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BRPI0912489B1 - CARBON NANOTUBE CONJUGATE TO INHIBIT PATHOGEN INFECTION STRUCTURES IN VEGETABLES - Google Patents

CARBON NANOTUBE CONJUGATE TO INHIBIT PATHOGEN INFECTION STRUCTURES IN VEGETABLES Download PDF

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BRPI0912489B1
BRPI0912489B1 BRPI0912489-6A BRPI0912489A BRPI0912489B1 BR PI0912489 B1 BRPI0912489 B1 BR PI0912489B1 BR PI0912489 A BRPI0912489 A BR PI0912489A BR PI0912489 B1 BRPI0912489 B1 BR PI0912489B1
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carbon nanotubes
vegetables
structures
conjugate
inhibit
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BRPI0912489-6A
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Orlando Ladeira Luiz
Rodrigues Leonardo
Corrêa Junior Ary
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Universidade Federal De Minas Gerais
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Priority to BRPI0912489A priority Critical patent/BRPI0912489B8/en
Priority to PCT/BR2010/000411 priority patent/WO2011079356A1/en
Priority to ARP100104959A priority patent/AR079751A1/en
Publication of BRPI0912489A2 publication Critical patent/BRPI0912489A2/en
Publication of BRPI0912489B1 publication Critical patent/BRPI0912489B1/en
Publication of BRPI0912489B8 publication Critical patent/BRPI0912489B8/en

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    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
    • A01N63/00—Biocides, pest repellants or attractants, or plant growth regulators containing microorganisms, viruses, microbial fungi, animals or substances produced by, or obtained from, microorganisms, viruses, microbial fungi or animals, e.g. enzymes or fermentates
    • A01N63/60—Isolated nucleic acids
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B82—NANOTECHNOLOGY
    • B82Y—SPECIFIC USES OR APPLICATIONS OF NANOSTRUCTURES; MEASUREMENT OR ANALYSIS OF NANOSTRUCTURES; MANUFACTURE OR TREATMENT OF NANOSTRUCTURES
    • B82Y15/00—Nanotechnology for interacting, sensing or actuating, e.g. quantum dots as markers in protein assays or molecular motors
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
    • A01N25/00—Biocides, pest repellants or attractants, or plant growth regulators, characterised by their forms, or by their non-active ingredients or by their methods of application, e.g. seed treatment or sequential application; Substances for reducing the noxious effect of the active ingredients to organisms other than pests
    • A01N25/34—Shaped forms, e.g. sheets, not provided for in any other sub-group of this main group
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P3/00—Drugs for disorders of the metabolism

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  • Wood Science & Technology (AREA)
  • Dentistry (AREA)
  • Pest Control & Pesticides (AREA)
  • Agronomy & Crop Science (AREA)
  • Crystallography & Structural Chemistry (AREA)
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  • Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
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  • Saccharide Compounds (AREA)

Abstract

conjugado de nanotubos de carbono para inibir estruturas de infecção de patógenos em vegetais. a presente invenção descreve a construção e uso de um sistema hibrido envolvendo a conjugação de nanotubos de carbono e oligonucleotideos. em seu aspecto mais geral relata processo e metodologia para a inibição ou controle de pragas e infecções de patógenos em vegetais, em especial, em culturas de importante interesse comercial tais como: feijão, soja, café e eucalipto, o conjugado oligonucleotídeo-nanotubos de carbono é utilizado como agente de internalização celular carreando uma seqúência específica de ácido nucléico, também denominado de oligonucleotídeo, de fora para dentro do citoplasma da célula do patógeno.carbon nanotube conjugate to inhibit plant pathogen infection structures. The present invention describes the construction and use of a hybrid system involving the conjugation of carbon nanotubes and oligonucleotides. in its most general aspect reports process and methodology for the inhibition or control of pests and pathogen infections in plants, especially in crops of important commercial interest such as: beans, soybeans, coffee and eucalyptus, the oligonucleotide-carbon nanotube conjugate. It is used as a cell internalizing agent carrying a specific nucleic acid sequence, also called an oligonucleotide, from outside into the pathogen's cell cytoplasm.

Description

(54) Título: CONJUGADO DE NANOTUBOS DE CARBONO PARA INIBIR ESTRUTURAS DE INFECÇÃO DE PATÓGENOS EM VEGETAIS (51) Int.CI.: C12N 15/113; C12N 15/82; B82Y 5/00 (73) Titular(es): UNIVERSIDADE FEDERAL DE MINAS GERAIS (72) Inventor(es): LUIZ ORLANDO LADEIRA; LEONARDO RODRIGUES; ARY CORRÊA JUNIOR(54) Title: CONJUGATE OF CARBON NANOTUBES TO INHIBIT STRUCTURES OF PATHOGEN INFECTION IN VEGETABLES (51) Int.CI .: C12N 15/113; C12N 15/82; B82Y 5/00 (73) Holder (s): FEDERAL UNIVERSITY OF MINAS GERAIS (72) Inventor (s): LUIZ ORLANDO LADEIRA; LEONARDO RODRIGUES; ARY CORRÊA JUNIOR

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CONJUGADO DE NANOTUBOS DE CARBONO PARA INIBIRCONJUGATE OF CARBON NANOTUBES TO INHIBIT

ESTRUTURAS DE INFECÇÃO DE PATÓGENOS EM VEGETAIS”STRUCTURES OF PATHOGEN INFECTION IN VEGETABLES ”

CAMPO DA INVENÇÃOFIELD OF THE INVENTION

A presente invenção descreve a construção e uso de um sistema híbrido envolvendo a conjugação de nanotubos de carbono e oligonucleotídeos. Em seu aspecto mais geral relata processo e metodologia para a inibição ou controle de pragas e infecções de patógenos em vegetais, em especial, em culturas de importante interesse comercial tais como: feijão, soja, café e eucalipto. O conjugado oligonucleotídeo-nanotubos de carbono é utilizado como agente de internalização celular carreando uma seqüência específica de ácido nucléico, também denominado de oligonucleotídeo, de fora para dentro do citoplasma da célula do microrganismo. O objetivo desta internalização é permitir que o oligonucleotídeo passe para o citoplasma do microrganismo e interfira nos mecanismos de síntese protéica regulada pelo RNA mensageiro do microrganismo que como resultado leva a inibição de estruturas de infecção, causando a morte do microrganismo ou diminuição dos efeitos nocivos do agressor ao hospedeiro.The present invention describes the construction and use of a hybrid system involving the conjugation of carbon nanotubes and oligonucleotides. In its most general aspect, it reports a process and methodology for the inhibition or control of pests and infections of pathogens in vegetables, especially in crops of important commercial interest such as: beans, soybeans, coffee and eucalyptus. The oligonucleotide-carbon nanotube conjugate is used as a cell internalizing agent carrying a specific sequence of nucleic acid, also called oligonucleotide, from outside into the cytoplasm of the microorganism's cell. The purpose of this internalization is to allow the oligonucleotide to pass into the microorganism's cytoplasm and interfere with the protein synthesis mechanisms regulated by the microorganism's messenger RNA which, as a result, leads to the inhibition of infection structures, causing the death of the microorganism or reducing the harmful effects of aggressor to the host.

ESTADO DA TÉCNICATECHNICAL STATUS

Nanotubos de Carbono são estruturas quase unidimensionais formadas por ligações carbono-carbono em hibridização sp2 na forma de tubos cujo diâmetro pode variar de 1 nm (10'9 m) a 100 nm e comprimento típico da ordem de 104 vezes seu diâmetro (HERBST, M.H.; MACEDO, M.I.F. & ROCCO, A.M. Tecnologia dos Nanotubos de carbono: tendências e perspectivas de uma área multidisciplinar. Quím.Nova. 27 (6): 986-992. 2004; IIJIMA, S & ICHIHASHI, T.Carbon nanotubes are almost one-dimensional structures formed by carbon-carbon bonds in sp2 hybridization in the form of tubes whose diameter can vary from 1 nm (10 ' 9 m) to 100 nm and a typical length of the order of 10 4 times their diameter (HERBST, MH; MACEDO, MIF & ROCCO, AM Technology of carbon nanotubes: trends and perspectives of a multidisciplinary area. Quim.Nova. 27 (6): 986-992. 2004; IIJIMA, S & ICHIHASHI, T.

