comprising two amine groups.

34. The material according to claim 33, wherein the linker moiety is derived from a diamine of the formula H2N-X-NH2, wherein X is a group selected from -C1-C10alkylene, -C2-C10alkenylene, -C2-C10alkynylene, -C1-C5alkylene-O-C1-C5alkylene, -C1-C5alkylene-C6-C10arylene, -C6-C10arylene, -C2-C5alkenylene-C6-C10arylene and -C2- C5alkynylene-C6-C10arylene, and -C3-C6hctcroarylcnc.

35. The material according to any one of claims 27 to 34, wherein the linker moiety comprises between 1 and 5 carbon atoms.

36. The material according to any one of claims 27 to 35, wherein the linker moiety comprises a group selected from -C1-C5alkylene, -C2-C5alkenylene, -C2-C5alkynylene, - C1-C5alkylene-O-C1-C5alkylene.

37. The material according to claim 27, wherein the linker moiety is derived from a dicarboxylic acid selected from ethanedioic acid, propanedioic acid, butanedioic acid, pentanedioic acid, hexanedioic acid, heptanedioic acid, octanedioic acid, nonanedioic acid, decanedioic acid and substituted forms thereof.

38. The material according to claim 37, wherein the linker moiety is a succinyl linker, or is derived from succinic acid (butanedioic acid).

39. The material according to any one of claims 23 to 38, wherein each of the naturally occurring water soluble antimicrobial agents is selected from salicylic acid (SA), jasmonic acid (JA), oleanolic acid (OA), rosmarinic acid (RA), bonianic acid (BA), ursolic acid (UA), and betulinic acid (TA).

40. The material according to any one of claims 23 to 38, comprising SA.

41. The material according to any one of claims 23 to 39, formed of an antimicrobial agent different from SA.

42. The material according to any one of claims 23 to 41, comprising JA.

43. The material according to any one of claims 23 to 42, comprising OA.

44. The material according to any one of claims 23 to 43, being selected from SA-SA, SA-JA, SA-OA, SA-RA, SA-BA, SA-UA, SA-TA, JA-JA, JA-OA, JA-RA, JA-BA, JA- UA, JA-TA, OA-OA, OA-RA, OA-BA, OA-UA, OA-TA, RA-RA, RA-BA, RA-UA, RA-TA, BA-BA, BA-UA, BA-TA, UA-UA, UA-TA, TA-TA, SA-L-SA, SA-L-JA, SA- L-OA, SA-L-RA, SA-L-BA, SA-L-UA, SA-L-TA, JA-L-JA, JA-L-OA, JA-L-RA, JA-L- BA, JA-L-UA, JA-L-TA, OA-L-OA, OA-L-RA, OA-L-BA, OA-L-UA, OA-L-TA, RA- L-RA, RA-L-BA, RA-L-UA, RA-L-TA, BA-L-BA, BA-L-UA, BA-L-TA, UA-L-UA, UA-L-TA, and TA-L-TA, wherein each of SA, JA, OA, RA, BA, UA, TA is as defined in claim 17, L is a linker moiety as defined in any one of claims 39 to 43 and each ofis a hydrolysable or labile covalent bond.

45. An antimicrobial formulation comprising a material according to any one of claims 1 to 44.

46. The formulation according to claim 45, being an aqueous formulation.

47. The formulation according to claim 45 or 46, being a plant-protection formulation.

48. A method of treating or preventing growth of plant pathogens or herbivorous insects, the method comprising applying to a surface infested or prone to infestation by the pathogens or insects a formulation comprising at least one material according to any one of claims 1 to 44 or an antimicrobial formulation according to any one of claims 45 to 47.

49. The method according to claim 48, wherein the surface is a plant, plant part, or to a region in a vicinity of the plant or plant part.

50. The method according to claim 48, wherein the formulation is applied onto a surface of a seed, or a bulb, or a tuber, or an agricultural product by forming a coating thereon.

51. A method of treating a soil region to reduce microbial infections in plant populations therein, the method comprising treating or delivering to said soil region a formulation comprising a material according to any one of claim 1 to 44.

52. The method according to claim 51, wherein the formulation is delivered via irrigation.

53. A plant protection kit comprising a solid or a liquid formulation of at least one material according to any one of claims 1 to 44, wherein the solid or liquid formulation is configured for addition into a commercial or premade pesticide or fertilizer formulation; and instructions of use.

54. An antimicrobial material according to any one of claims 1 to 44, the antimicrobial material being in a form suitable for preventing or eradicating or diminishing growth of a plant microbial source selected from bacteria, viruses, fungi and / or herbivorous insects.

55. The material according to claim 54, for use pre-harvest or post-harvest.

56. The material according to claim 54, for direct application onto a plant or plant part.

57. The material according to any one of claims 54 to 56, wherein the dimer is a homodimer or a heterodimer of SA.

58. The material according to any one of claims 54 to 56, wherein the dimer is a homodimer or a heterodimer of OA.

59. The material according to any one of claims 54 to 56, wherein the dimer is a homodimer or a heterodimer of J A.

60. The material according to any one of claims 54 to 59, wherein the dimer is a heterodimer.

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