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WO2017186872A1 - Procédé pour identifier des agents phytopathogènes biologiquement actifs - Google Patents

Procédé pour identifier des agents phytopathogènes biologiquement actifs Download PDF

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Publication number
WO2017186872A1
WO2017186872A1 PCT/EP2017/060117 EP2017060117W WO2017186872A1 WO 2017186872 A1 WO2017186872 A1 WO 2017186872A1 EP 2017060117 W EP2017060117 W EP 2017060117W WO 2017186872 A1 WO2017186872 A1 WO 2017186872A1
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Prior art keywords
plant
rna
sample
dna
crop
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Ceased
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PCT/EP2017/060117
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German (de)
English (en)
Inventor
Inge Broer
Jana HUCKAUF
Roland SÖFFING
Andreas PRELWITZ
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Universitaet Rostock
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Universitaet Rostock
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Publication of WO2017186872A1 publication Critical patent/WO2017186872A1/fr
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6888—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms
    • C12Q1/6895—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms for plants, fungi or algae

Definitions

  • the present invention relates to analysis methods for seed and food control.
  • it relates to a method of detecting biologically active plant pathogens in plant crops comprising the steps of providing a sample of plant crops presumably containing plant pathogens, determining RNA present in biologically active plant pathogens and providing the detection of plant pathogens in the crop in Cases where RNA could be determined.
  • the invention also relates to a kit for carrying out the method.
  • the invention relates to a method for determining the efficiency of an anti-plant pathogen treatment of plant crops comprising providing a first sample of plant crop before and a second sample of plant crop after the anti-plant pathogen treatment, determining the amount of DNA and RNA present in biologically active plant pathogens in the first and second samples of plant crop and forming a ratio of the amount of DNA to RNA in the first and second samples and relating the ratios of first and second samples to each other; wherein the ratio of the amount of DNA to RNA in the second sample versus the ratio of the amount of DNA to RNA in the first sample is an indicator of the proportion of biologically active plant pathogens in the plant crop.
  • fungi such as Fusarium and Steinbrand can destroy whole crops, as they not only lead to yield losses, but , by the formation of mycotoxins, that cereals are no longer allowed for use as food or feed.
  • fungi also reduce the shelf life and apparently also the germination capacity of seeds.
  • the invention thus relates to a method for the detection of biologically active plant pathogens in plant crops comprising:
  • RNA RNA could be determined.
  • the present method in addition to the aforementioned steps, may also include further steps. These steps may be: Harvesting of plants for the processing of the harvested crops to the vegetable crop. The processing of the harvested plants to the vegetable crop. Treatment, such as the anti-plant pathogen treatment of plant crops described elsewhere, before and after the detection of biologically active plant pathogens. The storage of vegetable crops. The destruction of plant crops after successful detection of biologically active plant pathogens.
  • the method according to the invention may also comprise the detection of DNA from which the RNA present in biologically active plant pathogens is transcribed.
  • the detection of the DNA can be done before or after the detection of the RNA. If DNA is detected, the presence of the biologically active or inactive plant pathogens in the sample is likely. If the detection of the RNA is provided, the presence of biologically active plant pathogens is likely.
  • the method according to the present invention may preferably be automated.
  • the sample treatment and / or the detection can be supported by suitable analysis robots and nucleic acid analyzers, such as PCR analyzers.
  • suitable analysis robots and nucleic acid analyzers such as PCR analyzers.
  • the sequence analysis and the identification of the pathogen pathogen can be performed computer-aided.
  • detection includes the qualitative, semi-quantitative and / or quantitative determination of the presence and / or amount of biologically active plant pathogens in the sample
  • determination of the presence in the context of the invention also includes determination of the identity of the plant Because of the specific detection methods described here for the RNA present in biologically active plant pathogens, it is thus possible to determine the presence and identity of the plant pathogen in the sample and its amount, how the RNA is determined and how the detection can be demonstrated in detail , is described in more detail elsewhere.
  • vegetable crop in the sense of the invention comprises any parts of a plant that can be harvested, including whole plants, sprouts or plant parts such as leaves, stems, roots, fruits and seeds.