Single-shell carbon nanotubes of 1-nm diameter. Nature. 363: 603-605. 1993; IIJIMA, S. Helical microtubules of graphitic carbon. Nature. 354: 56-58. 1991). Os nanotubos de carbono podem ser produzidos com uma única parede de carbono sendo denominados de nanotubos de carbono de parede única (SWNT) ou com múltiplas paredes de carbono concêntricas denominados de nanotubos de carbono de múltiplas paredes (MWNT) (SINHA, N. & YEOW,Single-shell carbon nanotubes of 1-nm diameter. Nature. 363: 603-605. 1993; IIJIMA, S. Helical microtubules of graphitic carbon. Nature. 354: 56-58. 1991). Carbon nanotubes can be produced with a single carbon wall being called single-wall carbon nanotubes (SWNT) or with multiple concentric carbon walls called multi-wall carbon nanotubes (MWNT) (SINHA, N. & YEOW ,

J.T.W. Carbon Nanotubes for biomedical applications. IEEE Transactions onJ.T.W. Carbon Nanotubes for biomedical applications. IEEE Transactions on

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Nanobioscience. 4(2): 180-195. 2005). Os nanotubos de carbono possuem alta rigidez estrutural, alta biocompatibilidade, baixa citotoxicidade e com seu diâmetro na faixa de alguns nanômetros são pequenos o suficiente para atravessar passivamente a parede celular e membrana citoplasmática de células, carreando assim biomoléculas ligadas a sua superfície do meio extracelular para o intracelular (KOSTARELOS, K.; LACERDA, L.; PASTORIN, G.; WU, W.; WIECKOWSKI, S.; LUANGSIVILAY, J.; GODEFROY, S.; PANTAROTTO, D.; BRIAND, J.; MULLER.S.; PRATO, M. & BIANCO, A. Cellular uptake of functionalized carbon nanotubes is independent of functional ío group and cell type. Nature. 2: 108-113. 2007; ZHANG, Z.; YANG, X.; ZHANG, Y.; ZENG, B.; WANG, S.; ZHU, T.; RODEN, R.B.S.; CHEN, Y. & YANG, R. Delivery of telomerase reverse transcriptase small interfering RNA in complex with positively charged single-walled carbon nanotubes suppresses tumor growth. Clin.Cancer Res. 12 (16): 4933-4939. 2006; KAM, N.W.S. & DAI, H.Nanobioscience. 4 (2): 180-195. 2005). Carbon nanotubes have high structural rigidity, high biocompatibility, low cytotoxicity and with a diameter in the range of a few nanometers are small enough to passively pass through the cell wall and cytoplasmic membrane of cells, thus carrying biomolecules attached to their surface from the extracellular medium to the intracellular (KOSTARELOS, K .; LACERDA, L .; PASTORIN, G .; WU, W .; WIECKOWSKI, S .; LUANGSIVILAY, J .; GODEFROY, S .; PANTAROTTO, D .; BRIAND, J .; MULLER. S .; PRATO, M. & BIANCO, A. Cellular uptake of functionalized carbon nanotubes is independent of functional group and cell type. Nature. 2: 108-113. 2007; ZHANG, Z .; YANG, X .; ZHANG, Y .; ZENG, B .; WANG, S .; ZHU, T .; RODEN, RBS; CHEN, Y. & YANG, R. Delivery of telomerase reverse transcriptase small interfering RNA in complex with positively charged single-walled carbon nanotubes suppresses tumor growth.Clin.Cancer Res. 12 (16): 4933-4939. 2006; KAM, NWS & DAI, H.

Carbon nanotubes as intracellular protein transporters: generality and biological functionality. J.Am.Chem.Soc. 127: 6021-6026. 2005; KAM, N.W.S.; JESSOP, T.C.; WENDER, P.A. & DAI, H. Nanotube molecular transporters: Internalization of carbon nanotube-protein conjugates into mammalian cells. J.Am.Chem.Soc. 126: 6850-6851. 2004). A ligação ou imobilização de biomoléculas a parede externa de nanotubos de carbono pode ser feita através de dois processos:Carbon nanotubes as intracellular protein transporters: generality and biological functionality. J.Am.Chem.Soc. 127: 6021-6026. 2005; KAM, N.W.S .; JESSOP, T.C .; WENDER, P.A. & DAI, H. Nanotube molecular transporters: Internalization of carbon nanotube-protein conjugates into mammalian cells. J.Am.Chem.Soc. 126: 6850-6851. 2004). The attachment or immobilization of biomolecules to the outer wall of carbon nanotubes can be done through two processes:

- Imobilização covalente - neste caso uma molécula ponte tem uma de suas extremidades ligada covalentemente à parede do nanotubo de carbono e a biomolécula ligada covalentemente à outra extremidade da molécula ponte (CHEN, S.; SHEN, W.; WU, G.; CHEN, D. & JIANG, Μ. A new approach to the functionalization of single-walled carbon nanotubes with both alkyl and carboxyl groups. Chem.Phys.Letters. 402: 312-317. 2005; HE, P. & URBAN, M.W. Controlled phospholipids functionalization of single-walled carbon nanotubes. Biomacromolecules. 6: 2455-2457. 2005; WANG, Y.; IQBAL, Z. & MALHOTRA, S.V. Functionalization of carbon nanotubes with amines and enzymes.- Covalent immobilization - in this case a bridge molecule has one end attached covalently to the wall of the carbon nanotube and the biomolecule linked covalently to the other end of the bridge molecule (CHEN, S .; SHEN, W .; WU, G .; CHEN , D. & JIANG, Μ. A new approach to the functionalization of single-walled carbon nanotubes with both alkyl and carboxyl groups. Chem.Phys.Letters. 402: 312-317. 2005; HE, P. & URBAN, MW Controlled phospholipids functionalization of single-walled carbon nanotubes. Biomacromolecules.6: 2455-2457. 2005; WANG, Y .; IQBAL, Z. & MALHOTRA, SV Functionalization of carbon nanotubes with amines and enzymes.

Chem.Phys.Letters. 402: 96-101. 2005; PANTAROTTO, D.; PARTIDOS, C.D.; GRAFF, R.;HOEBEKE, J.; BRIAND, J.; PRATO, M. & BIANCO, A. Synthesis, structural characterization, and immunological properties of carbon nanotubesChem.Phys.Letters. 402: 96-101. 2005; PANTAROTTO, D .; PARTIDOS, C.D .; GRAFF, R.; HOEBEKE, J .; BRIAND, J .; PRATO, M. & BIANCO, A. Synthesis, structural characterization, and immunological properties of carbon nanotubes

3/15 functionalized with peptides. J.Am.Chem.Soc. 125 (20): 6160-6164. 2003;3/15 functionalized with peptides. J.Am.Chem.Soc. 125 (20): 6160-6164. 2003;

POMPEO, F. & RESASCO, D.E. Water solubilization of single-walled carbon nanotubes by functionalization with glucosamine. Nanoletters. 2 (4): 369-373.POMPEO, F. & RESASCO, D.E. Water solubilization of single-walled carbon nanotubes by functionalization with glucosamine. Nanoletters. 2 (4): 369-373.

2002).2002).

- Imobilização não-covalente - neste caso a biomolécula adsorve à parede do nanotubo de carbono de modo não-covalente ficando fracamente aderida a sua parede por forças de Van der Walls (BECKER, M.L.; FAGAN, J.A.; GALLANT, N.D.; BAUER, B.J.; BAJPAI, V.; HOBBIE, E.K.; LACERDA, S.H.; MIGLER, K.B. & JAKUPCIAK, J.P. Length-dependent uptake of DNA-wrapped single-walled ío carbon nanotubes. Adv.Mater. 19: 939-945. 2007; GIGLIOTTI, B.; SAKIZZIE, B.; BETHUNE, D.S.; SHELBY, R.M. & CHA, J.N. Sequence-independent helical wrapping of single-walled carbon nanotubes by long genomic DNA. Nanoletters. 6 (2): 159-164. 2006; ZHANG, Z.; YANG, X.; ZHANG, Y.; ZENG, B.; WANG, S.; ZHU, T.; RODEN, R.B.S.; CHEN, Y. & YANG, R. Delivery of telomerase reverse transcriptase small interfering RNA in complex with positively charged single-walled carbon nanotubes suppresses tumor growth. Clin.Câncer Res. 12 (16): 4933-4939. 2006; KAM, N.W.S. & DAI, H. Carbon nanotubes as intracellular protein transporters: generality and biological functionality. J.Am.Chem.Soc. 127: 6021-6026. 2005; SIRDESHMUKH, R.; TEKER, K. &- Non-covalent immobilization - in this case, the biomolecule adsorbes to the carbon nanotube wall in a non-covalent way, being weakly adhered to its wall by Van der Walls forces (BECKER, ML; FAGAN, JA; GALLANT, ND; BAUER, BJ ; BAJPAI, V .; HOBBIE, EK; LACERDA, SH; MIGLER, KB & JAKUPCIAK, JP Length-dependent uptake of DNA-wrapped single-walled carbon nanotubes. Adv.Mater. 19: 939-945. 2007; GIGLIOTTI, B .; SAKIZZIE, B .; BETHUNE, DS; SHELBY, RM & CHA, JN Sequence-independent helical wrapping of single-walled carbon nanotubes by long genomic DNA. Nanoletters. 6 (2): 159-164. 2006; ZHANG, Z .; YANG, X .; ZHANG, Y .; ZENG, B .; WANG, S .; ZHU, T .; RODEN, RBS; CHEN, Y. & YANG, R. Delivery of telomerase reverse transcriptase small interfering RNA in complex with positively charged single-walled carbon nanotubes suppresses tumor growth.Clin.Cancer Res. 12 (16): 4933-4939. 2006; KAM, NWS & DAI, H. Carbon nanotubes as intracellular protein transporters: generality and biological functionality. J.Am.Chem.Soc. 127: 6021-6026. 2005; SIRDESHMUKH, R .; TEKER, K. &

PANCHAPAKESAN, B. Biological functionalization of carbon nanotubes. Mat. Res. Soc. Symp. Proc. 823: W4.1.1-W4.1.6. 2004; CHEN, R.J.;PANCHAPAKESAN, B. Biological functionalization of carbon nanotubes. Mat. Res. Soc. Symp. Proc. 823: W4.1.1-W4.1.6. 2004; CHEN, R.J .;

BANGSARUNTIP, S.; DROUVALAKIS, K.A.; KAM, N.W.S.; SHIM, M.; LI, Y.; KIM,W.; UTZ, P.J. & DAI, H. Noncovalent functionalization of carbon nanotubes for highly specific electronic biosensors. PNAS. 100 (9): 4984-4989. 2003).BANGSARUNTIP, S .; DROUVALAKIS, K.A .; KAM, N.W.S .; SHIM, M .; LI, Y .; KIM, W .; UTZ, P.J. & DAI, H. Noncovalent functionalization of carbon nanotubes for highly specific electronic biosensors. PNAS. 100 (9): 4984-4989. 2003).

