Crop disease management

Abstract

A method for supporting crop disease management is disclosed. The method comprising profiling a species community associated with a location by, subjecting an environmental sample of the location to DNA sequencing, identifying and quantifying at least one fungal and / or bacterial species of the species community present in the environmental sample based on the DNA sequencing, and identifying and / or quantifying the presence of at least one genetic variance of the identified species based on the DNA sequencing. Based on the profiled species community, determining an effective crop disease management strategy.

Description

[0001] CROP DISEASE MANAGEMENT

[0002] Cross Reference to Related Applications

[0003] This application claims the benefit of the following applications filed by the same applicant, the entire contents of which are incorporated herein by this reference for all purposes: PCT / EP2023 / 051107 (WO 2023 / 139112), filed on 18 January 2023, and entitled “RESISTANCE AND VIRULENCE DIAGNOSTICS"; and PCT application, PCT / EP2023 / 051106 (WO 2023 / 139111 ), filed on 18 January 2023, entitled “CROP PATHOGEN MONITORING AND POPULATION PREDICTION”.

[0004] Technical Field

[0005] The present disclosure relates to methods for supporting crop disease management.

[0006] Background

[0007] Plant pathogens may cause reduced plant growth, plant assimilation or even impairment of the vital functions of the plant resulting in a reduction or loss of plant productivity. Examples of plant pathogens include viruses, bacteria, nematodes, insects, and fungi, and they are capable of reproducing within or on its host spreading from one plant to another. Symptoms of a plant disease include change in color, shape, or function of the plant, for example the fungi Zymoseptoria tritici causes pale brown to greenish-grey oval lesions in the leaves of wheat. In case of moderate to high disease pressure, it can cause significant loss in plant productivity.

[0008] All species of plants, cultivated and wild, can be susceptible to disease. For farmers, plant pathogens may reduce yield and quality of agricultural production causing substantial economic loss. Use of disease control strategies reduce the negative effects of pathogens, but if not used properly can lead to resistant pathogen populations (e.g. to fungicides or to cultivars). As such, there has been extensive research and development in disease control strategies and resistance management for controlling plant diseases including crop rotation, appropriate planting date and plant density, control of field moisture, breeding plants with greater resistance to pathogens, and application of pesticides or biopesticide.

[0009] Disease control strategies and resistance management are typically based on experience and historical data from previous seasons.

[0010] Summary

[0011] According to an aspect of the present disclosure, a method for supporting crop disease management is provided. The method comprising profiling a species community associated with a location by subjecting an environmental sample of the location to DNA sequencing, identifying and quantifying at least one fungal and / or bacterial species of the species community present in the environmental sample based on the DNA sequencing, and identifying and / or quantifying the presence of at least one genetic variance of the identified species based on the DNA sequencing. Thereafter, based on the profiled species community, determining an effective crop disease management strategy.

[0012] The effective crop disease management strategy may be for promoting plant health and may be considered to include any method or process for managing crop diseases, for example, disease control strategies including determining adaptation to a disease control strategy. Disease control strategies may comprise management of a species or species community of fungi and / or bacteria, for example, by application of a plant protection product such as a pesticide, using biological control such as biological control agents, timing or intervals of application of a plant protection product / biological control, and / or selection of an optimal or particular resistant crop in order to control the development, reproduction and / or viability of at least one species in the species community whilst minimizing resistance development. Advantageously, by using the example methods described herein, disease control strategies can be tailored to manage the identified species taking into account any resistance and / or genetic variance(s) and quantities rather than employing disease control strategies based on historical data of previous seasons.

[0013] Another example of determining an effective crop disease management strategy may comprise determining an indication of disease risk to a plant. To explain further, by determining an indication or level of disease risks to a plant at a location, an outbreak of the disease can be controlled or even prevented. Further examples of crop disease management strategy are described herein.

[0014] In one example, a plurality of species in a species community associated with a location may be profiled such that a plurality of fungal and / or bacterial species of the species community are identified and quantified including any genetic variance of the identified and quantified species.

[0015] The method may comprise profiling the species community at time intervals and / or at a plurality of locations.

[0016] The method may comprise determining a resistance profile to a disease control strategy and / or virulence profile of the identified species based on the identified and / or quantified presence of at least one genetic variance of the identified species, and profiling a species community based on the resistance profile and / or virulence profile.

[0017] In one example, the operation of subjecting an environmental sample of the location to DNA sequencing may comprise a first DNA sequencing process for identifying and quantifying at least one fungal and / or bacterial species of the 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 variance of the identified species based on the DNA sequencing.

[0018] The DNA sequencing as described herein may be configured to provide a plurality of genetic variances and associated quantity of the identified species in a single read or experiment.

[0019] In one example, subjecting the sample to DNA sequencing may comprise using a sequencer capable of sequencing at least 200 base pairs in a single read or experiment.

[0020] The sample to DNA sequencing may comprise using a sequencer configured to process at least 100 reads in a single experiment.

[0021] In one example, subjecting the sample to DNA sequencing may comprise using nanopore sequencing technology or single-molecule real-time sequencing.

[0022] The environmental sample may be obtained from air, soil, plant material and / or water.

[0023] In one example, environmental samples may be obtained from a plurality of locations, and each environmental sample is subjected to the DNA sequencing so as to profile the species community across the plurality of locations. The method may further comprise interpolating information from the profiled species community of the plurality of locations in order to estimate a profile of a species community in a new location. The method may also or alternatively comprise forecasting a change in the profiled species community.

