92794

BREVET D'INVENTION Bl

(21) N° de dépôt: 92794 (51) Int. Cl.: A01N 25/00, A01N 25/34, C12N 11/14 (22) Date de dépôt: 07/08/2015

Priorité: © Inventeur(s): PRINTZ BRUNO - 57330 ESCHERANGE (France)

© Date de mise à disposition du public: Θ Mandataire(s): LECOMTE & PARTNERS SÀRL- Θ Date de délivrance: 14/02/2017 1016 LUXEMBOURG (Luxembourg)

(g) Titulaire(s): LUXEMBOURG INSTITUTE OF SCIENCE AND TECHNOLOGY (LIST) - 4362 ESCH/ALZETTE (Luxembourg)

© Pest protective apparatus for controlling plant pathogens. 57) Abstract: The invention is directed to a pest protective apparatus adapted for controlling plant pathogens. Said pest protective apparatus comprises at least one fibrous network and/or at least one fibrous cover. Said pest protective apparatus is remarkable in that said at least one fibrous network and/or said at least one fibrous cover comprises a system in which at least one precursor is transformed into a biopesticide and/or repellent . 1/17 92794 Pest protective apparatus for controlling plant pathogens

Description

Technical field

[0001] The invention is directed to a pest protective apparatus comprising a system which allows the transformation of a into a biopesticide and/or repellent product which will subsequently control plant pathogens. Background art

[0002] Pest management is currently largely performed with synthetic chemicals, but the development of alternative and eco-friendly methods to control pests are encouraged. [0003] Patent application published US 2012/0079625 A1 relates to a method for protecting living plants from harmful via a sheet-like structure impregnated with an which is from synthetic origin. [0004] The field of the biocontrol includes various techniques of integrated pest management such as macro-organisms, micro-organisms, pheromones, kairomones and natural substances originating from animals, minerals and plants to control pest and pathogens. In 2014, Sahayaraj K. summarized the current use of the nanotechnologies in the domain of the plant protection. The few data available on this topic brings to the conclusion that only few research all over the world are currently working with this emerging problematic although there is an urgent need of modern approaches of pest management (Sahayaraj K., Adv. Plant Biopest, (chapter 14), 2014, 279-293). [0005] Substances from various plant families have been reported to exert a repelling activity against fungus, bacteria, nematodes or insects and can be successfully used in the treatment/control of pests (Murthy N.B.K., et at., Indian J. Exp. Bioi., 1974, 12, 208-209 and Regnault-Roger C., et. ai., Biopesticides d’origine végétale (2ème edition), 2008.) In particular, sulphur containing molecules such as those produced among other by the families and members of the genus Allium which are enzymatically produced from non-toxic precursors are toxic at low level for a wide range of organisms (Ahuja I., eta!., Agron. Sustain. Dev., 2010, 30, 2/17 92794 311-348; Auger J., et at., Ecologie, 1994, 93-101; Cutler H.G., et al., Biologically Active Natural Products: Agrochemicals, 1999). [0006] The mixing of biopesticide precursor with active in liquid form to produce repellent is known to the art and has been proposed for the production of products such as , or , and/or mixture (WO 2015/013808 A1). Also known in the art is the use of a two-part pesticide precursor system comprising a glucosinolate concentrate on one side and an active myrosinase complex on the other side both in dry form. When both components are mixed with , the glucosinolate breakdown products are released to control pests (US 2015/0005172 A1). However, the lifespan of active enzymes in the environment is relatively low, which considerably hinders the reusability of the enzymes. In addition, the substrates are commonly hydrophilic, making them easily leachable and not accessible to the enzymes. [0007] Enhancing the lifespan of enzymes has been successfully performed in the domains of the biocatalysis, biofuels, -controlled drug delivery and the biosensors by immobilizing the enzymes into silica mesoporous material (Carlsson N., et al., Adv. Colloid Interface Sei., 2013, 205, 339- 360; Popat A., et ai., Nanoscale, 2011, 3, 2801-2818; Wang Y., et al., Chem. Mater., 2005, 17, 953-961). Mesoporous material relates to silica- based material with a pore diameter sufficient to allow the penetration of the enzyme. It can be in the form of SBA-15 (Santa Barbara-15), SBA-16, MCM-41 (Mobil Crystalline Materials-41) or all other type of mesoporous silica sieves/spheres/cages (FSM-16 (Folded Sheet Mesoporous-16), MCM-48, FDU-12 (Fudan University-12), MCF (Mesostructured Cellular Foam), SMS (Sponge Mesoporous Silica), mesoporous carbon, PMOs (Periodic Mesoporous Organosilica), Meso-MOFs (meso-Metal Organic Frameworks) (Zhou Z., et al., Top Catal., 2012, 55, 1081-1100). The pore structure may be folded-sheet, 2D hexagonal channels, cubic, spherical cages, mesocellular foam, sponge-like mesoporous silica, 3D cubic cages. The synthesis of the mesoporous silica is preferentially done in acidic conditions and with the triblock copolymer (Pluronic®P123) as template and tetraethyl orthosilicate (TEOS) as silica source. However, the use of 3/17 92794 other templates such as among other the triblock copolymer (Pluronic®F127), CTMA (cetyltrimethylammonium) (CnH2n+i(CH3)N+, n=8- 18), CTAB (cetyltrimethylammonium bromide) and other silica source including among other (3-aminopropyl)trimethoxysilane (APTMOS), sodium silicate or tetramethyl orthosilicate (TMOS) may also give interesting properties to the silica mesoporous material (Zhou Z., et a!., Top Catal., 2012, 55, 1081-1100). [0008] Enzyme immobilization into the silica matrix can be carried out through various techniques including cross-linking methods, covalent binding, physical adsorption, encapsulation and entrapment (CN 101451133 B). Carboxylethyl or aminopropyl functionalization of the mesoporous silica also appears as a good technique for improving the catalytic activities of the silica-based biocomposite (Lei C., et al., J. Am. Chem. Soc., 2002, 124, 11242-11243). Functionalization of the silica-material can further be used for covalent linking of the enzyme on the derivatized-silica. [0009] The synthesis of hydrophilic gels can result from the gelation of various natural compounds such as agarose, agar-agar, alginate, pectin, starch or gelatine taken alone or in mixes. They allow the diffusion of small hydrophilic molecules and prevent the diffusion of large particles such as silica mesoporous materials that retain blocked in the polymeric network. Microencapsulation of silica in alginate had remarkable advantages of sustained-release of compounds and stability under different pH values, different temperatures, and UV irradiation. The presence of the hydrophilic gel can further increase the stability of the material upon ageing and limit enzyme leaching (Coradin T., et al., Comptes Rendus Chim., 2003, 6, 147-152). Biocomposite incorporation in hydrophilic gels such as alginate showed much better performance due to the more homogeneous distribution of silica particles in the composite material (Xu S. et al., Ind. Eng. Chem. Res., 2006, 45, 511-517). [0010] The idea of the present invention is to merge all these parts (conception of a structure, using a precursor with active enzymes in liquid form to produce biopesticide and/or repellent product, enhancing the lifespan of said enzymes, use of hydrophilic gels) to produce for the first time a pest 4/17 92794