O feijoeiro é cultivado em todas as unidades da federação, sendo o quarto produto agrícola em área plantada e o sexto em valor da produção de grãos do país (IBGE - Fundação Instituto Brasileiro de Geografia e Estatística. Levantamento sistemático da produção agrícola: pesquisa mensal de previsão e acompanhamento das safras agrícolas no ano civil. Rio de Janeiro, v.21, n.1,The common bean is grown in all units of the federation, being the fourth agricultural product in planted area and the sixth in value of the country's grain production (IBGE - Brazilian Institute of Geography and Statistics Foundation. Systematic survey of agricultural production: monthly survey of forecast and monitoring of agricultural crops in the calendar year. Rio de Janeiro, v.21, n.1,

79p, 2009; ZIMMERMANN, M.J.DE; ROCHA, M.; YAMADA, T. Cultura do feijoeiro. Piracicaba: Associação Brasileira para a Pesquisa da Potassa e do Fosfato (POTAFOS), 689p. 1988). Entretanto, os Estados do Paraná, Minas79p, 2009; ZIMMERMANN, M.J.DE; ROCHA, M .; YAMADA, T. Bean culture. Piracicaba: Brazilian Association for the Research of Potash and Phosphate (POTAFOS), 689p. 1988). However, the States of Paraná, Minas Gerais

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Gerais, Bahia e São Paulo são, em ordem decrescente, os principais produtores do país (IBGE - Fundação Instituto Brasileiro de Geografia eGerais, Bahia and São Paulo are, in decreasing order, the main producers in the country (IBGE - Fundação Instituto Brasileiro de Geografia e

Estatística. Levantamento sistemático da produção agrícola: pesquisa mensal de previsão e acompanhamento das safras agrícolas no ano civil. Rio deStatistic. Systematic survey of agricultural production: monthly survey of forecast and monitoring of agricultural crops in the calendar year. River of

Janeiro, v.21, n.1, 79p, 2009).Janeiro, v.21, n.1, 79p, 2009).

Dentre os fatores responsáveis por baixar a produtividade do feijoeiro estão as doenças, das quais a ferrugem [Uromyces appendiculatus (Pers.) Unger] é considerada como de grande importância, causando danos da ordem de 45% podendo chegar a 100%, o que vai estar diretamente relacionado à severidade precoce da infecção (SANNAZZARO, A.M.; OLIVEIRA, S.H.F.; WUTKE, E.B.; CASTRO, J.L.; GALLO, P.B.; MARTINS, A.L.M.; BORTOLETO, N.; SABINO, J.C.; SILVEIRA, L.C.P.; SAKAI.M.; SAES, L.A.; PAULO, E.M.; KASAI, F.S.; DORNELLES, C.R.F. & BACCHI, G.S. Severidade de ferrugem em cultivares de feijoeiro no Estado de São Paulo. Arq.Inst.Biol. 70 (3): 323329. 2003; COELHO, R.R.; VALE F.X.R.; JESUS JUNIOR, W.C.; PAUL, P.A.; ZAMBOLIM, L. & BARRETO, R.W. Determinação das condições climáticas que favorecem o desenvolvimento da ferrugem e da mancha angular do feijoeiro. Fitopatol.Bras. 28 (5): 508-514. 2003; JESUS JUNIOR, W.C.; VALE, F.X.R.; COELHO, R.R.; HAU, B.; ZOMBOLIM, L.; COSTA, L.C. & BERGAMIN FILHO, A. Effects of angular leaf spot and rust on yield loss of Phaseolus vulgarís. Phytopathology. 91 (11): 1045-1053. 2001; FALEIRO, F.G.; RAGAGNIN, V.A.; VINHADELLI, W.S.; MOREIRA, M.A.; STAVELY, J.R. & BARROS, E.G. Resistência de linhagens de feijoeiro a quatro raças de Uromyces appendiculatus isoladas em Minas Gerais, Brasil. Fitopatol.Bras. 26 (1): 77-80. 2001; RODRIGUES, F.A.; FERNANDES, J.J. & MARTINS, M. Influência de semeaduras sucessivas de feijoeiro na severidade da mancha-angular e ferrugem e perdas na produção. Pesq. Agropec. Bras. 34 (8): 1373-1378. 1999; HALL, R. Compendium of bean disease. St. Paul: The American Phytopathological Society, 73p. 1991).Among the factors responsible for lowering the productivity of beans are diseases, of which the rust [Uromyces appendiculatus (Pers.) Unger] is considered to be of great importance, causing damage in the order of 45% and reaching 100%, which will be directly related to the early severity of the infection (SANNAZZARO, AM; OLIVEIRA, SHF; WUTKE, EB; CASTRO, JL; GALLO, PB; MARTINS, ALM; BORTOLETO, N .; SABINO, JC; SILVEIRA, LCP; SAKAI.M. ; SAES, LA; PAULO, EM; KASAI, FS; DORNELLES, CRF & BACCHI, GS Rust severity in common bean cultivars in the State of São Paulo. Arch.Biol. 70 (3): 323329. 2003; COELHO, RR; VALE FXR; JESUS JUNIOR, WC; PAUL, PA; ZAMBOLIM, L. & BARRETO, RW Determination of climatic conditions that favor the development of rust and angular leaf spot in common beans. Fitopatol.Bras. 28 (5): 508- 514. 2003; JESUS JUNIOR, WC; VALE, FXR; COELHO, RR; HAU, B .; ZOMBOLIM, L .; COSTA, LC & BERGAM IN FILHO, A. Effects of angular leaf spot and rust on yield loss of Phaseolus vulgarís. Phytopathology. 91 (11): 1045-1053. 2001; FALEIRO, F.G .; RAGAGNIN, V.A .; VINHADELLI, W.S .; MOREIRA, M.A .; STAVELY, J.R. & BARROS, E.G. Resistance of common bean lines to four breeds of Uromyces appendiculatus isolated in Minas Gerais, Brazil. Fitopatol.Bras. 26 (1): 77-80. 2001; RODRIGUES, F.A .; FERNANDES, J.J. & MARTINS, M. Influence of successive sowing of common bean on the severity of angular spot and rust and losses in production. Research Agropec. Bras. 34 (8): 1373-1378. 1999; HALL, R. Compendium of bean disease. St. Paul: The American Phytopathological Society, 73p. 1991).

U. appendiculatus é um fungo parasita obrigatório que se caracteriza por apresentar alta variabilidade patogênica. No mundo inteiro, mais de 250 raças já foram identificadas (HALEY, S.D.; MIKLAS, P.N.; AFANADOR, L.; KELLY,U. appendiculatus is a mandatory parasitic fungus that is characterized by high pathogenic variability. Worldwide, more than 250 breeds have been identified (HALEY, S.D .; MIKLAS, P.N .; AFANADOR, L .; KELLY,

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J.D. Random amplified polymorphic DNA (RAPD) marker variability between and within gene pools of common bean. Journal of the American Society forJ.D.Random amplified polymorphic DNA (RAPD) marker variability between and within gene pools of common bean. Journal of the American Society for

Horticultural Science, 119: 122-125, 1994). No Brasil várias raças fisiológicas diferentes já foram encontradas, confirmando sua variabilidade nas populações deste fungo presentes em nosso país (SANNAZZARO, A.M.; OLIVEIRA, S.H.F.; WUTKE, E.B.; CASTRO, J.L.; GALLO, P.B.; MARTINS, A.L.M.; BORTOLETO, N.; SABINO, J.C.; SILVEIRA, L.C.P.; SAKAI.M.; SAES, L.A.; PAULO, E.M.; KASAI, F.S.; DORNELLES, C.R.F. & BACCHI, G.S. Severidade de ferrugem em cultivares de feijoeiro no Estado de São Paulo. Arq.Inst.Biol. 70 (3): 323-329. 2003; FALEIRO, F.G.; RAGAGNIN, V.A.; VINHADELLI, W.S.;Horticultural Science, 119: 122-125, 1994). In Brazil, several different physiological breeds have been found, confirming its variability in the populations of this fungus present in our country (SANNAZZARO, AM; OLIVEIRA, SHF; WUTKE, EB; CASTRO, JL; GALLO, PB; MARTINS, ALM; BORTOLETO, N. ; SABINO, JC; SILVEIRA, LCP; SAKAI.M .; SAES, LA; PAULO, EM; KASAI, FS; DORNELLES, CRF & BACCHI, GS Severity of rust in common bean cultivars in the State of São Paulo. Biol. 70 (3): 323-329. 2003; FALEIRO, FG; RAGAGNIN, VA; VINHADELLI, WS;

MOREIRA, M.A.; STAVELY, J.R. & BARROS, E.G. Resistência de linhagens de feijoeiro a quatro raças de Uromyces appendiculatus isoladas em Minas Gerais, Brasil. Fitopatol.Bras. 26 (1): 77-80. 2001; RIOS, G.P.; ANDRADE, E.M. & COSTA, J.L.S. Avaliação da resistência de cultivares e linhagens do feijoeiro comum a diferentes populações de Uromyces appendiculatus. Fitopatol.Bras. 26 (2): 128-133. 2001; RODRIGUES, F.A.; FERNANDES, J.J. & MARTINS, M. Influência de semeaduras sucessivas de feijoeiro na severidade da manchaangular e ferrugem e perdas na produção. Pesq. Agropec. Bras. 34 (8): 13731378. 1999).MOREIRA, M.A .; STAVELY, J.R. & BARROS, E.G. Resistance of common bean strains to four breeds of Uromyces appendiculatus isolated in Minas Gerais, Brazil. Fitopatol.Bras. 26 (1): 77-80. 2001; RIOS, G.P .; ANDRADE, E.M. & COSTA, J.L.S. Evaluation of resistance of common bean cultivars and lines to different populations of Uromyces appendiculatus. Fitopatol.Bras. 26 (2): 128-133. 2001; RODRIGUES, F.A .; FERNANDES, J.J. & MARTINS, M. Influence of successive sowing of common bean on the severity of leaf spot and rust and losses in production. Research Agropec. Bras. 34 (8): 13731378. 1999).

