Early identification of pathogens in plants

Abstract

The present invention relates to the early detection of pathogens in plants and the identification of resistance markers of such pathogens against chemical or biological control agents. The subject of the present invention is a system, a method and a computer-readable medium for the early detection of the presence of pathogens at, on or in plants and the identification of resistance markers by means of sequence analysis.

Description

[0001] The present invention relates to the early identification of pathogens in, near or on plants or plant parts and the identification of markers of resistance of such pathogens to chemical or biological control agents. The subject of the present invention is a system, a method and a computer-readable medium for the early identification of the presence of pathogens in, near or on plants or plant parts and the identification of resistance markers by means of sequence analysis.

[0002] Early identification of pathogen infection of plants is extremely important. The earlier the infection is identified, the sooner appropriate measures can be taken to prevent the outbreak and / or spread of the disease and / or avoid damage to the plant.

[0003] WO2017 / 194276 describes a method for detecting plant diseases, which is based on the analysis of images of plant parts. However, plants may have been infected by pathogenic flora without showing visible symptoms. In addition, it is not possible to determine the resistance status of pathogenic flora with the help of such image analysis.

[0004] US20120088696A1 discloses a micro-electrochemical multiplex PCR platform, which can be used to amplify, study and quantify target nucleotides in real time and can be used, for example, for rapid detection of plant diseases. However, the platforms must be adapted specifically for identifying specific diseases and are relatively expensive in use.

[0005] Advantageously, not only is the pathogen recognized at an early stage, but also resistance to control agents can be determined already at an early stage in the pathogen population.

[0006] WO2019149626A1 relates to identifying resistance markers in the field.

[0007] The present invention is a supplement to the technical solution described in WO2019149626A1. The present invention combines the determination of resistance with the early identification of pathogens in plants. The technical solution proposed in this specification is efficient, adaptive and can introduce other information into the early identification of pathogens and resistance.

[0008] The first subject of the present invention provides a method comprising the following steps:

[0009] - Collecting samples from plants or the medium in which they grow,

[0010] - identifying a pathogen in said sample,

[0011] - identification of markers of resistance of said pathogen to control agents,

[0012] wherein the identification of the pathogen and the identification of the marker of resistance is performed by means of sequencing individual nucleic acids or individual peptides in the sample, wherein the nucleotide sequence or the amino acid sequence is determined when sequencing the individual nucleic acids or individual peptides, wherein the nucleotide sequence or the amino acid sequence is compared with a reference sequence, wherein the sequencing is performed in two stages, a first stage and a second stage,

[0013] ● wherein in said first stage, during the sequencing of each nucleic acid or each peptide the sequence determined respectively is compared with at least one pathogenic sequence, and

[0014] ■ in the event that the determined sequence is inconsistent with the at least one pathogenic sequence, further sequencing of the nucleic acid or the peptide is interrupted,

[0015] ■ In the case where the determined sequence is consistent with the pathogen sequence, the pathogen having the pathogen sequence is determined, and at least one resistance marker is determined based on the determined pathogen, and the process is switched to the second stage,

[0016] - wherein in said second stage the sequence of the nucleic acids or peptides in said sample is compared with said at least one resistance marker determined and / or with a non-resistance sequence.

[0017] Another subject of the invention is a system comprising:

[0018] - Sequencing unit,

[0019] - control and computing unit, and

[0020] at least one data storage device in which at least one pathogen sequence and at least one resistance marker are stored for at least one pathogen,

[0021] wherein the sequencing unit is configured to sequence nucleic acids or peptides in a sample of a plant or a medium in which the plant grows, and thereby determine the sequence of nucleotides or amino acids in the sample,

[0022] wherein the sequencing unit is configured to transmit the respectively determined sequence to the control and computing unit during the determination of the sequence,

[0023] The control and computing unit is configured to compare the sequence of each transmitted nucleic acid or peptide with at least one pathogen sequence in a first stage, and

[0024] ● in the event that the transmitted sequence is inconsistent with the at least one pathogen sequence, causing the sequencing unit to interrupt further sequencing of the corresponding nucleic acid or the corresponding peptide,

[0025] ● In the case where the transmitted sequence is consistent with the pathogen sequence, the pathogen having the pathogen sequence is determined, and at least one resistance marker is determined using the determined pathogen, and the second stage is switched,

[0026] The control and computing unit is configured to compare the sequence of the nucleic acid or peptide in the sample with the determined at least one resistance marker and / or non-resistance sequence in the second stage.

[0027] Another subject of the invention is a computer-readable (storage) medium comprising instructions which, when executed by a computer, cause the computer to perform the following steps:

[0028] - for a plurality of nucleic acids or peptides: receiving from a sequencing unit a continuously increasing sequence of nucleotides or amino acids in the corresponding nucleic acid or the corresponding peptide,

[0029] - in a first phase during the reception of the growing arrangement: detecting whether said arrangement of nucleotides or amino acids is contained one or more times in at least one pathogenic sequence,

[0030] - in the event that the arrangement is not contained in the pathogenic sequence: transmitting a signal to the sequencing unit in order to interrupt further sequencing of the nucleic acid or peptide,

[0031] - In the case where the arrangement is consistent with the pathogen sequence: determining the pathogen having the pathogen sequence, and determining at least one reference marker, and starting the second stage,

[0032] - In said second stage: said arrangement of nucleotides or amino acids is compared with said at least one reference marker and / or resistance-free sequence.

[0033] Another subject of the invention is a computer program comprising instructions which, when the computer program is executed by a computer, cause the computer to carry out the following steps:

[0034] - for a plurality of nucleic acids or peptides: receiving from a sequencing unit a continuously increasing sequence of nucleotides or amino acids in the corresponding nucleic acid or the corresponding peptide,

[0035] - in a first phase during the reception of the growing arrangement: detecting whether said arrangement of nucleotides or amino acids is contained one or more times in at least one pathogenic sequence,

[0036] - in the event that the arrangement is not contained in the pathogenic sequence: transmitting a signal to the sequencing unit in order to interrupt further sequencing of the nucleic acid or peptide,

[0037] - In the case where the arrangement is consistent with the pathogen sequence: determining the pathogen having the pathogen sequence, and determining at least one reference marker, and starting the second stage,

[0038] - In said second stage: said arrangement of nucleotides or amino acids is compared with said at least one reference marker and / or resistance-free sequence.

[0039] Preferred embodiments of the invention are found in the dependent claims, the description and the drawings.

[0040] The present invention will be explained in detail below without distinguishing between the inventive subject matter (method, system, computer-readable medium, computer program). The following explanation should apply in a similar manner to all inventive subject matter, regardless of the context (method, system, computer-readable medium, computer program) in which it is carried out.

[0041] When steps are mentioned in a certain order in this description or in the claims, this does not necessarily mean that the invention is limited to the mentioned order. Rather, it is conceivable that the steps can also be performed in a different order or also in parallel with each other; if one step is based on another step, it is necessarily necessary that the step based on the basis is performed later (which will become clear in the specific case). The mentioned order is thus a preferred embodiment of the invention.

[0042] The present invention provides means that allow the user to identify at an early stage the infection of a plant by a pathogen and the markers of resistance of the pathogen to control agents.

[0043] The term "pathogen" is understood to mean a microorganism or subcellular stimulant which causes health-damaging processes in other organisms. Pathogens may be, for example, algae, bacteria, parasites, fungi, prion proteins, protists, viruses or viroids. In this specification, the term "pathogen" is used synonymously with the term pathogen. Preferably, the present invention relates to the early detection of fungi that damage plants.