protective apparatus which is able to deliver in situ a constant flux of biopesticide and/or repellent product.

Summary of invention

Technical Problem

[0011] The invention has for technical problem to provide a pest protective apparatus which is producing a biopesticide and/or repellent product from a precursor which is harmless and easy to handle. Technical solution

[0012] The first object of the present invention is a pest protective apparatus, for controlling plant pathogens, comprising at least one fibrous network and/or at least one fibrous cover. Said pest protective apparatus is remarkable in that said at least one fibrous network and/or said at least one fibrous cover comprises a system in which at least one precursor is transformed into a biopesticide and/or repellent product. [0013] In one embodiment, said system is composed of fibres adapted to control plant pathogens which are coated with at least one preparation able to produce biopesticide and/or repellent for controlling plant pathogens. [0014] In one embodiment, said preparation able to produce biopesticide and/or repellent for controlling plant pathogens comprised at least one active enzyme. [0015] In one embodiment, said at least one active enzyme is immobilized within one nanoporous material. [0016] In one embodiment, said one nanoporous material is a mesoporous material. [0017] In one embodiment, said nanoporous material is embedded within one hydrophilic gel. [0018] In one embodiment, said hydrophilic gel is made of agarose, agar-agar, alginate, pectin, starch and/or gelatine, preferentially made of alginate. [0019] In one embodiment, said at least one active enzyme is one active enzyme selected from the group of glycosidase, lyase and/or lachrymatory-factor synthase, preferentially from the group of glycosidase. 5/17 92794 [0020] In one embodiment, said at least one enzyme from the group of glycosidase is thioglucosidase. [0021] In one embodiment, said at least one active enzyme immobilized within said nanoporous material is covalently bound to said nanoporous material, physically adsorbed to said nanoporous material, encapsulated within said nanoporous material or entrapped within said nanoporous material. [0022] In one embodiment, said at least one active enzyme immobilized within said nanoporous material is cross-linked with glutaraldehyde or with 1- ethyl-3-(3-dimethylaminopropyl)carbodiimide and /V-hydroxysuccinimide. [0023] In one embodiment, said nanoporous material is a silica-based mesoporous material. [0024] In one embodiment, said silica-based mesoporous material comprises tetraethyl orthosilicate and/or tetramethyl orthosilicate and/or methyltrimethoxysilane and/or (3-aminopropyl)trimethoxysilane. [0025] In one embodiment, said preparation has a pH comprised between 4 and 11. [0026] In one embodiment, said preparation further comprises said at least one precursor, at least one mineral and/or at least one . [0027] In one embodiment, said preparation is adapted to receive said at least one precursor, at least one mineral and/or at least one cofactor. [0028] In one embodiment, said at least one mineral and/or said at least one cofactor is phosphate buffer or ascorbic acid. [0029] In one embodiment, said phosphate buffer is at a concentration comprised between 10 mM and 500 mM, preferentially at a concentration comprised between 50 mM and 250 mM, more preferentially at a concentration comprised between 75 mM and 150 mM, even more preferentially at a concentration of 100 mM. [0030] In one embodiment, said ascorbic acid is at a concentration comprised between 50 μΜ and 1500 μΜ, preferentially at a concentration comprised between 150 pM and 1000 pM, more preferentially at a concentration comprised between 250 pM and 750 pM, even more preferentially at a concentration of 500 pM. 6/17 92794 [0031] In one embodiment, said pest protective apparatus further comprises means for distributing water to the fibrous network and/or fibrous cover, said means preferably comprising a cartridge configured for being in fluid connection with said at least one fibrous network and/or said at least one fibrous cover and for being fed with water. [0032] In one embodiment, said cartridge comprises at least one precursor which is transformed into a biopesticide and/or repellent product into said system of said at least one fibrous network and/or said at least one fibrous cover. [0033] In one embodiment, said cartridge further comprises at least one mineral and/or at least one cofactor. [0034] It is a second object of the present invention to disclose a method for controlling plant pathogens with a pest protective apparatus. Said method is remarkable in that said pest protective apparatus is in accordance with the first object of the present invention and in that said method comprises the first step of activating at least one precursor by addition of water to the system of said at least one fibrous network and/or said at least one fibrous cover to produce a biopesticide and/or repellent product for controlling plant pathogens; and the second step of applying the biopesticide and/or repellent product resulting from the first step to plants, preferentially vegetables and/or fruits, in order to prevent pest attacks. Said first step of activating at least one precursor may also be, for example, the catalytic splitting of said at least one precursor. [0035] In one embodiment, said at least one precursor is comprised in the pest protective apparatus, said pest protective apparatus preferentially also comprising at least one mineral and/or at least one cofactor. [0036] In one embodiment, said at least one precursor is added to said pest protective apparatus, said pest protective apparatus preferentially comprising at least one mineral and/or at least one cofactor. [0037] In one embodiment, said at least one precursor is added with also at least one mineral and/or at least one cofactor to said pest protective apparatus. 7/17 92794 Advantages of the invention

[0038] The invention is particularly interesting in that the precursor of the biopesticide and/or repellent product which is provided by the pest protective apparatus of the invention is chosen among the inactive and non-toxic forms of the potential organic derivatives. The biopesticide and/or repellent preparation which is coated onto the fibres of the fibrous network and/or fibrous cover further comprises immobilized enzyme which provides enhanced enzymatic activity compared to a non-immobilized enzyme.