O processo de infecção da ferrugem-do-feijão é complexo e depende do reconhecimento por parte do patógeno de estruturas particulares da superfície do hospedeiro. Um apressório deve ser produzido após o contato da ponta do tubo germinativo com o lábio do estômato que é a estrutura apropriada à penetração (CORRÊA JR., A. & HOCH, H.C. Identification of thigmoresponsive loci for cell differentiation in Uromyces germlings. Protoplasma. 186: 34-40. 1995; TERHUNE, B.T.; BOJKO, R.J. & HOCH, H.C. Deformation of stomatal guard cell lips and microfabricated artificial topographies during appressorium formation by Uromyces. Experimental Mycology. 17: 70-78. 1993; WYNN, E.K. Appressorium formation over stomates by the bean rust fungus: Response to a surface contact stimulus. Phytopathol. 66:136-146. 1976). O apressório é então o produto do reconhecimento do sítio de infecção e o seu correto posicionamento no hospedeiro é um estágio crucial para o sucesso da infecçãoThe bean rust infection process is complex and depends on the pathogen's recognition of particular structures on the host's surface. An appressorium must be produced after contact of the tip of the germ tube with the stoma's lip, which is the appropriate structure for penetration (CORRÊA JR., A. & HOCH, HC Identification of thigmoresponsive loci for cell differentiation in Uromyces germlings. Protoplasma. 186 : 34-40. 1995; TERHUNE, BT; BOJKO, RJ & HOCH, HC Deformation of stomatal guard cell lips and microfabricated artificial topographies during appressorium formation by Uromyces. Experimental Mycology. 17: 70-78. 1993; WYNN, EK Appressorium formation over stomates by the bean rust fungus: Response to a surface contact stimulus (Phytopathol. 66: 136-146. 1976). The appressorium is then the product of the recognition of the infection site and its correct positioning in the host is a crucial stage for the success of the infection.

6/15 e conseqüentemente o estabelecimento da doença (ALLEN, E. A., HAZEN, B.6/15 and consequently the establishment of the disease (ALLEN, E. A., HAZEN, B.

E„ HOCH, H. C„ KWON, Y„ LEINHOS, G. Μ. E., STAPLES, R. C„ STUMPF,E „HOCH, H. C„ KWON, Y „LEINHOS, G. Μ. E., STAPLES, R. C „STUMPF,

Μ. A. & TERHUNE, Β. T. Apressorium formatium in response to topographical signals by 27 rust species. Phytopathol. 81:323-331. 1991; HOCH, H. C. &Μ. A. & TERHUNE, Β. T. Apressorium formatium in response to topographical signals by 27 rust species. Phytopathol. 81: 323-331. 1991; HOCH, H. C. &

STAPLES, R. C. Structural and Chemical changes among the rust fungi during appressorium formation. Annual Review of Phytopathology. 25:231-247. 1987; STAPLES, R. C„ MACKO, V., WYNN, W. K. & HOCH, H. C. Terminology to describe the differentiation response by germlings of fungai spores. Phytopathol. 73: 380. 1983). Desta forma, qualquer interrupção no processo de ío reconhecimento do sítio de infecção ou desenvolvimento do apressório afeta o estabelecimento da doença.STAPLES, R. C. Structural and Chemical changes among the rust fungi during appressorium formation. Annual Review of Phytopathology. 25: 231-247. 1987; STAPLES, R. C „MACKO, V., WYNN, W. K. & HOCH, H. C. Terminology to describe the differentiation response by germlings of fungai spores. Phytopathol. 73: 380. 1983). In this way, any interruption in the process of recognition of the infection site or development of the appressorium affects the establishment of the disease.

Durante o desenvolvimento de um apressório em ferrugens, vários genes são transcritos (KULKARNI, R.D. & DEAN, R.A. Identification of proteins that interact with two regulators of appressorium development, adenylate cyclase and cAMP-dependent protein kinase A, in the rice blast fungus Magnaporthe grisea. Mol. Gen. Genomics. 270: 497-508. 2004; KWON, Y.H.; HOCH, H.C. & AIST, J.R. Initiation of appressorium formation in Uromyces appendiculatus'. organization of the apex, and the responses involving microtubules and apical vesicles. Can. J. Bot. 69: 2560-2573. 1991). Em U. appendiculatus (Pers.:Pers.)During the development of an appressorium in rust, several genes are transcribed (KULKARNI, RD & DEAN, RA Identification of proteins that interact with two regulators of appressorium development, adenylate cyclase and cAMP-dependent protein kinase A, in the rice blast fungus Magnaporthe grisea Mol. Gen. Genomics. 270: 497-508. 2004; KWON, YH; HOCH, HC & AIST, JR Initiation of appressorium formation in Uromyces appendiculatus'. Organization of the apex, and the responses involving microtubules and apical vesicles. J. Bot. 69: 2560-2573. 1991). In U. appendiculatus (Pers.:Pers.)

Ungler diversos estudos foram realizados, tentando investigar moléculas envolvidas no processo de diferenciação do tubo germinativo em apressório (YANIV, Z. & STAPLES, R. C. The purification and properties of the aminoacyltRNA from bean rust urediniospores. Biochem. Biophys. Acta. 232:717-725. 1971; RAMAKRISHNAN, L. & STAPLES, R. C. Evidence for a template RNA in resting urediniospores of the bean rust fungus. Contríb. B. Thompson Instit. 24:1197-1202. 1970). Uma família de genes expressos entre o período coincidente com a formação do apressório foi caracterizada (XUEI, X.; BHAIRI, S.; STAPLES, R.C. & YODER, O.C. Characterization of INF56, a gene expressed during infection structure development of Uromyces appendiculatus.Ungler several studies were carried out, trying to investigate molecules involved in the process of differentiating the germ tube into appressorium (YANIV, Z. & STAPLES, RC The purification and properties of the aminoacyltRNA from bean rust urediniospores. Biochem. Biophys. Acta. 232: 717- 725. 1971; RAMAKRISHNAN, L. & STAPLES, RC Evidence for a template RNA in resting urediniospores of the bean rust fungus (Contributed by B. Thompson Instit. 24: 1197-1202. 1970). A family of genes expressed between the period coinciding with the formation of the appressorium was characterized (XUEI, X .; BHAIRI, S .; STAPLES, R.C. & YODER, O.C. Characterization of INF56, a gene expressed during infection structure development of Uromyces appendiculatus.

Gene. 110: 49-55. 1992; BHAIRI, S.M.; STAPLES, R.C.; FREVE, P. & YODER, O.C. Characterization of an infection structure-specific gene from the rust fungus, Uromyces appendiculatus. Gene. 81: 237-243. 1989). Entretanto,Gene. 110: 49-55. 1992; BHAIRI, S.M .; STAPLES, R.C .; FREVE, P. & YODER, O.C. Characterization of an infection structure-specific gene from the rust fungus, Uromyces appendiculatus. Gene. 81: 237-243. 1989). However,

7/15 estudos de função e identificação desses genes são de difícil execução, uma vez que o isolamento tradicional de transformantes para características de infecção gera obrigatoriamente células incapazes de infectar, o que no caso de parasitas obrigatórios resulta em letalidade, pois estes fungos se multiplicam apenas nos tecidos vivos do hospedeiro.7/15 studies of function and identification of these genes are difficult to perform, since the traditional isolation of transformants for infection characteristics necessarily generates cells unable to infect, which in the case of mandatory parasites results in lethality, as these fungi multiply only in the host's living tissues.

A técnica de oligonucleotídeo anti-senso é usada extensivamente como ferramenta para inibir a expressão de mRNA alvos e permitir a elucidação da função de genes, ambos in vitro e in vivo (OEKELEN, D.V.; LUYTEN, W.H.M.L. AND LEYSEN, J.E. Ten years of antisense inhibition of brain G-protein-coupled receptor function. Brain Res. Rev. 42: 123-142. 2003). Foi demonstrado previamente, por um membro da equipe que apresenta este pedido de patente, que a microinjeção de sequências em anti-senso do gene INF24 resulta na inibição da formação de apressórios de U. appendiculatus in vitro (BARJA, F.; CORRÊA JR., A.; STAPLES, R.C. AND HOCH, H.C. Microinjected antisense Inf 24 oligonucleotides inhibit appressorium development in Uromyces. Mycol.Res. 102 (12): 1513-1518. 1998). No entanto, a técnica de microinjeção é de difícil execução e com baixa rentabilidade, o que inviabiliza a sua utilização em protocolos de controle de doença.The antisense oligonucleotide technique is used extensively as a tool to inhibit the expression of target mRNA and allow the elucidation of gene function, both in vitro and in vivo (OEKELEN, DV; LUYTEN, WHML AND LEYSEN, JE Ten years of antisense inhibition of brain G-protein-coupled receptor function (Brain Res. Rev. 42: 123-142. 2003). It was previously demonstrated by a member of the team submitting this patent application that the microinjection of antisense sequences of the INF24 gene results in the inhibition of the appressor formation of U. appendiculatus in vitro (BARJA, F .; CORRÊA JR. , A .; STAPLES, RC AND HOCH, HC Microinjected antisense Inf 24 oligonucleotides inhibit appressorium development in Uromyces. Mycol.Res. 102 (12): 1513-1518. 1998). However, the microinjection technique is difficult to perform and with low profitability, which prevents its use in disease control protocols.