[0044] The term "control agent" is understood to mean an agent that is effective in controlling pathogens and / or their spread. Examples of control agents are fungicides, insecticides and herbicides. Control agents typically include one or more active ingredients. An "active ingredient" is a chemical or biological substance that has a specific effect or causes a specific response in a pathogen.

[0045] The term "resistance" is understood to be a property of individual pathogens of a class, which manifests itself in the ability of these individuals to tolerate treatment with control agents which normally control this class of pathogens and to complete their development cycle normally or further.

[0046] The term "user" is understood to mean a person who implements the invention; a user is especially a person who utilizes the system of the invention and / or uses a computer with the computer program of the invention installed in order to implement the invention.

[0047] Two types of resistance can be distinguished; target-specific resistance, also called target site resistance, and metabolic resistance, also called non-target site resistance.

[0048] Target-specific resistance is usually caused by mutations at a single gene associated with the target of the control agent in the pathogen. The mutation can, for example, lead to stronger expression of the target protein or to changes in the site to which the active ingredient of the control agent binds. For this type of resistance, there are molecular biological tests.

[0049] Resistance not directly linked to the target (non-target site resistance) is attributed to all other mechanisms, such as slowed uptake of the control agent by the pathogen.

[0050] The invention makes it possible in particular to identify target site resistance at an early stage.

[0051] The pathogens / pathogens of interest in the present invention primarily infect plants, especially cultivated plants. The term "cultivated plants" is understood to mean plants that are purposefully cultivated as agricultural crops through human intervention. The cultivated plant parts grown can be suitable for human and / or animal consumption.

[0052] Basically, it is conceivable that the teachings of the present invention can also be applied to animals, in particular livestock. Therefore, another subject of the present invention is the application of the teachings discussed in this specification to animals, in particular livestock.

[0053] In a first step, at least one sample is collected. The term "collection" should not be understood in any limiting way. An example of a synonymous term is the term "sampling".

[0054] The sample is obtained from the plant or the medium in which the plant grows. The term "medium" includes not only soil, but also any other matrix that is in contact with the plant and supports the plant and / or supplies nutrients to it. In the case of hydroponic plants, the sample can also be obtained from the water surrounding the hydroponic plant.

[0055] Preferably, parts of the plant are sampled, such as leaves, flowers, stems, roots, pollen, epidermis and / or a part of the mentioned components and / or a plurality of the mentioned components and / or other components. Samples can also be taken from "secretions" such as nectar or resin. Preferably, various tissue types of the plant are taken and combined in order to cover a variety of different pathogens. Preferably, the plant is at an early stage of development at the time of sampling.

[0056] The developmental stage of a plant can be given in the form of so-called BBCH coding, for example. The abbreviation BBCH officially stands for the Federal Biological Research Center for Agriculture and Forestry, the Federal Plant Variety Office and the Chemical Industry (BiologischeBundesanstalt, das Bundessortenamt und die CHemische Industrie). The extended BBCH scale for uniformly coding the phenological developmental stages of monocotyledons and dicotyledons is the Federal Biological Research Center for Agriculture and Forestry (BBA), the Federal Plant Variety Office (BSA), the Agricultural Industry Alliance (IVA) and the Groβbeeren / Erfurt Vegetable and Horticultural Academy (see, for example, https: / / www.julius-kuehn.de / publikationsreihen-des-jki / bbch-skala / ).

[0057] Preferably, at the time of sampling, the plant is in the 1st main growth stage (leaves (main branches)) or the 2nd main growth stage (formation of lateral branches) or the 3rd main growth stage (main branches / stem growth / branching (main stem) growth or rosette growth) or the 4th main growth stage (development of vegetative plant parts or vegetative reproductive organs / inflorescence or panicle) or the 5th main growth stage (emergence of inflorescence) or the 6th main growth stage (flowering) of the BBCH scale. It has been found that fungi and / or fungal spores can already be detected in, near or on the surface of the plant at these early stages, however, the plant does not necessarily have already visible disease symptoms. Identification at such an early time point allows early treatment of the plant in order to inhibit the outbreak of the disease and / or the proliferation and / or spread of the pathogen and / or the transfer of the pathogen to neighboring plants or reduce the risk thereof.

[0058] The sampling can be performed automatically. "Automatically" means that the sampling is performed by a machine or by multiple machines without human assistance. Such a machine or device for automatic sampling is also referred to as a "sampling unit" in this specification.

[0059] The sampling can be carried out by means of or by means of a mobile device, which for example moves or is moved in and / or above the field where the plants are grown. It is conceivable, for example, to use (preferably unmanned) ground machines and / or (preferably unmanned) aerial vehicles (e.g. drones) and / or robots. In greenhouses, robots for automated sampling are suitable.

[0060] However, sampling can also be carried out by means of one or more devices which are built fixedly at one location.

[0061] It is also conceivable that the user (person) performs the sampling himself.

[0062] The sample is sent to a sequencing unit, which is configured to determine the sequence of the nucleic acid or peptide in the sample.

[0063] "Nucleic acid" is a macromolecule composed of various building blocks, namely nucleotides, which contain genetic information in organisms and subcellular bodies (such as viruses). Nucleic acids exist in two forms in nature: deoxyribonucleic acid (DNA) and ribonucleic acid (RNA). DNA is the genetic material of all organisms from single-cell bacteria to multicellular mammals. Some viruses use RNA instead of DNA as their genetic material. It is preferred to sequence the DNA strands in the sample.

[0064] "Peptide" is an organic compound containing peptide bonds between amino acids. Long peptide chains are also called proteins. Proteins are present in every cell and serve as molecular "tools" and perform different tasks depending on their specific structure, such as enabling cell movement, transporting metabolites, pumping ions, catalyzing chemical reactions or recognizing signal substances. In this specification the terms "peptide" and "protein" are used synonymously.

[0065] The term "sequencing" is understood to mean determining the arrangement of nucleotides in a nucleic acid or the arrangement of amino acids in a peptide (protein). Correspondingly, a sequence is the order of nucleotides or the arrangement of amino acids. The terms "sequence" and "arrangement" are used synonymously in this specification.

[0066] It is conceivable that the sample is prepared before being sent to sequencing. The cells in the sample are destroyed in such preparation, for example, to release the DNA molecule, then extract it and fragment it when appropriate. The relevant measures for preparing are described in the prior art (see, for example, RP Schaudies (Ed.): Biological Identification, Woodhead Publishing Series in Electronic and Optical Materials: Number 59, Elsevier 2014, ISBN 978-0-85709-501-5; Jianping Xu: Next-generation Sequencing, Caister Academic Press 2014, ISBN 978-1-908230-33-1; Vijai Bhadauria: Next-generation Sequencing and Bioinformatics for Plant Science, Caister Academic Press 2017, ISBN 978-1-910190-65-4). Sample processing may be performed using a sample preparation unit, which may be an integral part of the sampling unit and / or sequencing unit or may be a separate unit.

[0067] During sample preparation, specific regions of the existing nucleic acid can be amplified; however, amplification is not necessarily necessary. It may be meaningful to amplify regions that are characteristic of a species or that can uniquely and / or reliably identify a species. As an example of such a region, the ITS region can be mentioned.

