Crop disease management

Abstract

A method for supporting crop disease management is disclosed. The method comprises analyzing a population of species associated with a location by DNA sequencing an environmental sample at the location, identifying and quantifying at least one fungal and / or bacterial species of a population of species present in the environmental sample based on the DNA sequencing, and identifying and / or quantifying the presence of at least one genetic variation in the identified species based on the DNA sequencing. And determining an effective crop disease management strategy based on the analyzed species community.

Description

Cross-reference to related applications

[0001] This application claims the benefit of the following applications filed by the same applicant, the entire contents of which are incorporated herein by reference for all purposes: PCT / EP2023 / 051107 (WO 2023 / 139112) entitled “RESISTANCE AND VIRULENCE DIAGNOSTICS” filed on 18 January 2023; and PCT application PCT / EP2023 / 051106 (WO 2023 / 139111) entitled “CROP PATHOGEN MONITORING AND POPULATION PREDICTION” filed on 18 January 2023. Technical Field

[0002] This disclosure relates to methods for supporting crop disease management. Background Technology

[0003] Plant pathogens can cause reduced plant growth, impaired plant assimilation, or even damage to vital plant functions, leading to decreased or lost plant productivity. Examples of plant pathogens include viruses, bacteria, nematodes, insects, and fungi, and they are capable of multiplying within or on their host, spreading from one plant to another. Symptoms of plant diseases include changes in plant color, shape, or function; for example, the fungus *Zymoseptoria tritici* causes pale brown to greenish-gray oval lesions in wheat leaves. Under moderate to high disease stress, it can lead to significant losses in plant productivity.

[0004] All plant species, whether cultivated or wild, are susceptible to diseases. For farmers, plant pathogens can reduce agricultural yields and quality, resulting in significant economic losses. Disease control strategies can mitigate the negative impacts of pathogens, but improper application can lead to resistance development in pathogen populations (e.g., resistance to fungicides or cultivars). Therefore, extensive research and development have been undertaken into disease control strategies and resistance management for plant diseases, including crop rotation, appropriate planting dates and density, field water management, breeding plants with greater resistance to pathogens, and the application of pesticides or biopesticides.

[0005] Disease control strategies and resistance management are usually based on experience and historical data from previous planting seasons. Summary of the Invention

[0006] According to one aspect of this disclosure, a method for supporting crop disease management is provided. The method includes analyzing a location-related species community by: performing DNA sequencing on an environmental sample at that location; identifying and quantifying, based on the DNA sequencing, at least one fungal and / or bacterial species of the species community present in the environmental sample; and identifying and / or quantifying, based on the DNA sequencing, the presence of at least one genetic variation in the identified species. Subsequently, based on the analyzed species community, effective crop disease management strategies are determined.

[0007] Effective crop disease management strategies can be used to promote plant health and can be considered to include any methods or processes used to manage crop diseases, such as disease control strategies (including determining adjustments to disease control strategies). Disease control strategies can include managing species or species communities of fungi and / or bacteria by, for example, applying plant protection products such as pesticides, using biological controls such as biocontrol agents, setting the timing or interval for applying plant protection products / biological controls, and / or selecting optimal or specific resistant crops to control the development, reproduction, and / or viability of at least one species in the species community while minimizing resistance development. Advantageously, by using the example methods described herein, disease control strategies can be tailored to manage identified species (taking into account any resistance and / or genetic variation and its quantity, rather than adopting disease control strategies based on historical data from previous planting seasons).

[0008] Another example of identifying effective crop disease management strategies can be determining indicators of plant disease risk. To explain further, by determining indicators or levels of plant disease risk at a given location, disease outbreaks can be controlled or even prevented. This article describes further examples of crop disease management strategies.

[0009] In one instance, multiple species in a location-related species community can be analyzed to identify and quantify multiple fungal and / or bacterial species in that community, including any genetic variation in the identified and quantified species.

[0010] This method may include analyzing species communities at time intervals and / or at multiple locations.

[0011] The method may include determining the resistance and / or virulence characteristics of the identified species to disease control strategies based on the identification and / or quantification of at least one genetic variation in the identified species, and analyzing the species community based on the resistance and / or virulence characteristics.

[0012] In one instance, the operation of DNA sequencing of environmental samples at a location may include a first DNA sequencing process that identifies and quantifies at least one fungal and / or bacterial species of a species community present in the environmental sample based on DNA sequencing, and a second DNA sequencing process that identifies and / or quantifies the presence of at least one genetic variation in the identified species based on DNA sequencing.