Brief description of the drawings

[0039] Figure 1: Pest protective apparatus.

Description of an embodiment

[0040] The present invention relates to a pest management biocontrol system made of active biocatalysts able to produce natural repellents and/or biopesticides by means of controlled enzymatic reactions. [0041] As depicted on figure 1, the pest protective apparatus 100 of the present invention comprises at least one fibrous network and/or at least one fibrous cover 14. [0042] Said network and/or said cover 14 comprises a system 500 in which at least one precursor 8 is transformed into a biopesticide and/or repellent product 10. [0043] The system 500 is made of fibres 200 which are in fact coated with a preparation 300 which presents biopesticide and/or repellent properties. Such type of preparation comprises at least one active enzyme 4 which is capable to transform at least one substrate 8 into a biopesticide and/or repellent product 10, which will subsequently control plant pathogens. [0044] In order to enhance the lifespan of said at least one enzyme 4, the biocatalyst is immobilized within one nanoporous material 2, preferably into a mesoporous silica matrix, more particularly into a silica-based 8/17 92794

mesoporous material, the whole being glued or embedded into a hydrophilic gel 6. [0045] The mesoporous materials of the present invention are ordered silica- based mesoporous particles with a narrow pore size distribution, a well- defined pore geometry and a well-defined pore connectivity. [0046] The pore size distribution and the global geometry of the mesoporous material are defined according to the properties of the biopesticide­ producing enzymes and/or repellent-producing enzymes that are immobilized into the mesoporous structure. [0047] The pore size of the silica-based mesoporous material is comprised between 5 nm and 30 nm. [0048] The immobilization procedure should allow to optimize the catalytic activities of the enzymes in comparison with the free enzymes and to enhance their reusability in order to produce a constant and sufficient flux of biopesticide and/or repellent product allowing a sufficient biopesticide and/or repellent activity against the targeted pathogens. [0049] The silica-based mesoporous material will be synthesized in acidic conditions using a silica precursor that could be among other tetraethyl orthosilicate (TEOS), and/or tetramethyl orthosilicate (TMOS), and/or methyltrimethoxysilane (MTMOS), and/or (3-aminopropyl)triemthoxysilane (APTMOS), in combination with tri-block copolymer mixtures such as Pluronic®F127 and/or Pluronic®F123 as structure-directing agents. [0050] The mesoporous structure can be produced hydrothermally and/or by sol­ gel synthesis and can be functionalized. [0051] The enzymes that may be immobilized into the mesoporous material are those involved in the natural responses of the plants when facing a pathogen attack. The biopesticide and/or repellent compounds produced during the enzymatic reaction can be a bactericide, a fungicide, an insecticide and/or a nematicide. [0052] The enzyme from the glycosidases, oxidoreductases, the transferases, the , the lyases, the isomerases and the ligases can be immobilized into the mesoporous material. 9/17 92794