É conhecida a propriedade de nanotubos de carbono de parede única (SWNT) ou de múltiplas paredes (MWNT) em penetrar no interior de células de mamíferos, plantas, bactérias e fungos (LIU, Q.; CHEN, B.; WANG, Q.; SHI, X.; XIAO, Z.; LIN, J. & FANG, X. Carbon Nanotubes as Molecular Transporters for Walled Plant Cells. Nano Lett. 9 (3): 1007-1010. 2009; KOSTARELOS, K.; LACERDA, L.; PASTORIN, G.; WU, W.; WIECKOWSKI, S.; LUANGSIVILAY, J.; GODEFROY, S.; PANTAROTTO, D.; BRIAND, J.; MULLER.S.; PRATO, M. & BIANCO, A. Cellular uptake of functionalized carbon nanotubes is independent of functional group and cell type. Nature. 2: 108-113. 2007; KAM, N.W.S.; LIU, Z. & DAÍ, H. Functionalization of carbon nanotubes via cleavable disulfide bonds for efficient intracellular delivery of siRNA and potent gene silencing. J.Am.Chem.Soc. 127: 12492-12493. 2005; KAM, N.W.S.; JESSOP, T.C.; WENDER, P.A. & DAI, H. Nanotube molecular transporters: Internalization of carbon nanotube-protein conjugates into mammalian cells.The property of single-walled carbon (SWNT) or multi-walled (MWNT) carbon nanotubes in penetrating the interior of mammalian cells, plants, bacteria and fungi (LIU, Q .; CHEN, B .; WANG, Q. ; SHI, X .; XIAO, Z .; LIN, J. & FANG, X. Carbon Nanotubes as Molecular Transporters for Walled Plant Cells. Nano Lett. 9 (3): 1007-1010. 2009; KOSTARELOS, K .; LACERDA , L .; PASTORIN, G .; WU, W .; WIECKOWSKI, S .; LUANGSIVILAY, J .; GODEFROY, S .; PANTAROTTO, D .; BRIAND, J .; MULLER.S .; PRATO, M. & BIANCO , A. Cellular uptake of functionalized carbon nanotubes is independent of functional group and cell type.Nature. 2: 108-113. 2007; KAM, NWS; LIU, Z. & DAÍ, H. Functionalization of carbon nanotubes via cleavable disulfide bonds for efficient intracellular delivery of siRNA and potent gene silencing. J.Am.Chem.Soc. 127: 12492-12493. 2005; KAM, NWS; JESSOP, TC; WENDER, PA & DAI, H. Nanotube molecular transporters: Internalization of carbon nanotube -protein conjugates into mammalia n cells.

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J.Am.Chem.Soc. 126: 6850-6851. 2004). Essa propriedade permite a utilização dessa ferramenta como promissores carreadores de biomoléculas para o interior de células vivas e o estudo de sua função.J.Am.Chem.Soc. 126: 6850-6851. 2004). This property allows the use of this tool as promising carriers of biomolecules into living cells and the study of their function.

Moléculas biológicas, tais como proteína e ácidos nucléicos, interagem com facilidade à superfície de NTC e têm sido usadas como surfactantes dessas nanoestruturas com sucesso (ZHAO, X. & JOHNSON, J.K. Simulation of adsorption of DNA on carbon nanotubes. J.Am.Chem.Soc. 129: 1043810445. 2007; MALIK, S.; VOGEL, S; RÕSNER, H.; ARNOLD, K.; HENNRICH, F.; KÕHLER, A.; RICHERT, C. & KAPPES, M.M. Physical Chemical characterization of DNA-SWNT suspensions and associated composites. Composites Science and Technology. 67: 916-921. 2007; GIGLIOTTI, B.; SAKIZZIE, B.; BETHUNE, D.S.; SHELBY, R.M. & CHA, J.N. Sequenceindependent helical wrapping of single-walled carbon nanotubes by long genomic DNA. Nanoletters. 6 (2): 159-164. 2006; KAM, N.W.S.; LIU, Z. & DAÍ, H. Functionalization of carbon nanotubes via cleavable disulfide bonds for efficient intracellular delivery of siRNA and potent gene silencing.Biological molecules, such as protein and nucleic acids, interact easily on the surface of NTC and have been used as surfactants of these nanostructures with success (ZHAO, X. & JOHNSON, JK Simulation of adsorption of DNA on carbon nanotubes. J.Am.Chem .Soc. 129: 1043810445. 2007; MALIK, S .; VOGEL, S; RÕSNER, H .; ARNOLD, K .; HENNRICH, F .; KÕHLER, A .; RICHERT, C. & KAPPES, MM Physical Chemical characterization of DNA-SWNT suspensions and associated composites. Composites Science and Technology. 67: 916-921. 2007; GIGLIOTTI, B .; SAKIZZIE, B .; BETHUNE, DS; SHELBY, RM & CHA, JN Sequenceindependent helical wrapping of single-walled carbon nanotubes by long genomic DNA. Nanoletters. 6 (2): 159-164. 2006; KAM, NWS; LIU, Z. & DAÍ, H. Functionalization of carbon nanotubes via cleavable disulfide bonds for efficient intracellular delivery of siRNA and potent gene silencing .

J. Am.Chem.Soc. 127: 12492-12493. 2005; KAM, N.W.S. & DAI, H. Carbon nanotubes as intracellular protein transporters: generality and biological functionality. J.Am.Chem.Soc. 127: 6021-6026. 2005; KAM, N.W.S.; JESSOP, T.C.; WENDER, P.A. & DAI, H. Nanotube molecular transporters: Internalization of carbon nanotube-protein conjugates into mammalian cells. J.Am.Chem.Soc. 126: 6850-6851. 2004; CHEN, R.J.; BANGSARUNTIP, S.; DROUVALAKIS,J. Am.Chem.Soc. 127: 12492-12493. 2005; KAM, N.W.S. & DAI, H. Carbon nanotubes as intracellular protein transporters: generality and biological functionality. J.Am.Chem.Soc. 127: 6021-6026. 2005; KAM, N.W.S .; JESSOP, T.C .; WENDER, P.A. & DAI, H. Nanotube molecular transporters: Internalization of carbon nanotube-protein conjugates into mammalian cells. J.Am.Chem.Soc. 126: 6850-6851. 2004; CHEN, R.J .; BANGSARUNTIP, S .; DROUVALAKIS,

K. A.; KAM, N.W.S.; SHIM, M.; LI, Y; KIM.W.; UTZ, P.J. & DAI, H. Noncovalent functionalization of carbon nanotubes for highly specific electronic biosensors. PNAS. 100 (9): 4984-4989. 2003).K. A .; KAM, N.W.S .; SHIM, M .; LI, Y; KIM.W .; UTZ, P.J. & DAI, H. Noncovalent functionalization of carbon nanotubes for highly specific electronic biosensors. PNAS. 100 (9): 4984-4989. 2003).

Foram encontradas algumas patentes relacionadas à invenção:Some patents related to the invention were found:

O pedido de patente W02005104179 descreve o uso de nanotubos de carbono para análises de amostras químicas e biológicas, porém não relata o uso de nanotubos para introduzir substâncias no interior do citoplasma de células.Patent application W02005104179 describes the use of carbon nanotubes for analysis of chemical and biological samples, but does not report the use of nanotubes to introduce substances into the cell cytoplasm.

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O pedido de patente W02006078640 relata a introdução de substâncias em células causando alteração gênica, entretanto, não são utilizados nanotubos de carbono.Patent application W02006078640 reports the introduction of substances into cells causing gene alteration, however, carbon nanotubes are not used.

Todavia, não foi descrito no estado da técnica a inibição de fitopatógenos utilizando oligonucleotídeos conjugados com nanotubos de carbono.However, inhibition of phytopathogens using oligonucleotides conjugated to carbon nanotubes has not been described in the prior art.

PROBLEMAS DO ESTADO DA TÉCNICAPROBLEMS OF THE STATE OF THE TECHNIQUE

O controle da ferrugem é realizado através da utilização de cultivares resistentes à doença, práticas culturais e/ou a aplicação de defensivos agrícolas. A existência de uma grande variabilidade de raças de U.The rust control is carried out through the use of cultivars resistant to the disease, cultural practices and / or the application of pesticides. The existence of a great variability of U breeds.

appendiculatus circulante dificulta o controle da ferrugem. Esse fato, associado à utilização de defensivos agrícolas que levam a seleção de microrganismos fitopatogênicos resistentes aos compostos disponíveis mostra a necessidade de se buscar alternativas mais eficazes e menos poluentes para o controle da ferrugem em vegetais.circulating appendiculatus hampers rust control. This fact, associated with the use of pesticides that lead to the selection of phytopathogenic microorganisms resistant to the available compounds shows the need to seek more effective and less polluting alternatives for the control of rust in vegetables.

VANTAGENS DA TECNOLOGIATECHNOLOGY ADVANTAGES

A compreensão dos mecanismos de patogênese e sua manipulação abrem caminho para a obtenção de estratégias que permitem uma solução mais específica e menos impactante para o meio ambiente.Understanding the mechanisms of pathogenesis and its manipulation opens the way to obtain strategies that allow a more specific and less impactful solution for the environment.