[0013] As described in this article, DNA sequencing can be configured to provide multiple genetic variations and associated quantities of an identified species in a single read or experiment.

[0014] In one instance, DNA sequencing of a sample may include using a sequencer capable of sequencing at least 200 base pairs in a single read or experiment.

[0015] DNA sequencing of a sample may include using a sequencer configured to process at least 100 reads in a single experiment.

[0016] In one instance, DNA sequencing of a sample can include using nanopore sequencing technology or single-molecule real-time sequencing.

[0017] Environmental samples can be obtained from air, soil, plant material, and / or water.

[0018] In one example, environmental samples can be obtained from multiple locations, and DNA sequencing can be performed on each sample to analyze species communities at multiple locations. The method may further include interpolating information from the analyzed species communities at multiple locations to estimate the characteristics of species communities at new locations. The method may also, or alternatively, include predicting changes in the analyzed species communities.

[0019] In one instance, the method may further include instructing the user to be aware of identified plant disease risks. In another instance, the method may further include recommending to the user adjustments to identified location-specific disease control strategies.

[0020] Environmental samples may contain at least seed material, and the method may further include analyzing the species community present in the seed material to support seed authentication.

[0021] In one instance, the method may further include identifying allergens and / or toxin-producing species based on the analyzed species community.

[0022] According to another aspect of this disclosure, a method for supporting seed authentication is provided. The method includes analyzing a species community associated with a sample by: performing DNA sequencing on the sample; identifying and quantifying, based on the DNA sequencing, at least one fungal and / or bacterial species present in the species community of the sample; and identifying and / or quantifying, based on the DNA sequencing, the presence of at least one genetic variation in the identified species. The method further includes using the analyzed species community to authenticate seeds associated with the sample.

[0023] The sample may be a seed sample involving at least one seed, and the analyzed species community is used to identify the seed associated with that seed sample. Alternatively or alternatively, the sample may be from a crop production field used for seed production, and the analyzed species community is used to identify the seeds produced from that crop production field. Attached Figure Description

[0024] To better understand this disclosure, and to more clearly show how this disclosure can be implemented, reference will now be made to the following figures by way of example, wherein:

[0025] Figure 1 This is a flowchart of an example method disclosed herein;

[0026] Figure 2 This is a flowchart of an example method disclosed herein;

[0027] Figure 3 This is a flowchart of an example method disclosed herein; and

[0028] Figure 4 This is a flowchart of an example method disclosed herein. Detailed Implementation

[0029] In the following description, for illustrative purposes, numerous specific details of certain instances are set forth. References to “instance” or similar language in this specification mean that a particular feature, structure, or characteristic described in connection with that instance is included in at least one instance, but not necessarily in others.

[0030] Crop disease management (including fungal / bacterial disease control strategies and resistance management) needs to be carefully developed and implemented to protect plants and prevent pathogens from rapidly developing resistance to pesticides (which renders pesticides ineffective and reduces the options available for disease control). However, current crop disease management has limitations because plant diseases are often only visually identifiable after an outbreak, by which time plants may already be damaged; and disease control strategies and resistance management are based on experience from previous growing seasons and disease occurrences, rather than current or real-time plant health conditions. This means it is difficult to control unexpected pathogens, pathogen ratios, and pathogen genetic variation.

[0031] To manage crop diseases, a holistic understanding of the species community is required in real-time or near real-time. Examples disclosed herein provide methods for supporting crop disease management by analyzing location-related species communities. The analysis includes DNA sequencing of environmental samples to identify and quantify any fungal and / or bacterial species within the species community, and also to identify and quantify the presence of any genetic variation in the identified species. As explained in more detail below, this can be accomplished in real-time, near real-time, within 48 hours or days, to provide a current understanding of the species community. By understanding the species present in the species community and their associated genetic variation (including the corresponding quantity and frequency at a specific location and time), crop disease management can be supported for the current season, while also taking into account the development of resistance in any species to pesticides or cultivars. For example, disease control strategies can be tailored to the analyzed species community to control diseases and / or manage species resistance to pesticides. Another example includes determining the risk of disease outbreaks based on the analyzed species community even before the disease becomes prevalent. Furthermore, simultaneous analysis of multiple species within a species community can provide information on the most effective and sustainable disease management strategies for crops; in other words, it provides integrated crop management.

[0032] The terminology used in this article will now be described.