[0053] Preferred transferases are those belonging to the glycosidases class, i.e. the enzymes hydrolysing O-glycosyl and S-glycosyl compounds. [0054] Preferred lyases are, for example, carbon-sulfur lyases, in particular alliin lyase (also known as alliinase). [0055] Enzymes from the PF10604 family, i.e. the lachrymatory-factor synthase, can also be used in the present invention. [0056] A preferred glycosidase is thioglucosidase. This is the enzyme of choice which has been tested for transforming a precursor into a biopesticide and/or repellent product. [0057] The immobilization procedure can be performed through the following techniques known in the art: covalent binding, physical adsorption, encapsulation and entrapment. [0058] The immobilization includes the penetration of the enzymes inside the mesoporous material, through the nanopores and an eventual step of cross-linkage between the enzymes using gluturaldehyde or 1 -ethyl-3-(3- dimethylaminopropyl)carbodiimide and AAhydroxysuccinimide. [0059] The mesoporous biocatalyst will then be embedded in a hydrophilic gel 6 made of agarose, agar-agar, alginate, pectin, starch, gelatine taken alone or in mixes, preferentially alginate. [0060] The use of alginate allows a gelation without heating. Only CaCh must be added for the gelation step. [0061] The hydrophilic gel allows the diffusion of the products of the enzymatic reaction, the diffusion of the substrates of the enzymes and the confinement of all chemicals required to maintain the enzymes in a working status. [0062] The hydrophilic gel 6 may contain all minerals and cofactors indispensable for the enzymatic activities. This can be a phosphate buffer (pH = 6.1) at a concentration comprised between 10 mM and 500 mM, preferentially at a concentration of 100 mM and/or ascorbic acid at a concentration comprised between 50 μΜ and 1500 μΜ, preferentially at a concentration of 500 pM. 10/17 92794 [0063] In case the minerals and cofactors are not added before the gelation step, they can be added by diffusion within the gel by soaking into an aqueous buffer comprising these compounds. [0064] The pH of preparation 300 able to produce a biopesticide and/or a repellent for controlling plant pathogens is preferably comprised between 4 and 11. In particular, the pH of the hydrophilic gel allows an optimal activity of the immobilized enzymes. [0065] Upon addition of the substrate 8 for the enzyme 4 and upon addition of water, and, upon addition of minerals and cofactors for activating the enzyme if they are not formerly included within said hydrophilic gel 6, the enzyme 4 will proceed to the synthesis of a product 10 which will present biopesticide and/or repellent properties. This product 10 will be released and will act to eliminate (in case of biopesticide activity) and/or to keep away (in case of repellent activity) the pest and/or the plant pathogens from the living plants, such as bacteria, fungi, insects, bugs, and/or nematodes. [0066] The scheme on figure 1 represents in particular a precursor 8 composed of two chemical moieties, one being the biopesticide and/or the repellent product 10, the other being a compound 22. [0067] Preferentially, the product 10 of the enzymatic reaction is a volatile organic compound. [0068] The fibres 200 of the pest protective apparatus 100, which are coated with the preparation 300 able to produce biopesticide and/or repellent for controlling plant pathogens, are preferably natural fibres. Such natural fibres might be for example hemp, flax, nettle, cotton, jute, ramie, sisal, and/or any other. [0069] Preferentially, the fibres are hemp or flax. [0070] The substrate 8 of the enzyme 4 is able to diffuse within the fibre, in order to reach the preparation 300 able to produce biopesticide and/or repellent for controlling plant pathogens where said substrate 8 will be processed by the active enzyme 4. [0071] Those fibres 200 are further coated by a protective layer, which is a hydrophobic layer 12 in order to be protected from drying. 11/17 92794 [0072] Such hydrophobic layer is also configured to be permeable to the biopesticide and/or repellent product 10. [0073] The hydrophobic layer 12 may be (3-aminopropyl)triethoxysilane, (3- mercaptopropyl)triethoxysilane, succinic anhydride, alkylketene dimer, 3- isopropenyl-a-oc-dimethylbenzyl isocyanate, /n-phenylene bismaleimide, vinyl trialkoxysilane, 3-metacryloyloxy propyl trimetoxysilane and/or any other. [0074] The moiety 22, resulting from the cleavage of the precursor 8, stays therefore within the preparation 300 able to produce biopesticide and/or repellent and does not therefore pollute the environment. [0075] As depicted on figure 1, the pest protective apparatus 100 of the present invention may further comprise means for distributing water to said at least one fibrous network and/or said at least one fibrous cover 14, said means preferably comprising a cartridge 16. Said cartridge 16 is configured for being in fluid connection with said at least one fibrous network and/or said at least one fibrous cover 14 and for being fed with water. Said cartridge 16 may comprise at least one inlet 18 and at least one outlet 20. [0076] Depending whether the pest protective apparatus 100 comprises or does not comprise the cartridge 16, said apparatus 100 is configured either to transform at least one precursor 8, which is incorporated to the preparation 300 able to produce biopesticide and/or repellent by diffusion through the fibre 200, into a biopesticide and/or a repellent product 10; or to incorporate said at least one precursor 8 into said cartridge 16. [0077] Depending whether the pest protective apparatus 100 comprises or does not comprise the cartridge 16, the at least one mineral and/or the at least one cofactor indispensable for the enzyme activity are incorporated either directly within the hydrophilic gel 6 of the preparation 300 able to produce biopesticide and/or repellent; or within the cartridge 16. [0078] The enzymatic reaction is triggered by the addition of water, which plays the role of the solvent of the reaction, bringing subsequently the substrate 8 into contact with the fibres 200 of the preparation 300 able to produce biopesticide and/or repellent, and more particularly, into contact with the of the enzyme. 12/17 92794 [0079] The second utility of water is to bring to the enzyme the at least one mineral and/or the at least one cofactor indispensable for its activity. [0080] Water can come from the rain and/or from artificial means, such as for example a drain pump, a hosepipe and/or an irrigation system, and can be channelled to contact the preparation 300 able to produce biopesticide and/or repellent for controlling plant pathogens, triggering subsequently the enzymatic reaction. [0081] When the pest protective apparatus 100 comprises a cartridge 16, the flux of water reaches the cartridge 16 through the inlet 18 which is provided on the cartridge 16. [0082] Once the enzymatic reaction is over, the product 10, which presents biopesticide and/or repellent properties, is released. Generally, the product 10 is a volatile organic compound. [0083] This product 10 acts to eliminate (in case of biopesticide activity) and/or to keep away (in case of repellent activity) the pests and/or the plant pathogens from the living plants, such as bacteria, fungi, insects, bugs, and/or nematodes. [0084] The biopesticide and/or repellent product 10 resulting from the activation of the precursor 8 by addition of water to the pest protective apparatus 100 taught in the present invention is applied to plants, preferentially vegetables and/or fruits, in order to prevent pest attacks. Revendications 92794

1. Appareil (100) de protection contre les nuisibles, pour contrôler les pathogènes des plantes, comprenant au moins un réseau de fibres et/ou au moins une couverture fibreuse (14), caractérisé en ce que ledit au moins un réseau fibreux et/ou ladite au moins une couverture fibreuse (14) comprend un système (500) dans lequel au moins un précurseur (8) est transformé en un produit biopesticide et/ou répulsif (10).

2. Appareil (100) de protection contre les nuisibles selon la revendication 1, caractérisé en ce que ledit système est composé de fibres (200) adaptées à contrôler les pathogènes des plantes qui sont couvertes avec au moins une préparation (300) apte à produire un biopesticide et/ou un répulsif pour contrôler les pathogènes des plantes.

3. Appareil (100) de protection contre les nuisibles selon la revendication 2, caractérisé en ce que ladite préparation (300) apte à produire un biopesticide et/ou un répulsif pour contrôler les pathogènes des plantes comprend au moins un enzyme actif (4).

4. Appareil (100) de protection contre les nuisibles selon la revendication 3, caractérisé en ce que ledit au moins un enzyme actif (4) est immobilisé à l'intérieur d'un matériau nanoporeux (2).

5. Appareil (100) de protection contre les nuisibles selon la revendication 4, caractérisé en ce que ledit un matériau nanoporeux (2) est un matériau mésoporeux.

6. Appareil (100) de protection contre les nuisibles selon l'une quelconque des revendications 4-5, caractérisé en ce que ledit matériau nanoporeux (2) est intégré dans un gel hydrophile (6).

7. Appareil (100) de protection contre les nuisibles selon la revendication 6, caractérisé en ce que ledit gel hydrophile (6) est fait d'agarose, d'agar-agar, d'alginate, de pectine, d'amidon et/ou de gélatine, de préférence d'alginate.

8. Appareil (100) de protection contre les nuisibles selon l'une quelconque des revendications 3-7, caractérisé en ce que ledit au moins un enzyme actif (4) est un enzyme actif choisi du groupe de glycosidase, de lyase et/ou de synthase de facteur lacrymogène-synthase, de préférence du groupe de glycosidase.

9. Appareil (100) de protection contre les nuisibles selon la revendication 8, caractérisé en ce que ledit au moins un enzyme (4) du groupe de glycosidase est le thioglucosidase.