[0068] The ITS region (ITS: Internal Transcribed Spacer) is a nucleotide sequence frequently used in phylogenetic studies. The ITS region is a non-coding region between genes with highly variable sequences; it is therefore particularly suitable for determining phylogenetic relationships at the low taxonomic levels of species, genera, and families. Sequence analysis of this region can be used, in particular, for the identification of fungal genera and families (see, for example, TM Pryce et al.: Rapid Identification offi by sequencing the ITS 1 and ITS2 regions using an automated capillary electrophoresis System; Medical Mycology October 2003, 4 1, 369-381; RH Nilsson et al.: A Comprehensive, Automatically Updated Fungal ITS Sequence Dataset for Reference-Based Chimera Control in Environmental Sequencing Efforts, Microbes Environ. 2015 Jun; 30(2): 145-150; AJ Buehler et al.: Internal transcribed spacer (ITS) sequencing reveals considerable fungal diversity in dairy products, Dairy Sei. 2017; 100(11): 8814-8825; CLS Choch et al.: Nuclear ribosomal internal transcribed spacer (ITS) region as a universal DNA barcode marker for Fungi, PNAS April 17, 2012 109 (16) 6241-6246). By comparing the determined ITS sequence with the 5.8S rRNA gene sequence of the fungal strain to be identified, the sequence similarity used as the basis for taxonomic group association is determined. The nucleotide sequence of the ITS region is about 500-600 base pairs. In addition to the highly conserved regions whose sequences are identical for all fungi, there are also variable regions. These variable regions can be characteristic identification marks for species, genus or group of fungi. Thus, the analysis of these subsequences can achieve taxonomic association of fungal strains.

[0069] Because the ITS region appears repeatedly as a gene family with multiple hundreds to thousands of tandemly repeated copies respectively, the ITS region is overexpressed in a stoichiometric ratio for a single nuclear gene and is easily accessible in molecular phylogenetic studies. Another advantage of using the ITS region for molecular phylogenetic studies is determined by its position. The ITS region is located in the rRNA gene cluster. It is composed of two sequence fragments, i.e. internal transcribed spacer 1 (ITS-1) and internal transcribed spacer 2 (ITS-2), which are located between highly conservative rDNA genes 26S, 5.8S and 18S. Due to this position, the rDNA sequence fragments of highly conservative rDNA genes can be used as primer sequences. Therefore, ITS-1 and ITS-2 sequences are easily achieved by means of the amplification of PCR.

[0070] The sequencing of nucleic acids or peptides present in the sample is performed by a sequencing unit. Preferably, sequencing is performed by means of a method based on nanotechnology (see, for example, M. Loose et al.: Real-time selective sequencing using nanopore technology, Nature Methods 2016, Vol. 13 No. 9, 751-758). In nanopore sequencing, for example, nucleic acids are transported individually from enzymes (helicases) through nanometer-sized holes in a non-conductive polymer membrane. If a voltage is applied to this membrane, an ion flow through the hole is generated, the size of which depends on the bases in the hole. Therefore, the base arrangement and any modification of the bases can be measured by measuring the ion current during the translocation of DNA / RNA through the hole.

[0071] The sequencing of the present invention can be carried out in at least two stages, the first stage and the second stage. The first stage is used to understand whether there is a pathogen in the sample. Only when the pathogen is identified in the first stage, the second stage is carried out. The second stage is used to identify the markers of resistance of the identified pathogen to one or more control agents. In these two stages, the nucleic acid or peptide is sequenced and the nucleotide arrangement and amino acid arrangement are determined thereby. In these two stages, the determined sequence is compared with the reference sequence during the determination process. In the first stage, the determined arrangement is compared with at least one pathogen sequence. Preferably, it is compared with multiple pathogen sequences. If there is consistency between the determined nucleotide arrangement or amino acid arrangement and the pathogen sequence, it can be considered that the pathogen is contained in the sample. In this way, the pathogen contained in the sample can be determined. In the second stage, the determined arrangement is compared with at least one resistance marker observed in the past on the determined pathogen and / or with a non-resistance sequence. This comparison is used to identify the markers of resistance of the identified pathogen to one or more control agents. A non-resistance sequence is a nucleotide arrangement or amino acid arrangement of the pathogen that does not have a marker of resistance of the pathogen to the control agent. Pathogen sequences, resistance markers and non-resistance sequences are reference sequences that can be known, for example, from one or more databases.

[0072] First, the first stage should be examined in detail.

[0073] Usually, a large part of the nucleic acids or peptides present in the sample is attributed to plants, especially when the sample is a component of a plant. However, the determination of the sequence of plant nucleic acids or plant proteins is of secondary importance. Therefore, according to the invention, the sequencing of nucleic acids or peptides in the first stage of sequencing is interrupted as soon as it is found that the nucleic acids or peptides are not derived from pathogens (but, for example, from plants).

[0074] For this purpose, the sequencing result is compared with one or more pathogenic sequences during sequencing. That is, while determining the nucleotide arrangement in the nucleic acid or the amino acid arrangement in the peptide, it is checked whether the nucleotide arrangement or the amino acid arrangement is contained in one or more pathogenic sequences. This is explained with an example. First, the first nucleotide in the nucleic acid is determined, then the second, and so on. The arrangement of nucleotides grows with each nucleotide that continues to be determined. When another nucleotide in the arrangement has been determined, it is checked whether the (growing) arrangement is contained in one or more pathogenic sequences. For example, an arrangement of four nucleotides has a high probability of being contained in one or more positions of the pathogenic sequence. However, in the case of an arrangement of twenty nucleotides, it is already possible to make this arrangement no longer appear in any position in the pathogenic sequence. Once it is found that the arrangement of nucleotides determined during sequencing is not contained in any position of one or more pathogenic sequences, sequencing can be terminated; at this time, any determination of other nucleotides in the nucleotide arrangement is meaningless; it is already known that the nucleic acid currently being sequenced cannot be a pathogenic nucleic acid. This is also applicable to determining the amino acid arrangement in the peptides in the sample.

[0075] The at least one pathogen sequence used for comparison in the first stage of sequencing is a nucleotide sequence or amino acid sequence of a defined pathogen that is specific to the pathogen (or a defined pathogen group, such as a species or genus or subfamily or family or suborder or order or class). With the help of such a specific pathogen sequence, a specific pathogen (or pathogen group) can be derived. Such a pathogen sequence can, for example, be a nucleic acid sequence that completely or partially includes an ITS region. In any case, the pathogen sequence must be a sequence different from the sequence of the plant being the subject of the study.

[0076] In a preferred embodiment, one or more pathogen sequences are (automatically) selected based on information about plants, about sampling time points, about sampling locations, about environmental conditions (temperature, air pressure, air humidity, precipitation, etc.) during a defined time period (e.g., weather data and / or climate data) at sampling time points and / or before sampling time points, and / or about history (e.g., pathogens and / or diseases observed in the past). For example, certain diseases only occur in certain plants. When a sample of a certain plant is obtained, pathogen sequences of pathogens that can infect this plant and trigger the disease can be selected. In addition, pathogens usually only occur in certain regions and only under certain environmental conditions. The location of sampling provides information about regions and pathogens that usually occur in these regions. Environmental conditions at sampling time points and / or in a defined time period (e.g., four weeks or three weeks or two weeks or one week or several days) before sampling time points provide information about which pathogens can be observed or which pathogens can be excluded. A pathogen that has been observed in the past at or near the sampling location (either directly or indirectly, for example, by means of disease symptoms) may also occur in the present (if environmental conditions and the respective observed plants permit), and therefore a specific pathogen sequence of such a pathogen can (and should) be selected because it (the pathogen) has appeared in the past.

[0077] Thus, a preferred embodiment of the present invention comprises the following steps:

[0078] (1) Identify the plant

[0079] (2) Identifying at least one pathogen capable of infecting the plant (the pathogen has infected such plant in the past)

[0080] (3) determining at least one pathogen sequence of the at least one pathogen determined

[0081] (4) collecting a sample from the plant or the medium in which the plant grows,

[0082] (5) Identifying pathogens in the sample

[0083] (6) Determine at least one resistance marker of the identified pathogen

[0084] (7) Identify markers of resistance of the identified pathogen to the control agent.