[0033] The terms “crop” and “plant” are used interchangeably in this disclosure, and they refer to any type of plant that is cultivated or grows naturally under different conditions, such as open fields or greenhouses. For example, a crop or plant can be a cereal, fruit, vegetable, tree, flower, grass, and / or shrub.

[0034] As used in this article, field, or field of crops or plants, refers to an area or location where crops naturally grow or are cultivated under different conditions, such as protected (e.g., greenhouse) or open field conditions.

[0035] As used herein, “crop disease management strategy” means any method or process used to manage crop diseases, including but not limited to disease control strategies (including determining adjustments to disease control strategies), disease risk management (such as determining indicators of plant disease risk), and / or resistance management.

[0036] "Disease control strategy", "disease control measures" or "treatment procedures" should be understood as managing fungal and / or bacterial species or species communities by, for example, applying plant protection products such as pesticides, using biological controls such as biocontrol agents, setting the time or interval for applying plant protection products / biological controls, and / or selecting optimal or specific resistant crops in order to control the development, reproduction and / or viability of at least one species in the species community while minimizing the development of resistance.

[0037] As used in this article, “environmental sample” can be an air, soil, water, plant and / or seed sample.

[0038] "Genetic variation," "genetic variation," or "variation" in a species should be understood as a mutation in a DNA sequence that differs from the wild-type sequence. For example, in genetic variation, at least one nucleotide in the DNA sequence has been altered, deleted, or inserted.

[0039] A "genotype" refers to a unique combination of multiple genetic variations, gene variations, or variations in a plant pathogen. If multiple independent genetic variations can be grouped into different unique combinations, each of these unique combinations can express different susceptibility or virulence characteristics.

[0040] As used herein, a "plant pathogen" (also known as a "crop pathogen" or simply a "pathogen") refers to a living organism that negatively affects the structure, development, and / or important functions of a plant. For example, plant pathogens can be viruses, viroids, fungi, oomycetes, nematodes, bacteria, plant protoplasm, protozoa, algae, insects, and / or parasitic plants. The term plant pathogen should be understood to refer to an individual organism or several organisms of a plant pathogen.

[0041] As used in this article, “location” can refer to a specific direction, location, region, or area.

[0042] The term "species" as used herein should be understood as a group of organisms that can reproduce with each other in nature and produce fertile offspring. The use of "species" in this document should be understood as both a single group of organisms and multiple groups of organisms. Furthermore, "species" can include species that are pathogenic, neutral, and / or beneficial to crops.

[0043] The term "species community" used in this article should be understood as a group or set of species.

[0044] "Resistance," "fungicide resistance," or "pesticide resistance" refers to the ability of a plant pathogen to survive exposure to pesticides designed to control it. In other words, any reduced, partial, or diminished sensitivity of a species to a disease control agent is considered "resistance."

[0045] As used herein, “resistance” refers to the ability of a plant pathogen or population of plant pathogens to overcome and survive exposure to a pest control agent, fungicide, pest control agent class or fungicide class as defined by FRAC, or other chemical or biological agent used to control the development, viability and reproduction of plant pathogens.

[0046] As used in this article, “resistance management” refers to strategies that delay the development of resistance to plant control products by plant pathogens.

[0047] As used in this article, "virulence trait" refers to the ability of a plant pathogen or a group of plant pathogens to infect a host (such as a plant) and cause damage to the host. A host may possess some intrinsic resistance to a pathogen (resistance trait), for example, it may be a cultivar with host resistance.

[0048] Now refer to the attached diagram, Figure 1 An example method 100 is shown for supporting crop disease management by analyzing the species community of a location. Example method 100 may include collecting environmental samples 101 at the location, analyzing the species community of the environmental samples 110 by DNA sequencing, and determining effective crop disease management strategies 105. The operation of example method 100 will now be described in more detail.

[0049] As described above, example method 100 may include collecting environmental samples 101. Environmental samples may be air, soil, water, plant tissue, and / or seeds. In the case where the environmental sample is air, a spore trap can be used to detect airborne fungal spores and airborne bacteria. Spore traps can be fixed or mobile; they can be located in, near, or in non-agricultural locations (e.g., urban areas) to sample local air, or located high above ground level to sample regional airflow. One example of a spore trap that can be used is a high-capacity spore trap that actively pumps a large volume of air into the device per minute, depositing spores / bacteria into a collection tube. This type of sampler can be located above ground level, for example, 10 to 15 meters above ground, rather than directly in the field, to sample regional airflow. Another example of a spore trap that can be used is a passive or lightly active pumping spore trap, which handles a much smaller volume of air compared to a high-capacity spore trap. In addition, lightly active pumping spore traps collect spores and deposit them into tubes, filters, or boxes. This type of spore trap can be located at ground level, for example, 1 to 2 meters above the ground, i.e., in or near fields, to sample local air. Other spore traps can also be used to collect environmental samples, such as mobile spore traps mounted on vehicles (such as cars, tractors, or drones). Spore traps can be configured to collect samples at specific times and dates. Furthermore, the spore traps described herein are not limited to collecting only fungal spores; they can also be used to collect other airborne microorganisms from the environment, including bacteria.