10. Appareil (100) de protection contre les nuisibles selon l'une quelconque des revendications 3-9, caractérisé en ce que ledit au moins un enzyme actif (4) immobilisé à l'intérieur dudit matériau nanoporeux (2) est lié de manière covalente audit matériau nanoporeux (2), physiquement adsorbé sur ledit matériau nanoporeux (2), encapsulé à l'intérieur dudit 92794 matériau nanoporeux (2) ou piégé à l'intérieur dudit matériau nanoporeux (2). 11. Appareil (100) de protection contre les nuisibles la revendication 10, caractérisé en ce que ledit au moins un enzyme actif (4) immobilisé à l'intérieur dudit matériau nanoporeux (2) est réticulé avec du glutaraldéhyde ou avec du l-éthyle-3-(3-diméthyle-aminopropyle) carbodiimide et du /V-hydroxysuccinimide.

12. Appareil (100) de protection contre les nuisibles selon l'une quelconque des revendications 4-11, caractérisé en ce que ledit matériau nanoporeux (2) est un matériau mésoporeux à base de silice.

13. Appareil (100) de protection contre les nuisibles selon la revendication 12, caractérisé en ce que ledit matériau mésoporeux à base de silice comprend de l'orthosilicate de tétraéthyle et/ou de l'orthosilicate de tétraméthyle et/ou du méthyltriméthoxysilane et/ou du (3- aminopropyle)triméthoxysilane.

14. Appareil (100) de protection contre les nuisibles selon l'une quelconque des revendications 2-13, caractérisé en ce que ladite préparation (300) a un pH compris entre 4 et 11.

15. Appareil (100) de protection contre les nuisibles selon l'une quelconque des revendications 2-14, caractérisé en ce que ladite préparation (300) comprend en outre ledit au moins un précurseur (8), au moins un minéral et/ou au moins un cofacteur.

16. Appareil (100) de protection contre les nuisibles selon l'une quelconque des revendications 2-14, caractérisé en ce que ladite préparation (300) est adaptée pour recevoir ledit au moins un précurseur (8), au moins un minéral et/ou au moins un cofacteur.

17. Appareil (100) de protection contre les nuisibles selon l'une quelconque des revendications 15 ou 16, caractérisé en ce que ledit au moins un minéral et/ou ledit au moins un cofacteur est de l'acide ascorbique ou un tampon phosphate.

18. Appareil (100) de protection contre les nuisibles selon les revendications 15 et 17, caractérisé en ce que ledit tampon de phosphate est à une concentration comprise entre 10 mM et 500 mM, de préférence à une concentration comprise entre 50 mM et 250 mM, plus préférentiellement à une concentration comprise entre 75 mM et 150 mM, encore plus préférentiellement à une concentration de 100 mM.

19. Appareil (100) de protection contre les nuisibles selon les revendications 15 et 17, caractérisé en ce que ledit acide ascorbique est à une concentration comprise entre 50 pM et 1500 pM, de préférence à une concentration comprise entre 150 pM et 1000 pM, plus préférentiellement à une concentration comprise entre 250 pM et 750 pM, encore plus préférentiellement à une concentration de 500 pM.

20. Appareil (100) de protection contre les nuisibles selon l'une quelconque des revendications 1 à 19, caractérisé en ce que ledit appareil (100) de protection contre les nuisibles comprend en outre un moyen pour distribuer de l'eau sur le réseau fibreux et/ou la couverture fibreuse 92794 (14), lesdits moyens comprenant de préférence une cartouche (16) configurée pour être en communication fluidique avec ledit au moins un réseau fibreux et/ou ladite au moins une couverture fibreuse (14) et pour être alimenté avec de l'eau.

21. Appareil (100) de protection contre les nuisibles selon la revendication 20, caractérisé en ce que ladite cartouche (16) comprend au moins un précurseur (8) qui se transforme en un produit biopesticide et/ou répulsif (10) dans ledit système (500) dudit au moins un réseau fibreux et/ou de ladite au moins une couverture fibreuse (14).

22. Appareil (100) de protection contre les nuisibles selon l'une quelconque des revendications 20 à 21, caractérisé en ce que ladite cartouche (16) comprend en outre au moins un minéral et/ou au moins un cofacteur.

23. Une méthode de contrôle des pathogènes de plante avec un appareil (100) de protection contre les nuisibles, caractérisé en ce que ledit appareil (100) de protection contre les nuisibles est en accord avec l'une quelconque des revendications 1 à 22; et dans lequel ladite méthode comprend les étapes suivantes: a) l'activation d'au moins un précurseur (8) par addition d'eau au système (500) dudit au moins un réseau fibreux et/ou de ladite au moins une couverture fibreuse (14) pour produire un produit biopesticide et/ou répulsif pour contrôler les pathogènes des plantes; b) l'application du produit biopesticide et/ou répulsif résultant de l'étape (a) aux plantes, préférentiellement aux légumes et/ou aux fruits, afin de prévenir les attaques de nuisibles.

24. Méthode de contrôle des pathogènes des plantes selon la revendication 23, caractérisé en ce que ledit au moins un précurseur (8) est compris dans l'appareil (100) de protection contre les nuisibles, ledit appareil (100) de protection contre les nuisibles comprenant de préférence en outre au moins un minéral et/ou au moins un cofacteur.

25. Méthode de contrôle des pathogènes des plantes selon la revendication 23, caractérisé en ce que ledit au moins un précurseur (8) est ajouté audit appareil (100) de protection contre les nuisibles, ledit appareil (100) de protection contre les nuisibles comprenant de préférence au moins un minéral et/ou au moins un cofacteur.

26. Méthode de contrôle des pathogènes des plantes selon la revendication 23, caractérisé en ce que ledit au moins un précurseur (8) est ajouté avec également au moins un minéral et/ou au moins un cofacteur audit appareil (100) de protection contre les nuisibles. 92794 17/17 92794 Abstract

The invention is directed to a pest protective apparatus adapted for controlling plant pathogens. Said pest protective apparatus comprises at least one fibrous network and/or at least one fibrous cover. Said pest protective apparatus is remarkable in that said at least one fibrous network and/or said at least one fibrous cover comprises a system in which at least one precursor is transformed into a biopesticide and/or repellent product. National Application LE GOUVERNEMENT Number DU GRAND-DUCHÉ DE LUXEMBOURG Ministère de l'Économie SEARCH REPORT et du Commerce extérieur in accordance with Article 35.1 a) of the Luxembourg law on patents L0 1166 LU 92794 dated 20 July 1992 DOCUMENTS CONSIDERED TO BE RELEVANT