  • the term includes in addition to the primary crop within the meaning of the invention also directly obtained from the primary crop described above, vegetable Products. These include, for example, products such as dry preparations, eg dried leaves, powders which are obtained from the primary crop without further processing steps or pellets, for example pellets, which are used as animal feed.
  • frozen products or freeze-dried products come into consideration.
  • the crop preferably comes from a crop, and more preferably from a crop selected from the group: vegetables and lettuce plants such as tomatoes, lettuce, peppers, cabbages, legumes such as peas, beans, chickpeas, lentils, soybeans, peanuts or lupins, rape, soybeans , Corn, rice, sunflower, canola, safflower, poppy, mustard, hemp, castor, olive, calendula, punica, evening primrose, pumpkin, borage, field bean, vetches, alfalfa, clover, serradella, phacelia, oil radish, oil flax, ragweed, buckwheat , Grasses of various species or trees such as apple, pear, plum, quince, chestnut, cherry, coconut, walnut, hazelnut, Brazil nut, macadamia, cereals wheat, barley, oats, rye, millet, triticale or spelled.
  • vegetables and lettuce plants such as tomatoes, lettuce, peppers,
  • biologically active plant pathogens refers to a pathogen which is biologically active and which can cause damage and / or disease to the crop,
  • the plant pathogen is a bacterium, a virus or Particularly preferred is a fungus of the genus Fusarium or Tiüetia.
  • a biologically active within the meaning of the invention is the ability to transcribe RNA and subsequent translation, which can be provided by the plant pathogen itself, for example in cases where the plant pathogen is one cellular organism, ie a bacterium or a fungus, by the use of its own enzymes or in the case of viruses by the machinery of a plant host cell.
  • RNA preferably of messenger RNA (mRNA).
  • mRNA messenger RNA
  • the mRNA is synthesized during transcription by the enzyme RNA polymerase.
  • RNA polymerase During the so-called translation of the mRNA, according to the RNA sequence, a corresponding polypeptide or protein is synthesized from the amino acids available in the cell.
  • Both processes usually take place in the case of bacteria or fungi in biologically active organisms.
  • RNA may also be present in transiently resting organisms, such as spores. These are basically to be regarded as viable and thus belong to the biologically active plant pathogens in the context of the invention.
  • viruses the Scope of the invention targeted to viruses that are infectious, so either replicate in suitable cells of the crop or are capable of acute.
  • sample is understood to mean a subset of the crop that is used for the analysis. This subset should be dimensioned such that, based on its extent, there is a high probability that - if the crop is contaminated with biologically active plant pathogens Depending on the crop, it may be necessary to pre-treat the sample for the subsequent determination of the RNA Necessary pre-treatments are known to the person skilled in the art.
  • RNA which is present in biologically active plant pathogens can be carried out according to the invention by all methods suitable for the detection of RNA.
  • the RNA can hereby be detected directly or after conversion into THEN, for example by reverse transcription.
  • Methods for detecting nucleic acids, including RNA and especially mRNA are known to those of skill in the art and include methods known in the art, such as polymerase chain reaction, in situ hybridization, or nucleic acid sequencing techniques.
  • Plant pathogens may therefore preferably not be present in other plant pathogens by the presence of RNA transcribed from genes characteristic of the plant pathogen.
  • Such specific genes may either be specific to one species, ie they are only present in it (eg E.
  • coli or Tilletia indica or be specific for one genus, ie they are present for example in all enterobacteria or all species of the genus Tilletia or specific to an entire class, such as viruses.
  • Methods for the determination of genes which are specific for a particular plant pathogen are known to the person skilled in the art and include, for example, the polymerase chain reaction, sequencing (chain termination synthesis, semiconductor sequencing, etc.), sequence analysis of individual organisms, or evolutionarily conserved sequence comparison Genes within a genus.
  • the RNA of at least one gene which is specific for a particular plant pathogen is determined.
  • RNA of the at least one plant pathogen-specific gene by means of a polymerase chain reaction is particularly preferably carried out.
  • Polymerase chain reaction (PCR) in the sense of the invention means a method for amplifying DNA in vitro by means of the enzyme DNA polymerase. Details of the PCR are known to the person skilled in the art.
  • the term polymerase chain reaction also includes methods for amplification and quantitation of DNA and RNA, also called quantitative real-time PCR. Quantitative real-time PCR is a duplication method for nucleic acids based on the principle of conventional polymerase chain reaction (PCR) and additionally allows the quantification of the recovered DNA or RNA.
  • RNA For the quantification of RNA is a reverse transcription followed by quantitative PCR (in the same approach), which is correctly referred to as qRT-PCR or RT-qPCR.
  • quantitative PCR in the same approach
  • the necessary devices and method steps for performing a quantitative real-time PCR, as well as corresponding calculation models are known in the art.