[0050] Soil and water samples can be collected at the target location (such as a crop field). In some instances, soil and / or water samples can be collected at a distance from the target location to understand species communities in a wider location, region, or area.

[0051] Other types of environmental samples may also be used, and in some instances, plant material, including plant tissue and / or seed material, may be collected. In some instances, only a single type of sample (e.g., air) is used, while in others, different types of samples (e.g., air and soil) are used to obtain a more comprehensive understanding of the species community at the location.

[0052] In some instances, networks of sample collection nodes or sensors can be used, such as a network of spore traps as described above. These nodes can be configured to collect air samples at specific time intervals; for example, a spore trap can sample year-round, with collection windows set (e.g., 1 day, 2 days, 3 days, 1 week) for 24-hour sampling or collection time intervals set (e.g., from 6 a.m. to 6 p.m.). The node network can be distributed across fields, larger areas, or regions, including spanning one or several countries. In some instances, the network of sample collection nodes is not limited to spore traps but can also consist of different types of nodes used to collect different types of samples (e.g., air, soil, and water).

[0053] By collecting environmental samples from multiple locations and analyzing them, as described in more detail below, a more comprehensive understanding of the species communities associated with those locations can be obtained. For example, differences in species communities across different locations and how they change over time can be determined. This understanding can further support crop disease management because information from the analyzed species communities at multiple locations can be interpolated to estimate the characteristics of the species community at a new location. In other instances, analyzed species communities associated with multiple locations can be used to predict changes in species communities at one of multiple locations or at a new location.

[0054] For clarity, when the following description of example method 100 refers to “environmental sample”, it should be understood that this also applies to multiple environmental samples that may come from multiple locations as described above.

[0055] Now back Figure 1 After collecting environmental samples, the species communities in the environmental samples are analyzed 110. This is achieved by: performing DNA sequencing on the samples 102, and based on the DNA sequencing, identifying and quantifying at least one fungal and / or bacterial species in the species community, and also identifying and quantifying the presence of any or at least one genetic variation in the identified species (103).

[0056] It should be understood that the analyzed species communities may include beneficial and / or pathogenic organisms, both of which contribute to improved crop disease management. In particular, identifying beneficial species can indicate soil health and these beneficial species can be effective as biological control agents, and identifying pathogenic species and their variants can improve crop disease management by identifying appropriate chemical and biological disease control agents while taking into account resistance risks.

[0057] In some instances, the analysis of multiple or even all species within a species community of one or more environmental samples provides a comprehensive or holistic understanding of that species community. This offers numerous advantages, including the possibility of managing multiple pathogens or pests simultaneously rather than a single pathogen or pest, which contributes to effective integrated crop management.

[0058] In one example, environmental samples 101 can be collected at single or multiple locations at time intervals, and the species community can be analyzed by DNA sequencing as described herein. For example, operations 101, 102, and 103 of example method 100 can be repeated at different time intervals and / or at different locations. This enables the monitoring of species community changes over time and / or at multiple locations in terms of species, genetic variation, and / or quantity. Based on this, aspects including spatiotemporal quantification of diseases, spatiotemporal quantification of the species community's sensitivity to the mode of action of pest control agents, and / or spatiotemporal quantification of species community toxicity can be determined. Furthermore, monitoring species community changes over time and / or location can optimize agronomic decisions related to disease control strategies, including the type of plant protection products (such as pest control agents), the application of biocontrol agents, the timing or interval of application of plant protection products and biocontrol agents, and / or the selection of optimal or specific resistant crops.

[0059] To further analyze the species community, example method 100 may further include determining the resistance characteristics of identified species and associated genetic variations to disease control strategies and / or the virulence characteristics 104 of identified species and associated genetic variations, as determined in operations 102 and 103. This is in Figure 1 The dashed lines indicate that this operation is optional. Generating resistance traits may include associating at least one genetic variation and its corresponding amount with resistance levels to a pest control agent and / or a group of pest control agents, and / or generating toxicity traits may include associating at least one genetic variation and its corresponding amount with toxicity levels. Further analysis of species communities in environmental samples using resistance and / or toxicity traits enables the development of customized disease control strategies to manage species development, reproduction, and / or viability while minimizing resistance development.