Category Citation of document with indication, where appropriate, Relevant CLASSIFICATION OF THE of relevant passages to claim APPLICATION (IPC)

Y DATABASE WPI 1-26 INV. Week 197820 Α01Ν25/0Θ Thomson Scientific, London, GB; A01N25/34 AN 1978-35958A C12N11/14 XP002744693, -& JP S53 38627 A (ASAHI CHEM IND CO LTD) 8 April 1978 (1978-04-08) * abstract *

Y DATABASE WPI 1-26 Week 199012 Thomson Scientific, London, GB; AN 1990-088317 ΧΡΘΘ2744694, -& JP ΗΘ2 42921 A (HANNAKA T) 13 February 1990 (1990-02-13) * abstract *

Y US 5 163 247 A (WEBER ROBERT E [US] ET AL) 1-26 17 November 1992 (1992-11-17) * claims 1, 6, 12, 13 * TECHNICAL FIELDS SEARCHED (IPC) Y W0 94/23573 Al (DU PONT [US]; MIAN AZIZ 1-26 A01N AHMED [US]; PETERSON LARRY WAYNE [US]; C12N WYSONG) 27 October 1994 (1994-10-27) * claims 1, 8 * * page 6, line 17 - line 18 * * page 7, line 3 - line 16 *

Y DATABASE WPI 1-26 Week 199629 Thomson Scientific, London, GB; AN 1996-283367 XP002744695, -& JP H08 119812 A (SUMITOMO CHEM CO LTD) 14 May 1996 (1996-05-14) * abstract *

-/--

The present search report has been drawn up for all claims

Date of completion of the search Examiner 21 September 2015 Habermann, Jörg (P04C55) CATEGORY OF CITED DOCUMENTS T : theory or principle underlying the invention E : earlier patent document, but published on, or 03.82

X : particularly relevant if taken alone after the filing date Y : particularly relevant if combined with another D : document cited in the application 1503

document of the same category L : document cited for other reasons A : technological background O : non-written disclosure & : member of the same patent family, corresponding FORM P : intermediate document document EPO

page 1 of 2 National Application LE GOUVERNEMENT Number DU GRAND-DUCHÉ DE LUXEMBOURG Ministère de l'Économie SEARCH REPORT et du Commerce extérieur in accordance with Article 35.1 a) of the Luxembourg law on patents L0 1166 LU 92794 dated 20 July 1992 DOCUMENTS CONSIDERED TO BE RELEVANT

Category Citation of document with indication, where appropriate, Relevant CLASSIFICATION OF THE of relevant passages to claim APPLICATION (IPC)

Y EMRE BASTÜRK ET AL: "Covalent 1-26 immobilization of [alpha]- onto thermally crosslinked electrospun PVA/PAA nanofibrous hybrid membranes", JOURNAL OF APPLIED POLYMER SCIENCE, vol. 127, no. 1, 4 May 2012 (2012-05-04), pages 349-355, XP5504O389, ISSN: 0021-8995, DOI: 10.1002/app.37901 * abstract *

A SHIWEN WANG ET AL: "Mesosilica-coated 1-26 ultrafine fibers for highly efficient laccase encapsulation", NANOSCALE, vol. 6, no. 12, 1 January 2014 (2014-01-01), page 6468, XP55214038, ISSN: 2040-3364, DOI: 10.1039/c4nr01166j * the whole document *

Y DE 33 12 214 Al (HAARMANN & REIMER GMBH 1-26 TECHNICAL FIELDS [DE]) 11 October 1984 (1984-10-11) SEARCHED (IPC) * example 7 * * page 4, line 22 - page 5, line 26 * * page 9, line 15 - line 21 *

Y,D W0 2015/013808 Al (MPT PRODUCTS & 1-26 TECHNOLOGIES INC [CA]) 5 February 2015 (2015-02-05) * examples 1-3 * * claims 1, 2, 19-21 *

The present search report has been drawn up for all claims 1 Date of completion of the search Examiner 21 September 2015 Habermann, Jörg (P04C55) CATEGORY OF CITED DOCUMENTS T : theory or principle underlying the invention E : earlier patent document, but published on, or 03.82

X : particularly relevant if taken alone after the filing date Y : particularly relevant if combined with another D : document cited in the application 1503

document of the same category L : document cited for other reasons A : technological background O : non-written disclosure & : member of the same patent family, corresponding FORM P : intermediate document document EPO

page 2 of 2 ANNEX TO THE SEARCH REPORT ON LUXEMBOURG PATENT APPLICATION NO. LO 1166 LU 92794

This annex lists the patent family members relating to the patent documents cited in the above-mentioned European search report. The members are as contained in the European Patent Office EDP file on The European Patent Office is in no way liable for these particulars which are merely given for the purpose of information. 21-09-2015

Patent document Publication Patent family Publication cited in search report date member(s) date

JP S5338627 A 08-04-1978 NONE

JP H0242921 A 13-02-1990 NONE

US 5163247 A 17-11-1992 CA 2055182 Al 20-10-1992 US 5163247 A 17-11-1992

WO 9423573 Al 27-10-1994 AU 6696694 A 08-11-1994 CA 2160867 Al 27-10-1994 EP 0695122 Al 07-02-1996 JP ΗΘ8508994 A 24-09-1996 WO 9423573 Al 27-10-1994

JP ΗΘ8119812 A 14-05-1996 NONE

DE 3312214 Al 11-10-1984 NONE

W0 2015013808 Al 05-02-2015 US 2015037310 Al 05-02-2015 W0 2015013808 Al 05-02-2015 P0457

FORM

For more details about this annex : see Official Journal of the European Patent Office, No. 12/82 EPO LE GOUVERNEMENT DU GRAND-DUCHÉ DE LUXEMBOURG Ministère de l'Économie et du Commerce extérieur WRITTEN OPINION

File No. Filing date (day/month/year) Priority date (day/month/year) Application No. LO1166 07.08.2015 LU92794

International Patent Classification (IPC) INV. A01N25/00 A01N25Z34 C12N11/14

Applicant Luxembourg Institute of Science and Technology

This report contains indications relating to the following items:

El Box No. I Basis of the opinion □ Box No. II Priority □ Box No. Ill Non-establishment of opinion with regard to novelty, inventive step and industrial applicability □ Box No. IV Lack of unity of invention □ Box No. V Reasoned statement with regard to novelty, inventive step or industrial applicability; citations and explanations supporting such statement □ Box No. VI Certain documents cited □ Box No. VII Certain defects in the application El Box No. VIII Certain observations on the application

Examiner

Habermann, Jörg Form LU237A (Cover Sheet) (January 2007) Application No. WRITTEN OPINION LU92794

Box No. I Basis of the opinion

1. This opinion has been established on the basis of the latest set of claims filed before the start of the search.

2. With regard to any nucleotide and/or amino acid sequence disclosed in the application and necessary to the claimed invention, this opinion has been established on the basis of:

a. type of material:

□ a sequence listing

□ table (s) related to the sequence listing

b. format of material:

□ on paper

□ in electronic form

c. time of filing/furnishing:

□ contained in the application as filed.