  • Quantitative real-time PCR is often referred to as Real Time Detection PCR, or RTD-PCR for short, or simply as qPCR for the quantification of DNA or as qRT-PCR for the quantification of RNA.
  • Different agents can be used for the detection of DNA or RNA.
  • the quantification of the PCR products of the PCR products can be achieved, for example, by the use of DNA dyes (eg ethidium bromide or SYBR Green I) or the use of Förster resonance energy transfer (FRET).
  • FRET probes for example, two different, each with a FRET donor or FRET acceptor ("reporter") labeled oligonucleotides are used, which side by side bind to the target sequence and thus bring the fluorochromes in a FRET for sufficient proximity (often
  • Other examples of quantification of PCR products utilizing the FRET principle include LoopTag probes, TaqMan probes (also often referred to as hydrolysis probes), Molecular Beacons, Scorpion primers, Lux Primer or lanthanide-labeled probes
  • different calculation models can be used, which are known to the person skilled in the art.
  • Comparative analysis In most cases, relative quantification using the so-called Ct- Cycle (threshold cycle) or Cp (crossing point) value describing the cycle at which fluorescence first increases significantly above background fluorescence.
  • Ct- Cycle threshold cycle
  • Cp crossing point
  • qPCR quantitative real-time PCR
  • RNA molecules are used as agent for the detection of a particular RNA having nucleotide sequences which are complementary to those of the RNA or one of them are derived DNA and that can hybridize with RNA or derived DNA molecules.
  • the structure (sequence) of the nucleic acid molecules used for detection is thus given by the structure (sequence) of the RNA molecules to be detected.
  • the agent for detecting the RNA in the method according to the invention particularly preferably comprises a nucleic acid probe which specifically hybridizes with the RNA of the at least one gene which is specific for a particular plant pathogen or hybridizes to a nucleic acid derived therefrom.
  • derivative nucleic acids can be understood as meaning amplification products, but also modified nucleic acids, such as DNA transcribed in reverse from the RNA.
  • Suitable probes usually have a length of 50 to 500 base pairs, preferably 100 to 400 base pairs or 200 to 300 base pairs. They have a sequence which is substantially complementary to that of the nucleic acid to be detected.
  • the agent for detecting the RNA in the method according to the invention particularly preferably comprises nucleic acid primers which specifically allow the amplification of a nucleic acid fragment derived from the RNA of the at least one gene specific for a particular plant pathogen.
  • the nucleic acid primers used are preferably an upstream and a downstream oriented oligonucleotide (so-called 5 ' and 3 ' or forward and reverse primers).
  • Oligonucleotides suitable as primers preferably have a length of 8 to 30 base pairs, particularly preferably 10 to 25 base pairs, 12 to 20 base pairs or 15 to 18 base pairs. They have a sequence which is substantially complementary to that of the nucleic acid to be detected is in the binding region of the primer.
  • the RNA can be determined by a biologically active plant pathogen.
  • several RNAs of the same plant pathogen can be determined. This usually allows a more reliable detection, should at least one RNA.
  • the RNA of at least one gene is to be determined which is specific for a particular plant pathogen.
  • RNA present in biologically active plant pathogens could be determined, evidence of the presence of the biologically active plant pathogen is provided.
  • the detection in the sense of the method according to the invention may additionally comprise the detection of the amount of biologically active plant pathogen.
  • the amount of biologically active plant pathogen directly correlates with the amount of RNA detected, particularly when qPCR or qRT-PCR is used as the assay.
  • RNAs of the genes listed below for the respective plant pathogens can be determined in the method according to the invention:
  • the method according to the invention advantageously allows biologically active plant pathogens in plant crops to be detected on the basis of the amount of RNA of at least one gene which is specific for a particular plant pathogen.
  • RNA determines the methods and not the RNA or there is no correlation between DNA and RNA.
  • the detection of biologically active plant pathogens can only be achieved by the determination of RNA.
  • the inventive method is fast, efficient, inexpensive and can be used on the road without special infrastructure requirements. To date, the burden of plant crops with plant pathogens is essentially measured by time-consuming germ tests in the laboratory.
  • spores of fungi are used for germination and growth via nutrient media. After a defined period of 7 to 10 days, a qualitative evaluation is possible, but a quantification of the infestation is not given.
  • Another method is the filtration method, for example, adhering fungal spores are solved by shaking the seed. The spores are collected on a filter paper and then counted with a microscope.
  • the plant pathogen is a bacterium or a virus.
  • the plant pathogen is a fungus.
  • the fungus is of the genus Fusarium or Tilletia.
  • the RNA of at least one gene is determined which is specific for a particular plant pathogen.