[0085] Another preferred embodiment of the present invention comprises the following steps:

[0086] (1) Identify the plant

[0087] (2) Determining the environmental conditions to which the plant is exposed and / or has been exposed

[0088] (3) Identifying at least one pathogen capable of infecting the plant (the pathogen has infected such plant in the past)

[0089] (4) determining at least one pathogen sequence of the at least one pathogen determined

[0090] (5) collecting a sample from the plant or the medium in which the plant grows,

[0091] (6) Identifying pathogens in the sample

[0092] (7) Determine at least one resistance marker of the identified pathogen

[0093] (8) Identify markers of resistance of the identified pathogen to control agents.

[0094] For example, the plant can be determined and / or the environmental conditions can be determined by the user inputting the corresponding information into the system of the present invention. The plant can be specified, for example, in the following way: the user enters the name of the plant or the name of the plant species or the code of the plant / plant species (for example according to the International Code for Nomenclature of Cultivated Plants, abbreviation ICNCP) into the system of the present invention, or selects information about the plant from a list or menu, or selects the plant, for example, by means of a graphical display of the plant (for example a photo or image) on a monitor of the system of the present invention.

[0095] It is also conceivable that the user specifies the plants to be cultivated in the field and, if appropriate, additionally specifies the geographical coordinates of the field. The system of the invention can be configured to determine in a database the possible occurrence of pathogens at this plant species with the aid of the specified plants and, if appropriate, the geographical coordinates. With the aid of the geographical coordinates, for example, current weather data and / or past weather data can be determined. It is conceivable that a model is selected for pathogens that may occur at the plant, which model calculates the probability of such pathogens occurring at the specified plant in the current time period with the aid of weather data. It is conceivable that only pathogens whose probability of occurring at the specified plant in the current time period is greater than a threshold value (minimum probability) are observed in detail. The threshold value can be determined empirically or freely determined by the user.

[0096] Thus, a preferred embodiment of the present invention comprises the following steps:

[0097] (1) Identify the plant

[0098] (2) Identifying at least one pathogen capable of infecting the plant (the pathogen has infected such plant in the past)

[0099] (3) determining the environmental conditions to which the plant is or has been exposed,

[0100] (4) Calculating the probability of the at least one pathogen appearing on the plant at the current time point

[0101] (5) Compare the probability with a predefined threshold

[0102] (6) Determining the pathogen sequences of those determined pathogens whose probabilities are higher than the threshold

[0103] (7) collecting a sample from the plant or the medium in which the plant grows,

[0104] (8) Identifying pathogens in the sample

[0105] (9) determining at least one resistance marker for the identified pathogen,

[0106] (10) Identify markers of resistance of the identified pathogen to the control agent.

[0107] The one or more pathogenic sequences preferably exist as FM indexes. FM index is a compressed full-text substring index based on Burrows-Wheeler transformation and developed by P.Ferragina and G.Manzini (see, for example, P.Ferragina and G.Manzini: Opportunistic Data Structures with Applications, Proceedings of the 41st Annual Symposium on Foundations of Computer Science 2000; 390-398). The name FM index represents the full-text index in Minute space. FM index is an opportunistic data structure that can not only realize compression of input text but also realize fast substring query. FM index can be used to efficiently determine the number of patterns that occur within the compressed input text and locate the position of each occurrence. Query time and required storage space have sublinear complexity relative to the size of input data.

[0108] The comparison of the nucleotide arrangement or amino acid arrangement just determined with one or more pathogenic sequences can be performed by a control and computing unit. Such a control and computing unit can be one or more computers. The control and computing unit can be a component of a sequencing unit or a separate unit. The control and computing unit can access one or more data storages. At least one pathogenic sequence is stored in the at least one data storage. The control and computing unit loads the at least one pathogenic sequence into a working memory, receives the nucleotide arrangement (for nucleotides or in a fragment of multiple nucleotides) during its determination, and checks whether the (growing) nucleotide arrangement appears in the pathogenic sequence once or multiple times.

[0109] Techniques for using the (growing) arrangement of nucleotides in a sequence for comparison during nucleotide determination have been described in the prior art (see, for example, S. Kovaka et al.: Targeted nanopore sequencing by real-time mapping of raw electrical Signal with UNCALLED, https: / / doi.org / 10.1101 / 2020.02.03.931923).

[0110] Once it is found that the determined nucleotide or amino acid arrangement does not appear in any position in the at least one pathogen sequence, the sequencing of the corresponding molecule is interrupted and further sequencing of the molecule is stopped. The technology of interrupting the determination of nucleotide arrangement has been described in the prior art (see, for example, A. Payne et al.: Nanopore adaptive sequencing for mixed samples, whole exome capture and targeted panels, https: / / doi.org / 10.1101 / 2020.02.03.926956). In the case of nanopore technology, for example, the nucleic acid guided through the hole can be transported out of the hole by means of a voltage pulse and further sequencing is then interrupted.

[0111] If no nucleic acid or peptide with a pathogen sequence is sequenced within a predefined time period, it can be assumed that no pathogen is present in the sample. The predefined time period can be determined empirically. A notification that no pathogen was found in the sample can be output.

[0112] Once a nucleic acid or peptide sequence in a sample is found to be consistent with a specific pathogen sequence, it can be assumed that the corresponding pathogen is present in the sample. That is, the corresponding pathogen can be automatically derived from the knowledge of the pathogen sequence. For the pathogen thus determined, a database is used to check whether there is a known resistance to one or more control agents. "Known" means that the corresponding resistance has been observed in the pathogen community in the past. If at least one resistance has been observed, at least one resistance marker is determined.

[0113] A “resistance marker” is understood to be genetic information which provides information about whether a pest may, has just evolved or has already evolved resistance to a control agent.

[0114] For many pathogens, it is known which sequences indicate that the pathogen has evolved or has resistance to control agents (see, for example: S. Omrane et al.: Plasticity of the MFS1 Promoter Leads to Multidrug Resistance in the Wheat Pathogen Zymoseptoria tritici, https: / / doi.org / 10.1128 / mSphere.00393-17; Y. Choi et al.: Identification of Genes Related to Fungicide Resistance in Fusarium fufikuroi, Mycohiology. 2017, 45(2): 101-104; KJ Brent: Fungicie Resistance in crop pathogens: How can it he managed? ISBN 90-72398-07-6; KG Zulak et al.: Improved Detection and Monitoring of Fungicide Resistance in Blumeria graminis f.sp. hordei With High-Throughput Genotype Quantifi cation hy Digital PCR,Front Microbiol.2018,9:706; M.de miccolis et al.: Genetics of Fungicide Resistance, DOI:10.1007 / 978-4-431-55642-8_2; HBDeising et al.: Mechanisms and significance of fungicide resistance, Braz.J.Microbiol.2008, Vol.39No.2; L.Kanetis et al.: Fungicide resistance profile and genetic structure of Botrytis cinerea from greenhouse crops in Cyprus, Eur.J.Plant.Pathol.2017,147,527-540; JLBeckerman: Detection of Fungicide Resistance, DOI: 10.5772 / 55981; L.Yang et al.: Cross-resistance of the pathogenicfungus Alternaria alternata to fungicides with different modes of action, BCM Microbiol 2019, 19, 205). .

[0115] That is, a "resistance marker" is a nucleotide sequence or amino acid sequence found in nucleic acids or peptides of resistant populations and responsible for or contributing to resistance. This can be, for example, a target sequence in a resistant pathogen that has a variation against a non-resistant species of the same type. "Target" is defined here as the target point of the control agent at the pathogen. Genetic changes in the target sequence may cause the control agent to no longer be effective or to have a reduced effect.