□ filed together with the application in electronic form.

□ furnished subsequently.

3. □ In addition, in the case that more than one version or copy of a sequence listing and/or table relating thereto has been filed or furnished, the required statements that the information in the subsequent or additional copies is identical to that in the application as filed or does not go beyond the application as filed, as appropriate, were furnished.

4. Additional comments:

Box No. V Reasoned statement with regard to novelty, inventive step and industrial applicability; citations and explanations supporting such statement

1. Statement

Novelty Yes: Claims 1-26 No: Claims

Inventive step Yes: Claims No: Claims 1-26

Industrial applicability Yes: Claims 1-26 No: Claims

2. Citations and explanations

see separate sheet

Form LU237B (January 2007) Application No. WRITTEN OPINION LU92794

Box No. VIII Certain observations on the application see separate sheet

Form LU237B (January 2007) WRITTEN OPINION Application number (SEPARATE SHEET) LU92794

Reference is made to the following documents:

D1 DATABASE WPI Week 197820 Thomson Scientific, London, GB; AN 1978-35958A, XP002744693;

-& JP S53 38627 A (ASAHI CHEM IND CO LTD) 8 April 1978 (1978-04-08)

D2 DATABASE WPI Week 199012 Thomson Scientific, London, GB; AN 1990-088317, XP002744694;

-& JP H02 42921 A (HANNAKA T) 13 February 1990 (1990-02-13)

D3 US 5 163 247 A (WEBER ROBERT E [US] ET AL) 17 November 1992 (1992-11-17)

D4 WO 94/23573 A1 (DU PONT [US]; MIAN AZIZ AHMED [US]; PETERSON LARRY WAYNE [US]; WYSONG) 27 October 1994 (1994-10-27)

D5 DATABASE WPI Week 199629 Thomson Scientific, London, GB; AN 1996-283367, XP002744695;

-& JP H08 119812 A (SUMITOMO CHEM CO LTD) 14 May 1996 (1996-05-14)

D6 Emre Bastürk ET AL: "Covalent immobilization of [alpha]-amylase onto thermally crosslinked electrospun PVA/PAA nanofibrous hybrid membranes", Journal of Applied Polymer Science, vol. 127, no. 1,4 May 2012 (2012-05-04), pages 349-355, XP55040389, ISSN: 0021-8995, DOI: 10.1002/app.37901

D7 Shiwen Wang ET AL: "Mesosilica-coated ultrafine fibers for highly efficient laccase encapsulation", Nanoscale, vol. 6, no. 12,1 January 2014 (2014-01-01), page 6468, XP55214038, ISSN: 2040-3364, DOI: 10.1039/c4nr01166j

D8 DE 33 12 214 A1 (HAARMANN & REIMER GMBH [DE]) 11 October 1984 (1984-10-11)

D9 WO 2015/013808 A1 (MPT MUSTARD PRODUCTS & TECHNOLOGIES INC [CA]) 5 February 2015 (2015-02-05)

Form LU237-3 (separate sheet) (January 2007) (sheet 1) WRITTEN OPINION Application number (SEPARATE SHEET) LU92794

Re Item V

Reasoned statement with regard to novelty, inventive step or industrial applicability; citations and explanations supporting such statement

1 Novelty

The present application is directed to an apparatus comprising a fibrous network and/ or a fibrous cover characterised in that said fibrous network and/or fibrous cover comprise a system in which a precursor is transformed into a biopesticide product.

Document D1 discloses a pest protective apparatus for controlling weeds comprising a fibrous network characterised in that said fibrous network comprises a system which releases a herbicide. Said system comprises a herbicide which has been adsorbed onto clay, lime or gypsum to improve residual activity which has been coated onto the fibre which is cellulosic, such as cotton (abstract).

D1 does not disclose the presence of a system which transforms a precursor into a biopesticide.

Document D2 discloses a pest protective apparatus for controlling plant pathogenic fungi comprising a fibrous network characterised in that said fibrous network comprises a system which releases a fungicide, such as thiabendazole or copper 8- hydroquinolinolate. Said system comprising a fungicide has been coated onto the fibre which is natural, such as hemp (abstract).

D2 does not disclose the presence of a system which transforms a precursor into a biopesticide.

Document D3 discloses a pest protective apparatus for controlling plant pathogens comprising a fibrous network coated with a latex further including antimicrobial agent, a pesticide or a fungicide (claims 1,6,12 and 13).

D3 does not disclose the presence of a system which transforms a precursor into a biopesticide.

Document D4 discloses a network comprising a polymeric fiber wherein an agriculturally active ingredient has been uniformly dispersed (claims 1,8). The fibers are made of water-soluble polymers, such as polyvinyl alcohol, polyethylene oxide, hydroxypropyl cellulose, hydroxyethyl cellulose, carboxymethyl cellulose, polyvinyl pyrrolidone, polyacryl amide or polyacrylic acid (p. 7,1. 3-16). The agriculturally active ingredient may be Bacillus thuringiensis or a baculovirus (p. 6,1. 17-18). D4 does not disclose the presence of a system which transforms a precursor into a biopesticide.

Form LU237-3 (separate sheet) (January 2007) (sheet 2) WRITTEN OPINION Application number (SEPARATE SHEET) LU92794

Document D5 discloses a pest protective apparatus for controlling plant pathogenic insects comprising a fibrous network characterised in that said fibrous network comprises a system which releases an insecticide. Said system comprising an insecticide, such as fenpropathrin or bifenthrin has been coated onto the fibre which may be natural, such as cotton, silk, wool, cellulose or flax (abstract).