  • the detection of the RNA by means of polymerase chain reaction is a vegetable food or feed or vegetable seed.
  • the vegetable crop is obtained from a crop.
  • the crop is preferred selected from the group consisting of: wheat, barley, oats, rye, soy, barley, millet, spelled, rice and corn.
  • the invention also relates to a kit for the determination of plant pathogens in plant crops comprising at least one agent for detecting the RNA of at least one gene that is specific for a particular plant pathogen.
  • kits in the sense of the invention is a combination of components which are necessary for the determination of plant pathogens in plant crops, preferably with the method according to the invention.
  • the kit according to the invention comprises at least one agent for detecting the RNA of at least one gene which is specific for a This agent can be nucleic acid probes or primers which allow the specific detection of the RNA, and how these have to be formed depends on the RNA to be detected and thus on the plant pathogen to be detected
  • the kit may contain further components, for example for carrying out PCR reactions or hybridizations, such as, for example, buffers, enzymes or detection components, such as dyes, chromophores, FRET components or
  • the kit may contain information for carrying out the method in general and for determining the amount of biologically active plant pathogen in a sample. It also includes calibration means, such as calibration curves, etc.
  • the kit may also include standards for creating calibration curves, e.g. Samples with a defined number of biologically active plant pathogens.
  • the agent is (i) a nucleic acid probe that specifically hybridizes with the at least one gene that is specific for a particular plant pathogen, or (ii) nucleic acid primers that specifically amplify a fragment of the at least allow a gene that is specific to a particular plant pathogen.
  • the invention further relates to a method for determining the efficiency of an anti-plant pathogen treatment of plant crops comprising:
  • the present method in addition to the aforementioned steps, may also include further steps. These steps may include, in particular, the anti-plant pathogen treatment of plant crop, but also the destruction of plant crop after inefficient anti-plant pathogen treatment.
  • the method according to the present invention can preferably be automated as already described above.
  • the sample treatment and / or the detection can be supported by suitable analysis robots and nucleic acid analyzers, such as PCR analyzers.
  • the sequence analysis and the identification of the plant pathogen can be carried out computer-assisted.
  • anti-plant pathogen treatment serves for the decontamination of plant crops or the inactivation of plant pathogens that may be present in the vegetable crop
  • the treatment may be of chemical or physical origin.
  • chemical pickling processes, hot water pickling or electron treatment of plant crops, such as seeds may be mentioned here
  • electronic seed treatment the energy of the electrons is metered so that they can only slightly penetrate into the seed Penetration into the grain releases most of its energy from the electron, killing pathogens, such as fungal spores, bacteria or viruses, on the surface and in the seed coat, while not damaging the embryo further inside and its ability to germinate of the seed.
  • active ingredients used in chemical pickling processes are various approved chemical compounds. As is known to those skilled in the art, these are selected according to the area of use and plant variety.
  • active substances that are used in chemical pickling processes could be mentioned here: Tebuconazole, fludixonil, fluoxastrobin, prothioconazole, triazoxide, difenoconazole, silthiofam, prochloraz, triticonazole, pyrimethanil, triadimenol, fuberidazole, imazalil or cyproconazole.
  • Bacterial plant diseases are often treated with antibiotics, such as plantomycin or streptomycin sulfate. The use of antibiotics, however, is highly regulated and often only allowed in strictly controlled cases.
  • a hot water pickling for wheat seed may be for at least 10 minutes at a water temperature of about 51 ° C to 52 ° C.
  • the anti-plant pathogen treatment is carried out by an electron treatment, a chemical pickling process and / or a hot water pickling. It is clear to the person skilled in the art that the efficiency of an anti-plant pathogen treatment can depend on various factors, for example in the case of a hot water pickling of the exact water temperature and the amount of seed used or, in the case of electron treatment, of the energy of the electrons. According to the invention, the proportion of vegetable crop preferably correlates with the efficiency of the anti-plant pathogen treatment.
  • Effective anti-plant pathogen treatment is believed to have been inactivated when a statistically significant amount of the plant pathogens contained in the plant crop were inactivated. Whether the amount of plant pathogen that could be inactivated is statistically significant is easily determined by standard statistical methods, see, e.g. p-value determinations, Student's t-test, Mann-Whitney test, etc., see standard literature, e.g. Dowdy and Wearden, Statistics for Research, John Wiley & Sons, New York 1983 for more details.
  • DNA and / or RNA quantitation can be accomplished by methods previously described elsewhere herein.