[0060] As described above, based on the analyzed species community, example method 100 may further include identifying effective crop disease management strategies 105, see [link to example method]. Figure 1 Effective crop disease management strategies are used to promote plant health and can be considered to include any methods or processes for managing crop diseases, such as disease control strategies (including determining adjustments to disease control strategies). Disease control strategies can include managing species or communities of fungi and / or bacteria by, for example, applying plant protection products such as pesticides, using biological controls such as biocontrol agents, setting the timing or interval for applying plant protection products / biological controls, and / or selecting optimal or specific resistant crops to control the development, reproduction, and / or viability of at least one species in the species community while minimizing resistance development. Advantageously, by using the example methods described herein, disease control strategies can be tailored to manage identified species (taking into account any resistance and / or genetic variation and its quantity, rather than adopting disease control strategies based on historical data from previous planting seasons). Furthermore, in one instance, the adjusted disease control strategy can be sent or recommended to users, authorities, farmers, service providers, etc., who can then implement the disease control strategy accordingly.

[0061] Another example of establishing effective crop disease management strategies can be identifying indicators of plant disease risk. To explain further, by determining indicators or levels of plant disease risk at a given location, outbreaks can be controlled or even prevented. For example, indicators of plant disease risk can be provided to users, authorities, farmers, service providers, etc., so they can make optimal agronomic decisions on how to manage that risk.

[0062] In yet another instance, identifying effective crop disease management strategies can be relevant to supporting seed certification. In this instance, environmental samples can be seed material, such as one or more whole seeds and / or portions of one or more seeds. Analyzing the species community associated with the seed material can support the seed certification process. In yet another instance, environmental samples come from crop production fields used for seed production, and the analyzed species community is used to certify seeds produced from those crop production fields.

[0063] In another instance, example method 100 may further include identifying allergens and / or toxin-producing species based on analyzed species communities. For example, the analyzed species community may be used to identify allergens such as wheat sooty mold, other fungal allergens, bacteria, and / or the analyzed species community may be used to identify toxin-producing species. These allergens and / or toxins can then be managed through crop disease management as described herein, for example, by treating crops, and / or by alerting users and / or providing advice on how to manage the identified allergens and / or toxin-producing species.

[0064] In addition to the advantages mentioned above, Example Method 100 offers the advantage of significantly reduced time for generating and analyzing DNA sequencing data compared to other known methods that typically require weeks to identify and analyze species and variants (at which point the data no longer represent real-time disease progression). Example Method 100 enables quantitative and qualitative analysis of species communities at a location in real-time or near real-time, within 48 hours or days, allowing the analyzed species communities to represent the current species communities present at the environmental sample location. This provides opportunities for a clearer and more detailed understanding of potential disease risks and tailored disease control strategies, while also taking into account resistance management associated with the currently analyzed species communities at a specific location. Thus, one example of using Example Method 100 is identifying the most effective types of one or more pest control agents for controlling the currently analyzed species. In contrast, known crop disease management strategies in the prior art are based on past growing seasons and experience, theoretical mathematical models, and do not take into account the complexity of investigating and analyzing factors affecting crops, and are therefore less effective because they estimate the current species community.

[0065] As described herein, DNA sequencing is performed on environmental samples to analyze location-associated species communities, see example methods 100, 300, and 400. The DNA sequencing disclosed herein can be a single DNA sequencing procedure, or it can be two separate DNA sequencing procedures. In the case of two separate DNA sequencing procedures, the first DNA sequencing procedure is used to identify and quantify at least one fungal and / or bacterial species present in the species community in the environmental sample. Once the species have been identified and quantified, a second DNA sequencing procedure can be performed to identify and / or quantify the presence of at least one genetic variation in the identified species. Reference will now be made to... Figure 2 Examples of preparation methods and DNA sequencing method 200 that can be used for methods 100, 300, and 400 are described.