D5 does not disclose the presence of a system which transforms a precursor into a biopesticide.

Document D6 discloses a fibrous network made from thermally crosslinked electrospun polyvinyl alcohol/polyacrylic acid. Onto said fibrous network a-amylase has been immobilised by covalent linkage (abstract).

D6 does not disclose the presence of a system which transforms a precursor into a biopesticide.

Document D7 discloses a fibre comprising a coating with mesoporous silica and the enzyme laccase (an oxidase) adsorbed therein. The composition demonstrated long­ term activity and recycling stability (the whole document).

D7 does not appear to disclose a fibre or preparation able to control plant pathogens. However, the applicant is advised that at any time during later proceedings a third party may provide information that laccase is capable of transforming a certain substrate into a biopesticide.

Document D8 discloses immobilised myrosinase (a thioglucosidase). Especially preferred for the immobilisation of myrosinase are acidic inorganic ion exchangers, such a zeolites or aluminosilicates (p. 4,1. 22 - p. 5,1. 26). The stability of immobilised myrosinase is reported to be significantly enhanced (p. 9,1. 15-21). The immobilised myrosinase hydrolyses a solution comprising liberating and (example 7).

D8 does not disclose a fibrous network and/or a fibrous cover.

Document D9 discloses a method for controlling pest in agricultural settings with a composition comprising myrosinase and sinigrin provided in form of a liquid formulation. The enzyme acts on the precursor to liberate for example allyl isothiocyanate (examples 1-3; claims 1,2,19-21). D9 does not disclose a fibrous network and/or a fibrous cover.

The subject-matter of claims 1-26 appears new, since none of the documents D1-D9 discloses methods and/or products having the exact features claimed by the applicant.

Form LU237-3 (separate sheet) (January 2007) (sheet 3) WRITTEN OPINION Application number (SEPARATE SHEET) LU92794

2 Inventive Step

The present application does not meet the criteria of patentability, because the subject-matter of claims 1-26 does not involve an inventive step.

Document D5 is regarded as being the prior art closest to the subject-matter of present claim 1 and discloses a pest protective apparatus comprising a fibrous network characterised in that said fibrous network comprises a pesticide and repellent product, such as bifenthrin.

The subject-matter of claim 1 therefore differs from this known apparatus in that a system in which a precursor is transformed into a biopesticide and/or repellent product is comprised instead of the product.

The present application does not provide any experimental data but merely hypothetical considerations.

The problem to be solved by the present invention may therefore be regarded as provision of an alternative preparation.

The solution proposed in claim 1 of the present application cannot be considered as involving an inventive step.

The skilled artisan is aware from document D9 of the possibility to produce a biopesticide (allyl isothiocyanate) by enzymatic action on a precursor material. He is further aware that it is possible to support the enzyme used in D9 in a porous carrier material (D8).

There is however no particular motivation apparent from the prior art combine the teaching of D5 (indicating the use of a natural fibrous material as a carrier for a pesticide and/or repellent) with the teaching of D8 and D9 (describing a biopesticide produced by enzymatic action and a way of how to stabilise the enzyme by supporting it onto a porous carrier).

Nonetheless, an inventive step may not be recognised to the subject-matter of present claim 1. The applicant has not demonstrated any unexpected effect deriving from the agglomeration of features according to present claim 1. All features are singularly known in the art for their technical effects. The mere combination of such features without any unexpected additional effect deriving from said combination however may not be regarded as providing an inventive step. Therefore, an inventive step must be denied to the subject-matter of present claim 1. The same applies to the further claims 2-26 the features of which do not provide any further technical elements for which have been experimentally demonstrated any effects of a surprising nature.

Form LU237-3 (separate sheet) (January 2007) (sheet 4) WRITTEN OPINION Application number (SEPARATE SHEET) LU92794

Moreover, it must be said that the present application is a concept paper which appears to be based on some theoretical considerations involving knowledge available in the prior art. However, no concrete experimental data has been provided as to the actual workability and effect of the claimed subject-matter. In addition, most of the claims have been defined so broad and vague ("a precursor is transformed into a biopesticide", "fibres adapted to control plant pathogens", "preparation is able to produce biopesticide") that they actually constitute nothing more than the invitation to embark on a research programme in order to establish their exact scope and limitations. Therefore, for the reasons laid out above, an inventive step must be denied to the subject-matter of present claims 1-26.

3 Industrial Applicability

The subject-matter of the present application seems to be industrially applicable.

Re Item VIII

Certain observations on the application

Present claim 1 lacks clarity. The claim is directed to a product which has been defined in terms of a result its needs to achieve ("a precursor is transformed into a biopesticide or repellent"). This merely amounts to a statement of the underlying problem, without providing the detailed technical features necessary for achieving this result. In the present case it appears that it would have been possible to define the subject-matter of present claim 1 with the actual features the preparation should have. The way the claim has been configured presently amounts to nothing more than the invitation to embark on a research programme in order to identify a fibrous network and/or a fibrous cover and at least one precursor for any biopesticide product as well as a means for the desired transformation.

In addition, it is noted that the term "for" must be interpreted as "suitable for".

The subject-matter of present claim 13 is not clear. The skilled artisan does not understand, how the apparatus (100) may comprise silicates which contain a tetrasubstituted silicon compound of the specific list given. What the applicant potentially intended was that the apparatus comprises silica which comprises a linker unit covalently joining the mesoporous material and the enzyme, said linker unit being based on the tetrasubstituted silicon compounds mentioned in the list.

Present claim 23 lacks clarity. It is not clear how the method should work in absence of the precursor (8) - which apparently is not present, since claim 25 defines as a further method step the addition of said precursor (8).

Form LU237-3 (separate sheet) (January 2007) (sheet 5) WRITTEN OPINION Application number (SEPARATE SHEET) LU92794

The features following the terms "preferentially", "more preferentially" and/or "even more preferentially" in present claims 7, 8,18,19 and 23-25 are to be regarded as merely facultative and are not limiting the subject-matter of the claim in any way.

Figure 1 discloses two different elements with the reference number (200).

Form LU237-3 (separate sheet) (January 2007) (sheet 6)