  • the formation of a ratio of the two quantities in the first and second sample can be carried out with all suitable mathematical operations, which allow to express the proportion of RNA relative to DNA or total amount of nucleic acids (ie the sum of the amount of DNA and amount of RNA).
  • the ratio for the amounts of the first is formed by the same method as for the amounts of the second samples.
  • Particularly suitable Here is the formation of a quotient of DNA amount to RNA amount or vice versa. Both ratios may then preferably be related by comparison.
  • the proportion of plant crops correlates with the efficiency of the anti-plant pathogen treatment.
  • the ratio is the quotient of the amount of DNA and the amount of RNA (DNA / RNA) and a larger quotient in the second compared to the first sample is an indicator of a successful anti-plant pathogen treatment and a Small or unchanged quotient in the second compared to the first sample is an indicator of failed plant pathogen treatment.
  • the ratio is preferably the quotient of the amount of DNA and the amount of RNA (DNA / RNA).
  • a larger quotient in the second compared to the first sample is an indicator of a successful anti-plant pathogen treatment, since the amount of RNA is smaller relative to the amount of DNA. In this case, no significant amount of biologically active plant pathogens remained after treatment that could still produce RNA.
  • a small or unchanged quotient in the second compared to the first sample is an indicator of a failed anti-plant pathogen treatment, since the proportions remain unchanged or the proportion of RNA even increases. This is only possible if biologically active plant pathogens remain after treatment to a significant extent.
  • the anti-plant pathogen treatment comprises an electron treatment, a chemical pickling method and / or a hot water pickling.
  • Example 1 Establishment of a Quantitative Detection System of Fusarium RNA on Wheat Grains
  • ⁇ tubulin ubiquitin
  • elongation factor la elongation factor la.
  • the quantification of DNA and RNA is performed using quantitative real-time PCR (also referred to as qPCR for DNA and qRT-PCR for RNA).
  • RNA expressed by these genes serves as a standard to quantify the RNA from fungi.
  • a plasmid standard is used.
  • primers were derived which allow the best possible reaction. The primers were designed in such a way that the primers directed against Steinbrand and Fusaria had as few homologies as possible. Thus, the reaction against both fungi can be done in a multiplex PCR. Identification of the detection limit of the method
  • Wheat seeds are mixed with defined, ascending concentrations of fungal spores.
  • the DNA of the mixtures is isolated and analyzed for DNA and RNA levels using the PCR developed above.
  • the aim of the experiment is to determine the lowest yet detectable germinable contamination of the seed. At the same time, the reproducibility of the method should be determined in this approach.
  • the sampling should allow a reliable statement about the largest possible seed batch in order to keep the sample size as low as possible.
  • a sampling scheme has been developed, which starts with a high sample volume and then according to the measured variability allows a reduction up to a sample load, which still allows a significant statement.
  • 30 samples a 1 kg were taken from a seed lot and the samples thoroughly mixed. From each sample, 10 g were removed and combined to a mixed sample, which in turn was thoroughly mixed. 5 ⁇ 10 g were taken from this sample, the DNA and RNA were isolated and the amounts were amplified by PCR. This process was repeated nine times for the same batch. Based on the variability of the samples, it is now possible to determine which sampling scheme or sample volumes are needed to obtain representative and statistically significant results.
  • Example 3 Proof of the efficiency of the electron treatment
  • samples are taken from treated and untreated seeds and analyzed with the above-developed qPCR for DNA and a qRT-PCR for the RNA. From the ratio of DNA to RNA in the treated sample compared to the ratio of DNA to RNA in the untreated sample, the proportion of germinable spores can be determined. Electron treatment for decontamination using low-energy electrons
  • the supply of controlled infected seeds is done by inoculating wheat with the model pathogens Fusarium and Weizensteinbrand. Fresh inoculation material is obtained annually through deliberately infected wheat cultivation. Mushroom detection of pathogens in the plate test and microscope
  • the pathogens Fusarium and Weizensteinbrand are also detected by plate testing and microcopy ("classical proofs" in comparison to the qPCR method developed here)
  • a microscope with a magnification of 120x is used to determine the seed coat thickness and germinated spores can be detected with the same microscope
  • the spores of both pathogens must be stimulated to germinate and grow by means of nutrient media with the so-called plate test.Also artificially inoculated seed is stimulated with a growth chamber to germinate and germinate. or 4- Leaf stage under controlled conditions in growth promoted.
  • the model pathogens are also microscoped and analyzed in the plate test. This test provides information on possible interactions between the pathogen and the host plant, as the natural process of germination and growth takes place under controlled conditions.