[0066] Example method 200 may include the use of a sequencer capable of high-throughput sequencing of at least 200, 300, 400, or 500 base pairs. For example, Oxford nanopore sequencing technologies (such as MinION, GridION, or PromethION) or the PacBio Sequel system, which performs real-time sequencing of single molecules, provided by PACBIO, can provide the required capabilities. These technologies may be referred to as third-generation sequencers, and they offer high throughput when combined with larger sequencing genetic regions ranging from a few hundred base pairs to 10,000 base pairs or more. Technologies provided by Oxford Nanopore Technologies (Oxford, UK) include flow cells containing an array of micropores called nanopores embedded in a resistive membrane. Each nanopore corresponds to its own electrode connected to a channel and a sensor chip, which measures the current flowing through the nanopore. As molecules pass through the nanopore, the current is perturbed to produce a characteristic “curve,” or current intensity value. The curve is then decoded using a base calling algorithm to identify DNA. Base recognition is a computational process that translates curves into DNA sequences. Specific bioinformatics pipelines can be combined to enable the quantification of genetic variants within pathogen populations.

[0067] Prior to DNA sequencing of environmental samples, example method 200 may include sample information collection 202, which determines where and when the samples were collected, meteorological and / or crop parameters. Subsequently, sample processing 203 may be performed, including merging the collected samples into a single sample such that it represents the species community of the location. DNA may then be extracted 204 to produce a DNA sample. The target gene can then be amplified from the DNA sample using specific primers via single-step or multiplex PCR 205. DNA barcoding 206 may then be performed to prepare a sample library (e.g., multiple sites, collection windows, or collection intervals) so that species and / or genetic variants can be identified after sequencing of the samples. The next step may be sequencing preparation 207, followed by DNA sequencing 208 using a third-generation sequencer as described herein. DNA sequencing 208 determines nucleic acid sequences, and this data is then analyzed 209 by comparing the determined DNA sequencing data with reference DNA sequences (or databases) from the prepared library. By doing so, species communities are analyzed, including identifying and quantifying at least one fungal and / or bacterial species present in the species community in the environmental sample, and identifying and / or quantifying the presence of at least one genetic variation in the identified species. As described herein, in some instances, method 200 may be performed twice: once to identify and quantify at least one fungal and / or bacterial species present in the species community in the environmental sample, and a second time to identify and / or quantify the presence of at least one genetic variation in the identified species.

[0068] Furthermore, based on the results of the analysis, environmental samples can be further analyzed by generating resistance and / or toxicity characteristics as described in this paper.

[0069] The quantification of the presence of species and genetic variants can be expressed as frequency, which can be determined as the percentage of the entire species population or the entire species community present in an environmental sample. This frequency can also be expressed in alternative ways, such as heatmaps, box plots, or pie charts with or without association with a spectrum. In cases where a particular resistant individual is present at a high frequency within a pathogen population, the performance of the expected pest control agent is likely to be more significantly affected. Population adaptation to fungicides and / or fungicide classes is a consequence of population composition, where each variant may be associated with different resistance factors. An adaptation index can be obtained by integrating the percentage of a single genotype with its resistance factor. Similarly, populations with a high frequency of individuals possessing high virulence determinants are expected to infect resistant crops more effectively. Similar to the adaptation index, the virulence index of a population can be inferred from the frequency of a single genotype and its associated phenotype.

[0070] Now refer to Figure 3Another example method 300 for supporting crop disease management is described. Example method 300 may include any features and related advantages mentioned in connection with example method 100.

[0071] Example method 300 includes analyzing a location-related species community 310 by: performing DNA sequencing on an environmental sample at that location 302; identifying and quantifying at least one fungal and / or bacterial species present in the species community in the environmental sample based on the DNA sequencing; and identifying and / or quantifying the presence of at least one genetic variation in the identified species based on the DNA sequencing 303. Example method 300 further includes: determining effective crop disease management strategies based on the analyzed species community 305. By analyzing the species community in the environmental sample, part or even the entire fungal and / or bacterial community can be understood. The analyzed species community may contain beneficial and / or pathogenic organisms. Example method 300 enables the improvement or optimization of crop disease management strategies, such as those related to... Figure 1 A more detailed explanation, however, is provided as an example, that disease control strategies can be tailored to manage identified species (taking into account any genetic variation as well as quantity and / or resistance to pest control agents). Furthermore, beneficial organisms in terms of soil health and / or biological control agents can be analyzed using example method 300 and used to support crop disease management strategies.

[0072] In one instance, analysis of multiple species within a location-related species community identifies and quantifies various fungal and / or bacterial species within that community, including any genetic variation in identified and quantified species. By analyzing multiple or even all species within a species community in an environmental sample, a comprehensive understanding of that community can be obtained. This offers numerous advantages, including the possibility of managing multiple pathogens simultaneously rather than a single pathogen, thus providing integrated crop management solutions.