[0116] If no resistance is known for the identified pathogen, a notification can be output as to which pathogen was identified in the sample and for which no resistance is known.

[0117] Once the pathogen in the sample has been identified and at least one resistance marker for said pathogen has been determined, the second stage of sequencing is initiated.

[0118] In the second stage, the nucleic acids or peptides in the sample are further sequenced; however, in the second stage, the focus is on identifying markers of resistance of the identified pathogen to one or more control agents. To this end, the nucleotide or amino acid sequence determined during sequencing is compared with the at least one resistance marker and / or with a non-resistance sequence.

[0119] If no nucleotide or amino acid arrangement consistent with the sequence of the at least one resistance marker appears during sequencing, it can be considered that the pathogen identified in the sample does not have a known resistance. A notification can be output: what pathogen has been found and no signs of known resistance to the control agent have been found. However, if a deviation occurs relative to the sequence without resistance, it may be that there is (yet) unknown resistance and / or the pathogen has evolved (not yet known) resistance. In this case, a notification can be output: what pathogen has been found and signs of known resistance to the control agent have been found, but there is a deviation in the nucleotide or amino acid arrangement relative to the sequence without resistance, which may / should be further investigated.

[0120] If a nucleotide or amino acid sequence consistent with the sequence of a resistance marker appears during sequencing, this is a sign that a (known) resistance is present. A notification may be output stating that a pathogen was found in the sample, that the pathogen has a resistance marker and that the possible resistance may be known, for example through a consistent resistance marker.

[0121] Thus, in a preferred embodiment, the step of "identifying markers of resistance of the identified pathogen to the control agent" comprises the following sub-steps:

[0122] (1) comparing the nucleic acid or peptide sequence in the sample with the at least one resistance marker and / or non-resistance sequence determined

[0123] (2) in the case where the resistance marker does not appear in the nucleic acid or peptide sequence in the sample and the sequence of the sample corresponds to a non-resistance sequence: outputting a notification that no resistance marker was found for the identified pathogen,

[0124] (3) in the case where the resistance marker does not appear in the nucleic acid or peptide sequence in the sample and the sequence of the sample does not correspond to a non-resistance sequence: outputting a notification that no known resistance marker was found for the identified pathogen, but a deviation from a non-resistance sequence was found,

[0125] (4) When the resistance marker appears in one or more of the sequences: outputting a notification that a resistance marker has been found for the identified pathogen, and outputting a notification regarding the resistance.

[0126] It is conceivable that the sequence of a nucleic acid or peptide of a resistant pathogen differs from the corresponding sequence of a non-resistant pathogen by only one nucleotide or a few nucleotides or by only one amino acid or a few amino acids. On the other hand, errors always occur when sequencing various nucleic acids or peptides. Therefore, if a nucleic acid or peptide with a sequence according to a resistance marker is identified, it is theoretically possible that an error has occurred here. Therefore, it is conceivable that a resistance marker is considered to be present only when a minimum number of nucleic acids or amino acids with a sequence according to a resistance marker is found.

[0127] Thus, in a preferred embodiment, the step of "identifying markers of resistance of the identified pathogen to the control agent" comprises the following sub-steps:

[0128] (1) comparing the nucleic acid or peptide sequence in the sample with the at least one resistance marker determined,

[0129] (2) in the case where the resistance marker does not appear in the nucleic acid or peptide sequence in the sample or the resistance marker appears in a number of nucleic acids less than a predetermined minimum number: outputting a notification that no resistance marker is found for the identified pathogen,

[0130] (3) In the case where the resistance marker appears in a minimum number of sequences or appears in a number greater than the minimum number: outputting a notification that a resistance marker has been found for the identified pathogen, and outputting a notification regarding the resistance.

[0131] In a preferred embodiment of the present invention, in the second stage of sequencing, the nucleic acid or peptide sequence in the sample is not only compared with the resistance marker, but also compared with the corresponding sequence of the same non-resistant pathogen. This sequence is also referred to as the non-resistant sequence in this specification. This non-resistant sequence is also a reference sequence.

[0132] It is conceivable that a nucleic acid or peptide derived from a pathogen is found in a sample, but where its sequence corresponds neither to a resistance marker nor to a non-resistance sequence. It may be a mutation that may lead to a resistance that is still unknown in some cases. That is, by comparing the sequence of a nucleic acid or peptide in a sample with a non-resistance sequence, new resistances that develop in some cases can be identified at an early stage.

[0133] Thus, a preferred embodiment of the present invention comprises the following steps:

[0134] - Collect samples from plants or the medium in which they grow

[0135] - Identify the pathogen in the sample

[0136] - determining at least one resistance marker of the identified pathogen and determining the resistance-free sequence of the identified pathogen

[0137] - identifying a marker of resistance of the identified pathogen to the control agent by comparing the nucleic acid or peptide sequence in the sample with the at least one resistance marker determined and the non-resistance sequence

[0138] -For the case where a sequence or a number of sequences greater than or equal to a minimum number has neither identity with said at least one resistance marker nor identity with said non-resistance sequence: output a notification indicating that such a pathogen has been identified in said sample, which has mutations pointing to new and / or developed resistance.

[0139] Also for the case where a pathogen may be detected in the sample but the resistance of the pathogen is not known, a second stage of sequencing can be initiated, for example in order to analyze the target sequence at the pathogen and identify mutations indicating evolved resistance when appropriate. In such a case, the determined nucleotide or amino acid arrangement is not compared with the resistance marker, but with the target sequence. The target sequence is also the reference sequence. The target sequence, the non-resistance sequence and the reference marker are also referred to in this specification as sequences indicating resistance, because resistance or lack of resistance can be determined with the help of these sequence fragments.

[0140] Thus, a preferred embodiment of the present invention comprises the following steps:

[0141] - Collect samples from plants or the medium in which they grow

[0142] - Identify the pathogen in the sample

[0143] - Determine the target sequence of the identified pathogen

[0144] - Identifying markers of novel resistance of the identified pathogen to the control agent by comparing the nucleic acid or peptide sequence in the sample with the target sequence

[0145] - In the case where one sequence or a number of sequences greater than or equal to the minimum number show a variation of said target sequence: output indicating that such a pathogen is identified in said sample, which has a variation pointing to a new and / or developed resistance.

[0146] In a preferred embodiment, the frequency Q of nucleic acids or peptides having a sequence according to the resistance marker found in the sample is determined. R , and determining the frequency Q of nucleic acids or peptides found in the sample having a sequence according to the resistance-free sequence NR The determined frequency Q R and Q NR Divide (for example, Q R / Q NR ). It is conceivable that the presence of a resistance marker is assumed only when the ratio exceeds a predefined minimum value. The minimum value can be determined empirically.

[0147] Thus, a preferred embodiment of the present invention comprises the following steps:

[0148] - Collect samples from plants or the medium in which they grow

[0149] - Identify the pathogen in the sample

[0150] - determining at least one resistance marker of the identified pathogen and determining the resistance-free sequence of the identified pathogen

[0151] - comparing the nucleic acid or peptide sequence in the sample with the at least one resistance marker determined and the non-resistance sequence

[0152] - Determine the frequency Q of nucleic acids or peptides found in the sample having a sequence corresponding to the resistance marker R , determine the frequency Q of nucleic acids or peptides found in the sample that have sequences that match the resistance-free sequence NR , calculate the determined frequency Q R and Q NR and compares the ratio with a predefined minimum value.