A internalização de oligonucleotídeos anti-senso, através da utilização 20 do conjugado nanotubo-oligonucleotídeo, representa uma alternativa eficiente para análise funcional de genes em fungos filamentosos, especialmente daqueles que apresentam limitações com as técnicas convencionais, bem como uma ferramenta para o desenvolvimento de novas estratégias de controle da doença no campo.The internalization of antisense oligonucleotides, through the use 20 of the nanotube-oligonucleotide conjugate, represents an efficient alternative for functional analysis of genes in filamentous fungi, especially those that have limitations with conventional techniques, as well as a tool for the development of new ones. disease control strategies in the field.

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BREVE DECRIÇÃO DAS FIGURASBRIEF DESCRIPTION OF THE FIGURES

Figura 1 - Ultramicrografias de Microscopia de Força Atômica de (a) INF24 anti-senso adsorvidos em nanotubos de carbono e (b) oligonucleotídeos INF24 senso adsorvidos a nanotubos de carbono.Figure 1 - Ultramicrographs of Atomic Force Microscopy of (a) INF24 antisense adsorbed on carbon nanotubes and (b) INF24 sense oligonucleotides adsorbed to carbon nanotubes.

Figura 2 - Folhas de feijoeiro inoculadas com o fungo Uromyces appendiculatus após tratamento com (a) nanotubo de carbono, (b) água, (c) oligonucleotídeos INF24 anti-senso adsorvidos em nanotubos de carbono e (d) oligonucleotídeos INF24 senso adsorvidos a nanotubos de carbono.Figure 2 - Bean leaves inoculated with the fungus Uromyces appendiculatus after treatment with (a) carbon nanotube, (b) water, (c) INF24 antisense oligonucleotides adsorbed on carbon nanotubes and (d) INF24 sense oligonucleotides adsorbed to nanotubes of carbon.

DESCRIÇÃO DETALHADA DA TECNOLOGIADETAILED TECHNOLOGY DESCRIPTION

A presente invenção relata um processo de imobilização não covalente de uma seqüência especifica de genes a nanotubos de carbono (NTC) produzindo assim um sistema hibrido oligonucleotídeos-nanotubo de carbono para ser utilizado como agente de internalização gênica no combate de fitopatógenos. O objetivo desta internalização é permitir que o oligonucleotídeo passe para o citoplasma do patôgeno e interfira nos mecanismos de síntese protéica regulada pelo RNA mensageiro do patôgeno que como resultado leva a inibição de estruturas de infecção, causando a morte do patôgeno ou diminuição dos efeitos nocivos do agressor ao hospedeiro.The present invention relates to a non-covalent immobilization process of a specific sequence of genes to carbon nanotubes (NTC) thus producing a hybrid oligonucleotide-carbon nanotube system to be used as a gene internalizing agent in the fight against phytopathogens. The purpose of this internalization is to allow the oligonucleotide to pass into the cytoplasm of the pathogen and interfere with the protein synthesis mechanisms regulated by the pathogen's messenger RNA, which as a result leads to the inhibition of infection structures, causing the death of the pathogen or reducing the harmful effects of the pathogen. aggressor to the host.

Este sistema híbrido conjugado possui alta bioatividade, alta bioseletividade e especificidade uma vez que é construído pela imobilização de um oligonucleotídeo específico ao nanotubo de carbono.This hybrid hybrid system has high bioactivity, high bioselectivity and specificity since it is built by immobilizing an oligonucleotide specific to the carbon nanotube.

Como modelo biológico para comprovar e aferir a eficácia da invenção utilizou-se o fungo fitopatogênico Uromyces appendiculatus (Pers.:Pers.) Ungler que é um fungo parasita obrigatório, causador da ferrugem-do-feijoeiro (Phaseolus vulgaris L.).As a biological model to prove and measure the effectiveness of the invention, the phytopathogenic fungus Uromyces appendiculatus (Pers.:Pers.) Ungler was used, which is a mandatory parasitic fungus that causes bean rust (Phaseolus vulgaris L.).

A presente invenção pode ser mais bem entendida através dos seguintes exemplos, não limitantes de tecnologia:The present invention can be better understood through the following, non-limiting examples of technology:

Exemplo 1: Preparação dos conjugados oligonucleotídeo-NTCExample 1: Preparation of oligonucleotide-NTC conjugates

Os conjugados oligonucleotídeo e NTC foram obtidos a partir de uma solução de oligonucleotídeo INF24 e NTC funcionalizados (SWNTf ou MWNT/)The oligonucleotide and NTC conjugates were obtained from a functionalized oligonucleotide solution INF24 and NTC (SWNTf or MWNT /)

11/15 que foi sonicada em ultra-som de banho por trinta minutos à temperatura ambiente e armazenada à -20 °C até a utilização nos experimentos.11/15 that was sonicated in a bath ultrasound for 30 minutes at room temperature and stored at -20 ° C until use in the experiments.

Exemplo2: Análise de Microscopia de Força Atômica dos conjugadosExample2: Analysis of Atomic Force Microscopy of Conjugates

As análises das ultramicrografias de Microscopia de Força Atômica (Figura 1) demonstraram que SWNTf conjugados com oligonucleotídeo INF24 apresentaram em sua superfície estruturas helicoidais dispostas ordenadamente ao logo de sua superfície longitudinal compatível com a adsorção dos oligonucleotídeos sobre a estrutura de carbono. Estas estruturas não são observadas em nanotubos não conjugados com os oligonucleotídeos.The analysis of the atomic force microscopy ultramicrographs (Figure 1) showed that SWNTf conjugated with INF24 oligonucleotide had helical structures on its surface arranged in an orderly manner along its longitudinal surface compatible with the adsorption of the oligonucleotides on the carbon structure. These structures are not observed in nanotubes not conjugated to oligonucleotides.

Exemplo 3: Teste in vitro de inibição de apressóriosExample 3: In vitro appressorium inhibition test

A suspensão do conjugado oligonucleotídeo INF24 e NTC (SWNTf ou MWNT/) foi vertida sobre urediniosporos depositados em membranas plásticas contendo topografia indutiva ou discos de folhas de feijoeiro. Os substratos inoculados foram incubados por 4 h a 18 °C, fixados e o número de apressórios formados foi determinado em 100 tubos germinativos. No caso dos discos de folhas de feijoeiro o número de apressórios foi determinado em tubos germinativos escolhidos aleatoriamente e destes, os tubos germinativos que cresceram sobre estômatos.The suspension of the oligonucleotide conjugate INF24 and NTC (SWNTf or MWNT /) was poured onto urediniospores deposited on plastic membranes containing inductive topography or bean leaf discs. The inoculated substrates were incubated for 4 h at 18 ° C, fixed and the number of appressoria formed was determined in 100 germ tubes. In the case of bean leaf discs, the number of appressoria was determined in germinal tubes chosen at random and of these, the germinal tubes that grew on stomata.

Exemplo 4 - Nanotubo puro (SWNTf)Example 4 - Pure nanotube (SWNTf)

Urediniosporos de U. appendiculatus tratados com dispersões de SWNT com modificações químicas (SWNT/) germinaram e desenvolveram-se adequadamente sobre substrato plástico, mostrando eficiente formação de apressórios de 53,00 % ± 6,56 % e 46,67 % ± 3,05 %, não apresentando diferença estatística significante entre si e o controle (48,67 % ± 3,79 %). Além disso, não foi observado efeito deletério sobre a germinação e viabilidade dos tubos germinativos tratados com os diferentes SWNTf. A taxa de germinação, comprimento do tubo germinativo e sua morfologia não diferiram do controle. Exemplo 5 - Nanotubo puro (MWNT/)Urediniospores of U. appendiculatus treated with SWNT dispersions with chemical modifications (SWNT /) germinated and developed properly on plastic substrate, showing an efficient appressorium formation of 53.00% ± 6.56% and 46.67% ± 3, 05%, with no statistically significant difference between them and the control (48.67% ± 3.79%). In addition, no deleterious effect was observed on the germination and viability of the germ tubes treated with the different SWNTf. The germination rate, length of the germ tube and its morphology did not differ from the control. Example 5 - Pure nanotube (MWNT /)

Urediniosporos de U. appendiculatus tratados com MWNTf germinaram e cresceram sobre substrato plástico contendo ranhuras e iniciaram a formação de apressórios após contato com a topografia indutiva como relatadoUrediniospores of U. appendiculatus treated with MWNTf germinated and grew on plastic substrate containing grooves and started the formation of appressoria after contact with the inductive topography as reported

12/15 previamente e de maneira similar ao observado para tratamentos com SWNTf.12/15 previously and in a manner similar to that observed for treatments with SWNTf.

O percentual médio de formação de apressórios em U. appendiculatus incubados no controle (água destilada) e no tratamento de MWNTf foi de 27,33 % ± 9,35 % e 23,33 % ± 7,71 % respectivamente. Nenhuma interferência foi observada nos eventos que se processam durante a formação de apressórios após tratamento com MWNTf.The average percentage of appressiculatus formation in U. appendiculatus incubated in the control (distilled water) and in the treatment of MWNTf was 27.33% ± 9.35% and 23.33% ± 7.71% respectively. No interference was observed in the events occurring during the formation of appressoria after treatment with MWNTf.