[0073] In one instance, similar to example method 100, example method 300 may include analyzing species communities by time intervals and / or at multiple locations.

[0074] Example method 300 may further include determining the resistance and / or virulence characteristics of an identified species to disease control strategies based on the identification and / or quantification of at least one genetic variation in the identified species, and analyzing the species community based on the resistance and / or virulence characteristics.

[0075] DNA sequencing may include a reference Figure 2 The method described.

[0076] In one example, operation 302, which involves DNA sequencing of an environmental sample at a location, includes a first DNA sequencing process that identifies and quantifies at least one fungal and / or bacterial species present in the species community of the environmental sample based on the DNA sequencing, and a second DNA sequencing process that identifies and / or quantifies the presence of at least one genetic variation in the identified species based on the DNA sequencing. In another example, operation 302 employs only a single DNA sequencing process.

[0077] The DNA sequencing in operation 302 can be configured to provide multiple genetic variations and associated quantities of an identified species in a single read or experiment. Furthermore, DNA sequencing of a sample can include the use of a sequencer capable of sequencing at least 200 base pairs in a single read or experiment. In one example, DNA sequencing of a sample includes the use of a sequencer configured to process at least 100 reads in a single read or experiment. Additionally, DNA sequencing of a sample includes the use of nanopore sequencing technology or single-molecule real-time sequencing, examples of which are described herein.

[0078] Environmental samples can be obtained from air, soil, plant material and / or water, wherein the plant material can be plant tissue and / or seed material (including one or more whole seeds and / or a portion of one or more seeds).

[0079] In one instance, multiple environmental samples were obtained from multiple locations, and DNA sequencing was performed on each sample to analyze species communities at multiple locations. The analyzed species can be used to interpolate information to estimate the characteristics of species communities at new locations and / or predict changes in the analyzed species communities.

[0080] Based on the analyzed species community, indicators of plant disease risk can be identified, and example method 300 may further include alerting users (such as authorities, farmers, service providers, etc.) to the indicators. In another instance, adjustments to disease control strategies for a location can be determined, and example method 300 may further include recommending such adjustments to users (such as authorities, farmers, service providers, etc.).

[0081] In yet another instance, identifying effective crop disease management strategies can be relevant to supporting seed certification. In this instance, environmental samples can be seed material, such as one or more whole seeds and / or portions of one or more seeds. Analyzing the species community associated with the seed material can support the seed certification process. In yet another instance, environmental samples come from crop production fields used for seed production, and the analyzed species community is used to certify seeds produced from those crop production fields.

[0082] In another instance, example method 100 may further include identifying allergens and / or toxin-producing species based on analyzed species communities. For example, the analyzed species community may be used to identify allergens such as wheat sooty mold, other fungal allergens, bacteria, and / or the analyzed species community may be used to identify toxin-producing species. These allergens and / or toxins can then be managed through crop disease management as described herein, for example, by treating crops, and / or by alerting users and / or providing advice on how to manage the identified allergens and / or toxin-producing species.

[0083] As described above, example method 300 may include any features and related advantages of example method 100.

[0084] Now refer to Figure 4 Describe another example method 400 for supporting seed authentication. Example method 400 may include any features and related advantages mentioned in connection with example methods 100 and 300.

[0085] Figure 4 An example method 400 for supporting seed authentication is illustrated, comprising analyzing a species community associated with a sample 410 by: performing DNA sequencing on the sample 402, identifying and quantifying at least one fungal and / or bacterial species present in the species community in the sample based on the DNA sequencing, and identifying and / or quantifying the presence of at least one genetic variation in the identified species based on the DNA sequencing 403. The example method then includes using the analyzed species community to authenticate seeds associated with the sample 405. In example method 400, the sample may be a seed sample involving at least one seed, and the analyzed species community is used to authenticate seeds associated with that seed sample. In another example, the sample comes from a crop production field used for seed production, and the analyzed species community is used to authenticate seeds produced from that crop production field.

[0086] The methods described herein are not limited to specific plants or crops, growing conditions, geographic regions, plant pathogens, or plant protection products (such as pesticides). These methods can be applied to any cultivated or wild crop that requires management or monitoring of resistance development. For example, these methods can be applied to other pathosystems, such as *Phakopsora pachyrhizi*, *Corynespora cassiicola*, *Diaporthe*, and *Colletotrichum* species in soybean pathogens; *Pyrenophorateres*, *Rhynchosporium commune*, and *Ramularia collo-cygni* in barley; *Alternaria solani* and *Phytophthora infestans* in potatoes and tomatoes; *Pseudoperonospora cubensis* in gourds; *Plasmopara viticola*, *Uncinula necator*, and *Botrytis* in grapes; and *Peronospora* in onions. destructor), and sunflower downy mildew (Plasmopara halstedii) in sunflowers.