[0153] - In the case where the ratio is greater than or equal to the minimum value: outputting a notification that a resistance marker is found for the identified pathogen, and outputting a notification regarding the resistance.

[0154] What is described in the present description for comparing a nucleic acid sequence or a peptide sequence with the at least one pathogenic sequence otherwise applies analogously to comparing a nucleic acid sequence or a peptide sequence with the at least one resistance marker and / or non-resistance sequence and / or target sequence:

[0155] ●In order to carry out the second stage of sequencing as efficiently as possible and to prevent a large part of the sequencing from falling on non-target sequences of plant nucleic acids or peptides or pathogens, in the second stage of sequencing, the sequencing of a single nucleic acid or peptide may be interrupted once it is discovered that the nucleic acid just sequenced or the peptide just sequenced cannot be derived from the target sequence of the pathogen (because the determined arrangement does not appear in the pathogen target sequence).

[0156] - The at least one resistance marker and / or resistance-free sequence is preferably present as a FM index.

[0157] ●The control and computing unit can load the at least one resistance marker and / or the non-resistance sequence into a working memory and, during its determination, receive a (growing) nucleotide arrangement or amino acid arrangement and compare it with the at least one resistance marker and / or with the non-resistance sequence.

[0158] When implementing the present invention, output information and / or notifications can be performed, for example, in the form of pictures and / or texts and / or voice. Information and / or notifications can be displayed on a display screen (monitor), printed out by a printer, stored in a data storage device and / or transmitted to another computer via a network.

[0159] Information about the identified pathogen can be, for example, the name of the pathogen. The following information can also be output: in which sample the pathogen was found, and / or at which location the sample was collected and / or at which time point the sampling was performed and / or from which plant the sample was taken.

[0160] Information about the identified resistance may include the name of the resistance, the target affected by the resistance, and / or the name of the control agent(s) or active ingredient(s) to which the identified pathogen is resistant.

[0161] The results of the plant sample analysis of the present invention can be used in various ways. For the case where a pathogen has been identified in which no signs of resistance or new resistance have been found, a control agent or multiple control agents that can be used to control the pathogen can be proposed. For the case where a sign of known resistance has been identified in which the presence of resistance has been found, a control agent for which the resistance is ineffective can be proposed. Such a control agent can then also be the use of electrical energy, thermal energy (for example in the form of water vapor or flame), electromagnetic radiation (UV radiation, laser irradiation) and / or mechanical means to remove infected plants or plant parts.

[0162] Because the present invention can also be performed on-site in the field by an autonomous system, the system of the present invention can also include a control device for controlling pathogens, which can automatically apply the selected control agent after (automatically) selecting the control agent for controlling the identified pathogen.

[0163] The results of the analysis of pathogens and resistance according to the invention can also be recorded in a pathogen card. Such a "pathogen card" is a representation of a portion of the surface of the earth, wherein for a plurality of locations on the surface information is recorded about whether a pathogen has been observed at the corresponding location and whether a sign of (existing or evolved or possible) resistance to a control agent has been found in the pathogen. The pathogen card is preferably a digital pathogen card.

[0164] The term "digital" means that the card can be processed by a machine, usually a computer system.

[0165] "Processing" is understood to be known methods for electronic data processing (EDV).

[0166] The digital pathogen card is preferably a digital representation of a field or a plurality of adjacent fields or an area comprising a field. Preferably, separate pathogen cards are produced for various pathogens and / or for various control agents or control agent groups and / or for specific plants where pathogens may occur, the control agent groups having the same active ingredient or the same chemical / biological type of active ingredient (e.g. chemical structural class) or the same mechanism of action or the same target (target). Preferably, the separate digital pathogen cards can be associated with each other, that is to say virtually superimposed.

[0167] In a preferred embodiment, for each position on the digital pathogen card where one or more analysis results exist, one or more time points at which the corresponding analysis was performed are saved.

[0168] In a preferred embodiment, a plurality of digital pathogen cards are associated with each other such that they display the temporal evolution of the distribution of one or more resistances.

[0169] Preferably, the digital pathogen card can be combined with other digital cards, for example with digital cards about soil quality, about water management, about cultivated plants, about temperature (at a defined time and / or for a defined period of time, for example in the form of average temperature and / or minimum temperature and / or maximum temperature), about precipitation (at a defined time and / or for a defined period of time, for example in the form of average precipitation and / or minimum precipitation and / or maximum precipitation), about sunlight exposure, about atmospheric movement (wind direction and wind force), about infection with one or more pathogens that occurred in the past, about agricultural measures that have been taken (for example sowing, watering, ploughing, applying plant protection agents, giving nutrients and similar measures), etc. The parameter values ​​recorded in the digital card can be measured values ​​and / or predicted values.

[0170] The pathogen card can, for example, provide the farmer with valuable information about in which areas of his field (or his fields) the pathogen is present and which resistance is present or spread. The propagation pattern can be analyzed in order to investigate the causes of the appearance and / or spread of pathogens and / or resistance. In a number of analyses that follow one another in time, the farmer gains insights into the spread of pathogens and / or resistance. He can then take measures to prevent and control pathogens and / or resistance. In the pathogen card, the farmer can also tell whether an infection with a (resistant) pathogen has been observed in an adjacent field. This information can help him take preventive measures for his field.

[0171] Furthermore, the combination of detailed, almost real-time generated digital pathogen cards with other data (e.g. solar radiation, temperature, air humidity and wind direction) allows accurate prediction of the spread of resistant and non-resistant pathogens. This allows targeted control or preventive treatment of adjacent areas and targeted use of control agents.

[0172] In addition, targeted monitoring of genetic information in pathogen populations in the environment allows for estimation of future evolution of resistance to a particular control agent.

[0173] In another step of the method of the present invention, measures for controlling pathogens and / or evolved resistance can be suggested and / or implemented (based on the pathogen card). First, one or more control agents for controlling pathogens can be identified with the help of the pathogens that have been identified in the sample. This can be done, for example, by querying in one or more databases. Information about control agents and pathogens for which control agents are targeted can be stored in the one or more databases. For the case where the identified pathogen is resistant, it can be checked in a second step whether there is resistance to one or more of the control agents determined from the at least one database. The control agent or control agents to which the identified pathogen is not resistant can be identified. The plant can then be treated with (one of) the identified control agents. If the pathogen is resistant to all control agents determined by the at least one database, alternative measures for controlling pathogens and / or disease transmission can be identified, suggested and / or implemented, such as: complete mechanical removal of the plants involved, crop rotation, flame burning, destruction by freezing, destruction by electrical energy, destruction by electromagnetic energy (e.g., by means of UV light, laser) and / or similar measures.

[0174] The present invention can be implemented in whole or in part by means of a computer system. A "computer system" is a system for electronic data processing, which processes data by means of programmable calculation rules. Such a system usually includes a control and calculation unit and peripherals, which is usually also called a "computer", i.e. a unit including a processor for performing logical operations and a working memory for loading computer programs.

[0175] "Peripherals" in computer technology refer to all devices that are connected to a computer and are used to control the computer and / or serve as input and output devices. Examples are monitors (displays), printers, scanners, mice, keyboards, joysticks, drives, cameras, microphones, speakers, etc. Internal connectors and expansion cards are also considered peripherals in computer technology.

[0176] Computer systems today are generally divided into desktop PCs, portable PCs, laptops, notebook computers, netbook computers, tablet PCs and so-called handheld devices (eg smartphones); all of these systems can be used to implement the present invention.

[0177] Input into the computer system (e.g. for control by a user) takes place via input devices such as a keyboard, a mouse, a microphone, a touch-sensitive display and / or the like. Output takes place via one or more output units, which may in particular be a monitor (display screen), a printer and / or a data memory.