Exemplo 6 - Oligonucleotídeos purosExample 6 - Pure oligonucleotides

Urediniosporos de U. appendiculatus tratados somente com oligonucleotídeos INF24 senso e anti-senso emitiram tubo germinativo, io cresceram sobre substrato plástico e desenvolveram apressórios, 42,67 % ± 7,02 %, e 49,67 % ± 3,79 % respectivamente. Ambos não diferiram estatisticamente do controle e do tratado com SWNTf. Esses resultados estão em acordo com relatos na literatura, uma vez que, oligonucleotídeo anti-senso adicionado diretamente no meio de cultura não apresenta atividade biológica (BENNET & COWSERT, 1999).U. appendiculatus urediniospores treated only with INF24 sense and antisense oligonucleotides emitted a germ tube, grown on plastic substrate and developed appressorios, 42.67% ± 7.02%, and 49.67% ± 3.79% respectively. Both did not differ statistically from the control and from the treatment with SWNTf. These results are in agreement with reports in the literature, since antisense oligonucleotides added directly to the culture medium do not present biological activity (BENNET & COWSERT, 1999).

Exemplo 7 - MWNTf conjugados com oligonucleotídeo INF24 anti-sensoExample 7 - MWNTf conjugated to antisense INF24 oligonucleotide

Urediniosporos de U. appendiculatus tratados com MWNTf conjugados com oligonucleotídeo INF24 senso apresentaram valores médios de formação de apressórios de 29,17 % ± 8,06 %; 32,33 % ± 17,98 %; 26,33 % ± 9,62 % eUrediniospores of U. appendiculatus treated with MWNTf conjugated with INF24 sense oligonucleotide showed mean values of appressoria formation of 29.17% ± 8.06%; 32.33% ± 17.98%; 26.33% ± 9.62% and

26,50 % ± 8,04 %, nas proporções de 1:0,5; 1:1; 1:2 e 1:3 respectivamente.26.50% ± 8.04%, in the proportions of 1: 0.5; 1: 1; 1: 2 and 1: 3 respectively.

Contudo, estes valores não diferiram estatisticamente entre si, do controle e do MWNT.However, these values did not differ statistically from each other, from the control and from the MWNT.

Os urediniosporos tratados com MWNTf conjugados com oligonucleotídeo INF24 anti-senso tiveram a formação de apressório inibida. Nas proporções utilizadas, de 1:0,5; 1:1; 1:2 e 1:3 (MWNTflNF24), os valores médios de formação de apressórios observados foram de 28,33 % ± 11,27 %; 13,50 % ± 7,20 %; 10,83 % ± 4,71 % e 2,17 % ± 2,04 %, respectivamente. O tratamento de 1:1 apresentou diferença significativa (P<0,05) quando comparado com os tratamentos controle, MWNTf e 1:0,5. No tratamento de 1:2, observou-se diferença estatística significante quando comparado com o controle e 1:0,5 (P<0,01) e MWNTf (P<0,05).Urediniospores treated with MWNTf conjugated to antisense INF24 oligonucleotide had the formation of appressor inhibited. In the proportions used, 1: 0.5; 1: 1; 1: 2 and 1: 3 (MWNTflNF24), the mean values of appressoria formation observed were 28.33% ± 11.27%; 13.50% ± 7.20%; 10.83% ± 4.71% and 2.17% ± 2.04%, respectively. The 1: 1 treatment showed a significant difference (P <0.05) when compared to the control, MWNTf and 1: 0.5 treatments. In the 1: 2 treatment, a statistically significant difference was observed when compared to the control and 1: 0.5 (P <0.01) and MWNTf (P <0.05).

13/1513/15

O tratamento na proporção de 1:3 apresentou o menor percentual de formação de apressórios e diferiu do controle, MWNTf e 1:0,5 (P<0,001). A proporção 1:0,5 não diferiu significativamente dos tratamentos controle eThe treatment in the proportion of 1: 3 showed the lowest percentage of appressoria formation and differed from the control, MWNTf and 1: 0.5 (P <0.001). The 1: 0.5 ratio did not differ significantly from the control and

MWNTf. Os dados mostram que a proporção entre NTC e oligonucleotídeos influencia a eficiência da inibição da formação de estruturas de infecção. Não se observou em nenhum dos tratamentos sinal de morbidade celular (Granulação citoplasmática, vacuolização etc).MWNTf. The data show that the ratio between NTC and oligonucleotides influences the efficiency of inhibiting the formation of infection structures. No sign of cell morbidity (cytoplasmic granulation, vacuolization, etc.) was observed in any of the treatments.

Exemplo 8 - MWNTf conjugados com oligonucleotídeo INF24 anti-senso sobre discos de folha to Urediniosporos de U. appendiculatus tratados com MWNTf conjugado com INF24 senso apresentaram valores médios de formação de apressórios de 21,3 % ± 3,05 %. Já para aqueles que apresentaram contato com os estômatos os valores percentuais médios de formação de apressórios foi de 69,7 % ± 2,76 %. Estes valores não diferiram estatisticamente do controle (água destilada), INF24S, INF24RC e do MWNTf.Example 8 - MWNTf conjugated to antisense INF24 oligonucleotide on U. appendiculatus leaf discs treated with M24Tf conjugated to INF24 sense showed mean values of appressor formation of 21.3% ± 3.05%. For those who had contact with the stomata, the mean percentage values of appressoria formation was 69.7% ± 2.76%. These values did not differ statistically from the control (distilled water), INF24S, INF24RC and MWNTf.

No tratamento com MWNTf conjugado com INF24 anti-senso foi observado o menor percentual de formação de apressórios, com valores de 4,0 % ± 4,58 % e de 14,6 % ± 16,28 %, para o número de apressórios formados para o total de tubos germinativos e para os apressórios formados após o contato do tubo germinativo com o lábio do estômato respectivamente. Nas duas situações foi observado um efeito inibitório na formação de apressório de maneira similar ao descrito previamente (BARJA et al., 1998) e os valores de formação de apressórios diferiu dos observados nos tratamentos controle, INF24RC e MWNTf conjugado com INF24 senso (P<0,05) e do MWNTf (P<0,01) e dos tratamentos controle, INF24RC, MWNTf e MWNTf conjuagados com INF24 senso (P<0,001), respectivamente para as contagens aleatórias e aquelas que apresentaram contato com estômatos.In the treatment with MWNTf conjugated to anti-sense INF24, the lowest percentage of appressorium formation was observed, with values of 4.0% ± 4.58% and 14.6% ± 16.28%, for the number of appressoriums formed for the total number of germ tubes and appressoria formed after contact of the germ tube with the stoma's lip, respectively. In both situations an inhibitory effect on the formation of appressorium was observed in a similar way to that previously described (BARJA et al., 1998) and the values of appressorium formation differed from those observed in the control treatments, INF24RC and MWNTf combined with INF24 sense (P < 0.05) and MWNTf (P <0.01) and control treatments, INF24RC, MWNTf and MWNTf combined with INF24 sense (P <0.001), respectively for random counts and those that had contact with stomata.

Exemplo 9 - SWNTf conjugados com oligonucleotídeo INF24 sensoExample 9 - SWNTf conjugated to INF24 sense oligonucleotide

Urediniosporos de U. appendiculatus tratados com SWNTf conjugados com oligonucleotídeo INF24 senso apresentaram valores médios de formação de apressórios de 51,44 % ± 9,00 %; 56,00 % ± 5,78 %; 51,78 % ± 4,55 % e 49,11 % ± 4,00 %, nas doses de 50; 25; 12,5 e 6,25 % (v/v) respectivamente.Urediniospores from U. appendiculatus treated with SWNTf conjugated with INF24 sense oligonucleotide showed mean values of appressoria formation of 51.44% ± 9.00%; 56.00% ± 5.78%; 51.78% ± 4.55% and 49.11% ± 4.00%, in doses of 50; 25; 12.5 and 6.25% (v / v) respectively.

14/1514/15

Contudo, estes valores não diferiram estatisticamente entre si, do controle e doHowever, these values did not differ statistically from each other, from control to

SWNTf.SWNTf.

Exemplo 10 - SWNTf conjugados com oligonucleotídeo INF24 anti-sensoExample 10 - SWNTf conjugated to antisense INF24 oligonucleotide

Entretanto quando tratamos urediniosporos com SWNTf conjugados com oligonucleotídeo INF24 anti-senso em diferentes concentrações tiveram a formação de apressório inibida. Também se observou um efeito dose dependente nas concentrações usadas. Os tratamentos de 50 e 12,5 % (v/v) apresentaram valores médios de formação de apressórios de 36,00 % ± 4,98 % e 37,89 % ± 8,38 %, respectivamente. Estes tratamentos apresentaram ío diferença significativa (P<0,05) quando comparados com os tratamentos controle, SWNT/e 6,25 % (v/v).However, when we treated urediniospores with SWNTf conjugated with antisense INF24 oligonucleotide in different concentrations, the formation of appressorium was inhibited. A dose-dependent effect was also observed in the concentrations used. The treatments of 50 and 12.5% (v / v) presented mean values of appressoria formation of 36.00% ± 4.98% and 37.89% ± 8.38%, respectively. These treatments showed a significant difference (P <0.05) when compared to the control treatments, SWNT / and 6.25% (v / v).