[0087] It should be noted that the above examples are for illustrative purposes only and not for limiting the invention, and those skilled in the art will be able to devise many alternative examples without departing from the scope of the appended claims. The word "comprising" does not exclude the presence of elements or steps other than those listed in the claims, and "a / an" does not exclude multiple elements or steps. Any reference marks in the claims should not be construed as limiting their scope.

Claims

1. A method for supporting crop disease management, the method comprising: The following analysis was conducted on location-related species communities: DNA sequencing was performed on environmental samples from the aforementioned location. Based on the DNA sequencing, at least one fungal and / or bacterial species present in the species community of the environmental sample was identified and quantified. Based on the DNA sequencing, the presence of at least one genetic variation in the identified species can be determined and / or quantified. as well as Based on the analyzed species communities, effective crop disease management strategies were identified.

2. The method of claim 1, wherein multiple species in a location-related species community are analyzed to identify and quantify multiple fungal and / or bacterial species in the species community, including any genetic variation of the identified and quantified species.

3. The method according to any of the preceding claims, wherein the method comprises analyzing the species community at time intervals and / or at multiple locations.

4. The method according to any of the preceding claims, the method further comprising determining the resistance and / or virulence characteristics of the identified species to disease control strategies based on the identification and / or quantification of at least one genetic variation in the identified species, and analyzing the species community based on the resistance and / or virulence characteristics.

5. The method according to any of the preceding claims, wherein DNA sequencing of the environmental sample at the location comprises a first DNA sequencing process for identifying and quantifying at least one fungal and / or bacterial species of a species community present in the environmental sample based on the DNA sequencing, and a second DNA sequencing process for identifying and / or quantifying the presence of at least one genetic variation in the identified species based on the DNA sequencing.

6. The method according to any of the preceding claims, wherein the DNA sequencing is configured to provide multiple genetic variations and associated quantities of the identified species in a single read.

7. The method according to any of the preceding claims, wherein DNA sequencing of the environmental sample comprises using a sequencer capable of sequencing at least 200 base pairs in a single read.

8. The method according to any of the preceding claims, wherein DNA sequencing of the environmental sample comprises using a sequencer configured to process at least 100 reads in a single experiment.

9. The method according to any of the preceding claims, wherein DNA sequencing of the environmental sample comprises using nanopore sequencing technology or single-molecule real-time sequencing.

10. The method according to any of the preceding claims, wherein the environmental sample is obtained from air, soil, plant material and / or water.

11. The method according to any of the preceding claims, wherein environmental samples are obtained from multiple locations, and the DNA of each environmental sample is sequenced to analyze the species communities at the multiple locations.

12. The method of claim 11, wherein the step of the method includes interpolating information of the analyzed species communities at the plurality of locations to estimate the characteristics of the species communities at the new locations.

13. The method of claim 11, wherein the step of the method includes predicting changes in the analyzed species community.

14. The method according to any of the preceding claims, wherein the method step includes instructing the user to be alerted to an identified risk of plant disease.

15. The method according to any preceding claim, the method further comprising recommending to the user an adjusted, determined disease control strategy for the location.

16. The method according to any preceding claim, wherein the environmental sample comprises at least seed material, and the method includes analyzing the species community present in the seed material to support seed certification.

17. The method according to any one of claims 1 to 15, the method further comprising identifying allergens and / or toxin-producing species based on the analyzed species community.

18. A method for supporting seed authentication, the method comprising: The following analysis was conducted on the species communities associated with the samples: DNA sequencing was performed on the sample. Based on the DNA sequencing, at least one fungal and / or bacterial species present in the species community of the sample were identified and quantified. Based on the DNA sequencing, the presence of at least one genetic variation in the identified species can be determined and / or quantified. as well as The analyzed species communities were used to identify the seeds associated with the samples.

19. The method of claim 18, wherein the sample is a seed sample involving at least one seed, and the analyzed species community is used to identify the seed associated with the seed sample.

20. The method of claim 18, wherein the sample is from a crop production field used for seed production, and the analyzed species community is used to certify seeds produced from the crop production field.

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