[0178] The invention will be explained in more detail below with the aid of specific exemplary embodiments and drawings, without intending to restrict the invention to these exemplary embodiments, the features and / or feature combinations shown in these exemplary embodiments.

[0179] In the attached picture:

[0180] Figure 1 The embodiment of the system of the present invention is schematically shown. The system (10) comprises a sampling unit (11), a sequencing unit (12), a control and calculation unit (13), a data storage (14) and an output unit (15).

[0181] With the aid of the sampling unit (11) a sample can be taken from the plant P or the medium in which the plant P grows. The sample can be processed and sent to a sequencing unit (12).

[0182] The sequence of the nucleic acids present in the sample is determined with the aid of a sequencing unit (12).

[0183] The control and computing unit (13) is used to control the various components of the system (10) of the present invention and coordinate data flow and signal flow. The control and computing unit can be a general purpose computer (English general purpose computer) that has been configured accordingly with the help of the computer program of the present invention to perform the steps described herein.

[0184] The control and computing unit (13) is connected to a data memory (14) from which it can retrieve one or more pathogen sequences and one or more resistance-indicating sequences for comparison purposes.

[0185] The control and calculation unit (13) receives the growing nucleic acid sequence from the sequencing unit (12).

[0186] In a first phase, the control and computing unit (13) checks whether the growing sequence is contained in one or more pathogen sequences. As soon as it is found that the growing nucleic acid sequence is not contained in the at least one pathogen sequence, the control and computing unit (13) sends a signal to the sequencing unit (12). The sequencing of the nucleic acid is interrupted by the signal and the sequencing of a new nucleic acid is started. As soon as it is found that the nucleic acid sequence is consistent with the pathogen sequence, the control and computing unit (13) causes the output unit (15) to output information about the pathogen whose sequence is consistent with the nucleic acid sequence. In addition, the control and computing unit (13) switches to a second phase.

[0187] In the second stage, the control and computing unit (13) checks whether the nucleic acid sequence in the growing sample is consistent with the resistance marker, the non-resistance sequence and / or the target sequence. Once the nucleic acid sequence is found to be consistent with the sequence indicating resistance, the control and computing unit (13) causes the output unit (15) to output information about resistance.

[0188] Figure 2 The steps performed by a computer are schematically and exemplarily shown in the form of a flow chart, in the working memory of which the computer program of the present invention is loaded. These steps are divided into two phases Ph1 and Ph2. Steps (201), (202), (203), (204), (205) and (206) are associated with the first phase. Steps (208), (209), (210), (211), (212), (213) and (214) are associated with the second phase. Step (207) is assigned to the first phase and produces a transition from the first phase Ph1 to the second phase Ph2. The computer starts by executing the steps in phase Ph1 and switches to phase Ph2 when a defined condition (see below) exists.

[0189] (201) The computer loads multiple pathogen sequences P from a data storage device.

[0190] (202) The computer receives a continuously growing nucleic acid sequence S.

[0191] (203) The computer checks whether the growing nucleic acid sequence S is contained in the pathogen sequence P (S∈P?).

[0192] (206) If the growing sequence S is not included in the pathogen sequence P, the computer sends a signal that causes the sequencing of the currently sequenced nucleic acid to be terminated. Alternatively, a new nucleic acid is sequenced and the computer receives the growing sequence S of the new nucleic acid.

[0193] (204) If the growing sequence S is contained in the pathogen sequence P, it is checked whether it can be considered with the help of the current information that the sequence S can be associated with a specific pathogen from P (S=P?).

[0194] (205) When the current information is insufficient to conclude that the sequence originates from a specific pathogen, continue sequencing the nucleic acid.

[0195] (207) In the case where the current information is sufficient to conclude that the sequence is derived from a specific pathogen, the pathogen is identified in the corresponding sample. Information about the pathogen can be output and the computer sends a signal, which causes the sequencing of the currently sequenced nucleic acid to be terminated. In addition, the second stage of sequencing, Ph2, is switched.

[0196] (208) The computer loads at least one resistance-indicating sequence R of the identified pathogen from a data storage device, wherein the at least one resistance-indicating sequence may include a resistance marker, a non-resistance sequence and / or a target sequence.

[0197] (209) The computer receives a continuously growing number of new nucleic acid sequences S.

[0198] (210) The computer checks whether the growing sequence S is contained in the at least one resistance-indicating sequence R (S∈R?).

[0199] (213) In the event that the growing sequence S is not contained in the sequence indicating resistance, the computer sends a signal that causes the sequencing of the currently sequenced nucleic acid to be terminated. Alternatively, a new nucleic acid is sequenced and the computer receives the growing sequence S of the new nucleic acid.

[0200] (211) If the growing sequence S is contained in the sequence R indicating resistance, it is checked whether it can be assumed with the help of the current information that the pathogen has the corresponding resistance (S=R?).

[0201] (212) If the current information is insufficient to conclude that the pathogen is resistant, continue sequencing the nucleic acid.

[0202] (214) When the current information is sufficient to conclude that the pathogen has resistance, output information about resistance.

[0203] Figure 3 Another embodiment of the system of the present invention is schematically shown. The system (10) comprises a sequencing unit (12), a control and calculation unit (13), a data storage (14) and an output unit (15).

[0204] The control and computing unit (13) and the output unit (15) are components of a standard computer system, such as a laptop. The control and computing unit (13) receives data from the sequencing unit (12), wherein these data represent a growing sequence of nucleotides or amino acids. The control and computing unit (13) is also connected to a database (14) via a network, such as the Internet, represented here by the cloud (C). It is conceivable that there are a plurality of databases. It is also conceivable that the database is a component of the computer system. The control and computing unit (13) obtains at least one pathogen sequence and, if appropriate, at least one sequence indicating resistance from the at least one database.

[0205] It is conceivable that the at least one database may also provide data on environmental conditions, data on control agents, data on plants and plant species, data on pathogens and / or similar data. It is also conceivable that one or more models for calculating the probability of the presence of a pathogen at a plant at a current point in time based on information on the plant and / or on environmental conditions are stored in the at least one database, which models may be called up by the control and calculation unit (13) and used for calculation purposes.

Claims

1. A method comprising the following steps: - Collecting samples from plants or the medium in which they grow, - identifying a pathogen in said sample, - identification of markers of resistance of said pathogen to control agents, wherein the identification of the pathogen and the identification of the marker of resistance is accomplished by sequencing individual nucleic acids or individual peptides in the sample, wherein a growing nucleotide sequence or amino acid sequence is determined during the sequencing of the individual nucleic acids or individual peptides, wherein the nucleotide sequence or amino acid sequence is compared with a reference sequence, wherein the sequencing is performed in two stages, a first stage and a second stage, ● wherein in said first stage, during the sequencing of each nucleic acid or each peptide the sequence determined respectively is compared with at least one pathogenic sequence, and ■ in the event that the determined sequence is inconsistent with the at least one pathogenic sequence, further sequencing of the nucleic acid or the peptide is interrupted, ■ In the case where the determined sequence is consistent with the pathogen sequence, the pathogen having the pathogen sequence is determined, and at least one resistance marker is determined based on the determined pathogen, and the process is switched to the second stage, - wherein in said second stage the sequence of the nucleic acids or peptides in said sample is compared with said at least one resistance marker determined and / or with a non-resistance sequence.

2. The method according to claim 1, comprising the steps of: - Identify the plants; - identifying at least one pathogen capable of infecting said plant; - for the at least one pathogen determined, determining at least one pathogen sequence; - collecting a sample from said plant or the medium in which said plant is growing, - identifying pathogens in said sample; - determining at least one resistance marker of the identified pathogen, - Identify resistance markers of identified pathogens to control agents.