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Abstract

La présente invention concerne un procédé permettant d'identifier des agents phytopathogènes biologiquement actifs dans des récoltes de végétaux, qui comprend les étapes suivantes : fournir un échantillon de récolte de végétaux dont on présume qu'il contient des agents phytopathogènes, déterminer l'ARN présent dans des agents phytopathogènes biologiquement actifs et établir la preuve de la présence d'agents phytopathogènes dans les cas où la présence d'ARN a pu être établie. L'invention concerne également un kit pour la mise en œuvre du procédé selon l'invention. L'invention concerne en outre un procédé permettant de déterminer l'efficacité d'un traitement anti agents phytopathogènes appliqué à des récoltes de végétaux, ledit procédé comprenant les étapes suivantes : fournir un premier échantillon de récolte de végétaux avant traitement anti agents phytopathogènes et un second échantillon de récolte de végétaux après traitement anti agents phytopathogènes, déterminer la quantité d'ADN et d'ARN présente dans des agents phytopathogènes biologiquement actifs, dans le premier et le second échantillon de récolte de végétaux et mettre en relation mutuelle les rapports issus du premier et du second échantillon, le rapport de la quantité d'ADN à l'ARN dans le second échantillon comparativement au rapport de la quantité d'ADN à l'ARN dans le premier échantillon constituant un indicateur de la proportion d'agents phytopathogènes biologiquement actifs dans la récolte de végétaux.
PCT/EP2017/060117 2016-04-27 2017-04-27 Procédé pour identifier des agents phytopathogènes biologiquement actifs Ceased WO2017186872A1 (fr)

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Application Number Priority Date Filing Date Title
DE102016207147.5A DE102016207147A1 (de) 2016-04-27 2016-04-27 Verfahren zum Nachweis von biologisch aktiven Pflanzenpathogenen
DE102016207147.5 2016-04-27

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Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2000046397A1 (fr) * 1999-02-02 2000-08-10 Technion Research And Development Foundation Ltd. Analyse a base d'acide nucleique et jeu de detection de la contamination par alternaria d'un produit alimentaire
WO2003087297A2 (fr) * 2001-08-08 2003-10-23 North Carolina State University Jeux ordonnes de microechantillons contre les maladies infectieuses
BRPI1106770A2 (pt) * 2011-10-05 2014-02-11 Vitatec Ltda Kit, processo, marcador molecular de ácidos nucléicos e seu uso para detecção, identificação, medição e monitoramento de fitopatógenos
WO2015066341A2 (fr) * 2013-10-30 2015-05-07 Green Life Biotech, Llc Systèmes de quantification de pathogenèse et procédés de traitement de la maladie du verdissement des agrumes

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2000046397A1 (fr) * 1999-02-02 2000-08-10 Technion Research And Development Foundation Ltd. Analyse a base d'acide nucleique et jeu de detection de la contamination par alternaria d'un produit alimentaire
WO2003087297A2 (fr) * 2001-08-08 2003-10-23 North Carolina State University Jeux ordonnes de microechantillons contre les maladies infectieuses
BRPI1106770A2 (pt) * 2011-10-05 2014-02-11 Vitatec Ltda Kit, processo, marcador molecular de ácidos nucléicos e seu uso para detecção, identificação, medição e monitoramento de fitopatógenos
WO2015066341A2 (fr) * 2013-10-30 2015-05-07 Green Life Biotech, Llc Systèmes de quantification de pathogenèse et procédés de traitement de la maladie du verdissement des agrumes

Non-Patent Citations (3)

* Cited by examiner, † Cited by third party
Title
DATABASE WPI Week 201435, Derwent World Patents Index; AN 2014-H81803, XP002771035 *
MACKENZIE D J ET AL: "IMPROVED RNA EXTRACTION FROM WOODY PLANTS FOR THE DETECTION OF VIRAL PATHOGENS BY REVERSE TRANSCRIPTION-POLYMERASE CHAIN REACTION", PLANT DISEASE, THE AMERICAN PHYTOPATHOLOGICAL SOCIETY, US, vol. 81, no. 2, 1 January 1997 (1997-01-01), pages 222 - 226, XP001100054, ISSN: 0191-2917 *
YI FANG, R P RAMASAMY: "Current and Prospective Methods for Plant Disease Detection", BIOSENSORS, vol. 4, 6 August 2015 (2015-08-06), pages 537 - 561, XP002771036 *

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