No tratamento de 25 % (v/v), observou-se o menor valor médio de formação de apressório, 31,44 % ± 1,83 %, que apresentou diferença estatisticamente significativa quando comparado com o controle, SWNTfe 6,25 % (P<0,01). A dose de 6,25 % (v/v) apresentou valor médio de apressórios formados de 52,67 % ± 3,46 % e não diferiu significativamente do controle e SWNTf. Os dados mostraram que conjugados SWNTMNF24RC foram eficazes na inibição da formação de apressórios em U. appendiculatus.In the treatment of 25% (v / v), the lowest mean value of appressorium formation was observed, 31.44% ± 1.83%, which showed a statistically significant difference when compared to the control, SWNTfe 6.25% ( P <0.01). The dose of 6.25% (v / v) showed a mean value of appressoria formed of 52.67% ± 3.46% and did not differ significantly from the control and SWNTf. The data showed that SWNTMNF24RC conjugates were effective in inhibiting appressor formation in U. appendiculatus.

Exemplo 11: Teste de inibição de U. appendiculatus ln vivo, urediniosporos de U. appendiculatus infectam as plantas de feijão e desenvolvem lesões na superfície das folhas 7 dias após a inoculação. Pontos amarelados, característicos da fase inicial do desenvolvimento da doença são observados nesse período. Esses pontos evoluem para lesões pulverulentas, de coloração marrom, coalescentes e com liberação dos urediniosporos de U. appendiculatus em plantas suceptíveis e sem tratamento de controle.Example 11: Inhibition test of U. appendiculatus in vivo, urediniospores of U. appendiculatus infect bean plants and develop lesions on the leaf surface 7 days after inoculation. Yellowish spots, characteristic of the initial stage of the development of the disease are observed in this period. These spots evolve into powdery, brown, coalescent lesions with the release of U. appendiculatus urediniospores in susceptible plants and without control treatment.

A inibição da ferrugem causada por U. appendiculatus foi realizada vertendo suspensão do conjugado oligonucleotídeo INF24 e NTC sobre a face adaxial de folhas primárias de plantas de feijão 24 h antes da inoculação de urediniosporos. As plantas inoculadas foram incubadas sob molhamento foliar por 18 h a temperatura ambiente, após o aparecimento dos sintomas características da doença o número de lesões foi determinado. Esses pontosThe rust inhibition caused by U. appendiculatus was carried out by pouring suspension of the oligonucleotide conjugate INF24 and NTC on the adaxial face of primary leaves of bean plants 24 h before the inoculation of urediniospores. The inoculated plants were incubated under leaf wetting for 18 h at room temperature, after the appearance of the characteristic symptoms of the disease the number of lesions was determined. These points

15/15 evoluem para lesões pulverulentas, de coloração marrom, coalescentes e com liberação dos urediniosporos de U. appendiculatus em plantas suceptíveis e sem tratamento de controle. Esses sintomas também são observados em plantas tratadas com MWNTf e MWNTf conjugado com INF24 senso e inoculadas com urediniosporos de U. appendiculatus, mostrando que esses tratamentos não interferem no processo de desenvolvimento da infecção das plantas.15/15 evolve into powdery, brown, coalescent lesions with the release of U. appendiculatus urediniospores in susceptible plants and without control treatment. These symptoms are also observed in plants treated with MWNTf and MWNTf conjugated with INF24 sense and inoculated with U. appendiculatus urediniospores, showing that these treatments do not interfere in the process of developing the plant infection.

Na avaliação visual (Figura 2) das folhas de feijoeiro, as plantas tratadas com MWNTf (a), controle (b) e MWNTf conjugadas com INF24 senso (d) ío apresentaram quantidade de pústulas/folha de 356,7 ± 367,54; 261,0 ± 212,87 e 131,8 ± 61,97, respectivamente. No entanto, observamos uma diminuição significativa da quantidade da doença nas plantas tratadas com MWNTf conjugada com INF24 anti-senso (Figura 2c), com valor médio de 31,5 ± 36,47, que apesar de menor, quando comparados com as folhas tratadas com MWNTf 15 e MWNTf conjugada com INF24 senso ou Controle, não diferiu estatisticamente desses tratamentos. Adicionalmente nenhum efeito tóxico foi observado nas plantas tratadas com MWNTf e os seus conjugados com INF24. As plantas não apresentaram manchas cloróticas, lesões na lâmina foliar ou senescência das folhas tratadas até 8 dias após o tratamento.In the visual evaluation (Figure 2) of the bean leaves, the plants treated with MWNTf (a), control (b) and MWNTf conjugated with INF24 sense (d) io presented a number of pustules / leaf of 356.7 ± 367.54; 261.0 ± 212.87 and 131.8 ± 61.97, respectively. However, we observed a significant decrease in the amount of the disease in plants treated with MWNTf conjugated with anti-sense INF24 (Figure 2c), with an average value of 31.5 ± 36.47, which, although lower, when compared to treated leaves with MWNTf 15 and MWNTf combined with INF24 sense or Control, did not differ statistically from these treatments. In addition, no toxic effects were observed in plants treated with MWNTf and its conjugates with INF24. The plants did not present chlorotic spots, lesions on the leaf blade or senescence of the treated leaves until 8 days after treatment.

1/21/2

Claims (8)

REIVINDICAÇÕES 1- CONJUGADO DE NANOTUBOS DE CARBONO PARA INIBIR ESTRUTURAS DE INFECÇÃO DE PATÓGENOS EM VEGETAIS, caracterizada por compreender a conjugação de oligonucleotídeos com nanotubos de carbono.1- CONJUGATE OF CARBON NANOTUBES TO INHIBIT STRUCTURES OF PATHOGEN INFECTION IN VEGETABLES, characterized by understanding the conjugation of oligonucleotides with carbon nanotubes. 2- CONJUGADO DE NANOTUBOS DE CARBONO PARA INIBIR ESTRUTURAS DE INFECÇÃO DE PATÓGENOS EM VEGETAIS, de acordo com a reivindicação 1, caracterizado pelos nanotubos serem selecionados do grupo compreendendo SWNTA ou MWNTf.2- CONJUGATE OF CARBON NANOTUBES TO INHIBIT STRUCTURES OF PATHOGEN INFECTION IN VEGETABLES, according to claim 1, characterized by the nanotubes being selected from the group comprising SWNTA or MWNTf. 3- CONJUGADO DE NANOTUBOS DE CARBONO PARA INIBIR ESTRUTURAS DE INFECÇÃO DE PATÓGENOS EM VEGETAIS, de acordo com as reivindicações 1 e 2, caracterizada pelos nanotubos apresentarem em sua superfície estruturas helicoidais dispostas ordenadamente ao logo de sua superfície longitudinal compatível com a adsorção dos oligonucleotídeos sobre a estrutura de carbono.3- CONJUGATE OF CARBON NANOTUBES TO INHIBIT STRUCTURES OF PATHOGEN INFECTION IN VEGETABLES, according to claims 1 and 2, characterized by the nanotubes having on their surface helical structures arranged neatly along their longitudinal surface compatible with the adsorption of oligonucleotides on the carbon structure. 4- CONJUGADO DE NANOTUBOS DE CARBONO PARA INIBIR ESTRUTURAS DE INFECÇÃO DE PATÓGENOS EM VEGETAIS, de acordo com a reivindicação 1, caracterizado pelos oligonucleotídeos serem selecionados do grupo compreendendo sequências senso e anti-senso do gene INF24.4- CONJUGATE OF CARBON NANOTUBES TO INHIBIT STRUCTURES OF PATHOGEN INFECTION IN VEGETABLES, according to claim 1, characterized by oligonucleotides being selected from the group comprising sense and antisense sequences of the INF24 gene. 5- CONJUGADO DE NANOTUBOS DE CARBONO PARA INIBIR ESTRUTURAS DE INFECÇÃO DE PATÓGENOS EM VEGETAIS, de acordo com as reivindicações 1 a 4, caracterizado por ser capaz de penetrar no citoplasma da células de patógenos.5- CONJUGATE OF CARBON NANOTUBES TO INHIBIT STRUCTURES OF PATHOGEN INFECTION IN VEGETABLES, according to claims 1 to 4, characterized by being able to penetrate the cytoplasm of pathogen cells. 6- CONJUGADO DE NANOTUBOS DE CARBONO PARA INIBIR ESTRUTURAS DE INFECÇÃO DE PATÓGENOS EM VEGETAIS, de acordo com as reivindicações 1 a 5, caracterizada por interferir nos mecanismos de síntese protéica regulada pelo RNA mensageiro do patógeno.6- CONJUGATE OF CARBON NANOTUBES TO INHIBIT STRUCTURES OF PATHOGEN INFECTION IN VEGETABLES, according to claims 1 to 5, characterized by interfering in the mechanisms of protein synthesis regulated by the pathogen's messenger RNA. 2/22/2 7- CONJUGADO DE NANOTUBOS DE CARBONO PARA INIBIR ESTRUTURAS DE INFECÇÃO DE PATÓGENOS EM VEGETAIS caracterizado pelo seu uso no controle de doenças no campo.7- CONJUGATE OF CARBON NANOTUBES TO INHIBIT STRUCTURES OF INFECTION OF PATHOGENS IN VEGETABLES characterized by its use in the control of diseases in the field. 8- CONJUGADO DE NANOTUBOS DE CARBONO PARA INIBIR 5 ESTRUTURAS DE INFECÇÃO DE PATÓGENOS EM VEGETAIS, caracterizado pela inibição do desenvolvimento da doença causada pelo fungo U. appendiculatus inoculado em espécies vegetais susceptíveis.8- CONJUGATE OF CARBON NANOTUBES TO INHIBIT 5 PATHOGEN INFECTION STRUCTURES IN VEGETABLES, characterized by inhibition of the development of the disease caused by the fungus U. appendiculatus inoculated in susceptible plant species. 1/11/1 FIGURASFIGURES
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