3. The method according to claim 1, comprising the steps of: - Identify the plants; - determining the environmental conditions to which the plant is and / or has been exposed; - identifying at least one pathogen capable of infecting said plant; - for the at least one pathogen determined, determining at least one pathogen sequence; - collecting a sample from said plant or the medium in which said plant is growing, - identifying pathogens in said sample; - determining at least one resistance marker of the identified pathogen, - Identify resistance markers of identified pathogens to control agents.

4. The method according to claim 1, comprising the steps of: - Identify the plants; - identifying at least one pathogen capable of infecting said plant; - determining the environmental conditions to which the plant is or has been exposed, - calculating the probability of the presence of at least one pathogen in the plant at the current point in time; - comparing said probability with a predefined threshold; - determining the pathogen sequences of those determined pathogens whose probability is higher than the threshold; - collecting a sample from said plant or the medium in which said plant is growing, - identifying pathogens in said sample; - determining at least one resistance marker of the identified pathogen, - Identify resistance markers of identified pathogens to control agents.

5. The method according to any one of claims 1 to 4, wherein the step of identifying the resistance marker of the identified pathogen to the control agent comprises the following sub-steps: - comparing the nucleic acid or peptide sequence in said sample with said at least one resistance marker determined, - in the case where the resistance marker is not present in any of the nucleic acid or peptide sequences of the sample: outputting a notification indicating that no resistance marker was found for the identified pathogen, - In the case where the resistance marker appears in one or more of the sequences: outputting a notification that a resistance marker has been found for the identified pathogen, and outputting a notification regarding the resistance.

6. The method according to any one of claims 1 to 4, wherein the step of identifying the resistance marker of the identified pathogen to the control agent comprises the following sub-steps: - comparing the nucleic acid or peptide sequence in said sample with said at least one resistance marker determined, - in the case where the resistance marker does not appear in any of the nucleic acid or peptide sequences in the sample or the number of nucleic acids in which the resistance marker appears is less than a predefined minimum number: outputting a notification that no resistance marker is found for the identified pathogen, - For a sequence in which the resistance marker appears in a minimum number or in a number greater than the minimum number: outputting a notification that a resistance marker has been found for the identified pathogen, and outputting a notification regarding the resistance.

7. The method according to any one of claims 1 to 4, comprising the following steps: - Collecting samples from plants or the medium in which they grow; - identifying pathogens in said sample; - determining at least one resistance marker of the identified pathogen and determining the resistance-free sequence of the identified pathogen; - identifying a resistance marker of the identified pathogen to the control agent by comparing the nucleic acid or peptide sequence in the sample with the at least one resistance marker determined and the non-resistance sequence; -For the case where a sequence or a number of sequences greater than or equal to a minimum number has neither identity with said at least one resistance marker nor identity with said non-resistance sequence: output a notification indicating that such a pathogen has been identified in said sample, which has mutations pointing to new and / or developed resistance.

8. The method according to any one of claims 1 to 4, comprising the following steps: - Collecting samples from plants or the medium in which they grow; - identifying pathogens in said sample; - determining the target sequence of the identified pathogen; - identifying new resistance markers of the identified pathogen to the control agent by comparing the nucleic acid or peptide sequences in the sample with the target sequence; - In the case where one sequence or a number of sequences greater than or equal to a minimum number show a variation relative to said target sequence: outputting a notification indicating that such a pathogen with a variation was identified in said sample, which has a variation pointing to new and / or developed resistance.

9. The method according to any one of claims 1 to 4, comprising the following steps: - Collecting samples from plants or the medium in which they grow; - identifying pathogens in said sample; - determining at least one resistance marker of the identified pathogen and determining the resistance-free sequence of the identified pathogen; - comparing the nucleic acid or peptide sequences in the sample with the at least one resistance marker determined and with the non-resistance sequences; - Determine the frequency Q of finding a nucleic acid or peptide having a sequence corresponding to the resistance marker in the sample R , determine the frequency Q of nucleic acids or peptides found in the sample that have sequences that match the resistance-free sequence NR , calculate the determined frequency Q R and Q NR and comparing the ratio with a predefined minimum value; - In the case where the ratio is greater than or equal to the minimum value: outputting a notification indicating that a resistance marker is found for the identified pathogen, and outputting a notification regarding the resistance.

10. The method according to any one of claims 1 to 4, wherein the sample of the plant is one or more leaves of the plant.

11. The method according to any one of claims 1 to 4, wherein the plant is in main growth stage 1 or 2 or 3 to 6 according to the BBCH scale.

12. The method according to any one of claims 1 to 4, wherein the pathogenic sequence is a DNA nucleotide sequence of a fungus, bacteria or virus.

13. The method according to any one of claims 1 to 4, further comprising the following steps: - creating and / or updating a pathogen card, wherein in the pathogen card a location is registered, wherein at the location a sample of the plant was taken, wherein for the location an indication is given whether a pathogen has been identified in the plant, and in case a pathogen has been identified in the plant, an indication is given whether the pathogen is resistant to a control agent, and - Output the pathogen card.

14. The method according to claim 13, wherein outputting the pathogen card is displaying the pathogen card on a display screen and / or outputting the pathogen card to a printer and / or transmitting the pathogen card to a device for automatically executing measures to prevent and treat the identified pathogens.

15. The method according to any one of claims 1 to 4, further comprising the following steps: - identification of control agents to be used against the identified pathogens, taking into account any resistance that may have been identified, - taking measures for controlling pathogens of said plants using the identified control agents.

16. A system comprising: - Sequencing unit, - control and computing unit, and at least one data storage device in which at least one pathogen sequence and at least one resistance marker are stored for at least one pathogen, wherein the sequencing unit is configured to sequence nucleic acids or peptides in a sample of a plant or a medium in which the plant grows, and thereby determine the sequence of nucleotides or amino acids in the sample, wherein the sequencing unit is configured to transmit the respectively determined sequence to the control and computing unit during the determination of the sequence, The control and computing unit is configured to compare the sequence of each transmitted nucleic acid or peptide with at least one pathogen sequence in a first stage, and ● in the event that the transmitted sequence is inconsistent with the at least one pathogen sequence, causing the sequencing unit to interrupt further sequencing of the corresponding nucleic acid or the corresponding peptide, ● In the case where the transmitted sequence is consistent with the pathogen sequence, the pathogen having the pathogen sequence is determined, and at least one resistance marker is determined using the determined pathogen, and the process is switched to the second stage, The control and computing unit is configured to compare the sequence of the nucleic acid or peptide in the sample with at least one determined resistance marker and / or non-resistance sequence in the second stage.

17. A computer-readable (storage) medium comprising instructions which, when executed by a computer, cause the computer to perform the following steps: - for a plurality of nucleic acids or peptides: receiving from a sequencing unit a continuously increasing sequence of nucleotides or amino acids in the corresponding nucleic acid or the corresponding peptide, - in a first phase during the reception of the growing arrangement: detecting whether said arrangement of nucleotides or amino acids is contained one or more times in at least one pathogenic sequence, - in the event that the arrangement is not contained in the pathogenic sequence: transmitting a signal to the sequencing unit in order to interrupt further sequencing of the nucleic acid or peptide, - In the case where the arrangement is consistent with the pathogen sequence: determining the pathogen having the pathogen sequence, and determining at least one reference marker, and starting the second stage, - In said second stage: said arrangement of nucleotides or amino acids is compared with said at least one reference marker and / or resistance-free sequence.

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