[0024] In one example, the method may further comprise alerting a user of a determined indication of disease risk to a plant. In another example, the method may further comprise recommending a user of a determined adaptation to a disease control strategy in the location.

[0025] The environmental sample may comprise at least seed material and the method may further comprise profiling the species community present in the seed material so as to support seed certification.

[0026] In one example, the method may further comprise identifying allergens and / or toxin producing species based on the profiled species community.

[0027] According to another aspect of the present disclosure, a method for supporting seed certification is provided. The method comprises profiling a species community associated with a sample by subjecting a sample to DNA sequencing, identifying and quantifying at least one fungal and / or bacterial species of the species community present in the sample based on the DNA sequencing, and identifying and / or quantifying the presence of at least one genetic variance of the identified species based on the DNA sequencing. The method further comprises using the profiled species community to certify seeds associated with the sample. The sample may be a seed sample relating to at least one seed, and the profiled species community is used for certifying seeds associated with the seed sample. Additionally or alternatively, the sample may be from a crop production field for producing seeds, and the profiled species community is used for certifying seeds produced from the crop production field.

[0028] Brief description of the drawings

[0029] For a better understanding of the present disclosure, and to show more clearly how it may be carried into effect, reference will now be made, by way of example, to the following drawings in which:

[0030] Figure 1 is a flow diagram of an example method of the present disclosure;

[0031] Figure 2 is a flow diagram of an example method of the present disclosure;

[0032] Figure 3 is a flow diagram of an example method of the present disclosure; and

[0033] Figure 4 is a flow diagram of an example method of the present disclosure.

[0034] Detailed Description

[0035] In the following description, for purposes of explanation, numerous specific details of certain examples are set forth. Reference in the specification to "an example" or similar language means that a particular feature, structure, or characteristic described in connection with the example is included in at least that one example, but not necessarily in other examples.

[0036] Crop disease management including disease control strategies and resistance management of fungi / bacteria need to be carefully devised and implemented to protect the plant and to avoid fast selection of resistance of pathogens to pesticides leading to ineffectiveness and diminishing options available for controlling a disease. However, crop disease management today is limited in that plant diseases are typically identified visually after breakout at which point the plant may already be damaged, and disease control strategies and resistance management are based on experience and occurrences of diseases in previous seasons rather than current or real-time plant health meaning that unexpected pathogens, ratio of pathogens, and genetic variances of pathogens are difficult to control.

[0037] To manage crop diseases, a holistic understanding of the species community in realtime or near real-time needs to be obtained. Examples of the present disclosure provide methods for supporting crop disease management by profiling a species community associated with a location. The profiling comprises DNA sequencing an environmental sample so as to identify and quantify any fungal and / or bacterial species of the species community, and also to identify and quantify the presence of any genetic variances of the identified species. This may be done in real-time, near real-time, within 48 hours or a few days as explained in more detail below so that a current understanding of the species community can be obtained. By knowing the species present in a species community and their associated genetic variances including respective quantity and frequency at a specific location and at a given time, crop disease management can be supported within the current season whilst also considering resistance development of any species to pesticides or cultivars. For example, disease control strategies can be adapted to the profiled species community to control a disease and / or manage resistance of species to pesticides. Another example includes determining risks of disease outbreak based on the profiled species community even before the disease has taken hold. Furthermore, the simultaneous profiling of multiple species in a species community can inform about the most effective and sustainable disease management strategy for the crop, in other words provide integrated crop management.

[0038] Terminologies used herein will now be described.

[0039] The terms “crop” and “plant” are interchangeably used in the present disclosure and they refer to any type of plant that is cultivated or growing naturally under different conditions like open field or greenhouses. For example, a crop or a plant may be a cereal, fruit, vegetable, tree, flower, grass and / or bush.

[0040] As used herein, a field, or a field of crop(s) or plant(s) refers to an area or location where crop grows naturally or is cultivated under different conditions like protected (e.g. greenhouse) or open field conditions.

[0041] “Crop disease management strategy” as used herein refers to any method or process for managing crop disease, including but not limited to disease control strategies including determining adaptation to a disease control strategy, disease risk management such as determining an indication of disease risk to a plant, and / or resistance management.

[0042] “Disease control strategy”, “disease control measure” or “treatment program” is to be understood as management of a species or species community of fungi and / or bacteria, for example, by application of a plant protection product such as a pesticide, using biological control such as biological control agents , timing or intervals of application of a plant protection product / biological control, and / or selection of an optimal or particular resistant crop in order to control the development, reproduction and / or viability of at least one species in the species community whilst minimizing resistance development.

[0043] An “environmental sample” as used herein may be an aerial, soil, water, plant and / or seed sample.

[0044] A “genetic variance”, “gene variance” or a “variance of a species” is to be understood as a mutation in the DNA sequence different from the wild type sequence. For example, in a genetic variance at least one nucleotide in a DNA sequence has been changed, deleted or inserted.

[0045] “Genotype” refers to the unique combination of multiple genetic variances, gene variances or variances of a plant pathogen. If multiple independent genetic variations can be brought together into different unique combinations, then each of these unique combinations might express a different sensitivity or virulence profile.

[0046] As used herein “plant pathogen”, also known as “crop pathogen” or just “pathogen”, refers to a living organism negatively affecting the structure, development and / or vital functions of a plant. For example, a plant pathogen may be a virus, viroids, fungi, oomycetes, nematodes, bacteria, phytoplasm, protozoa, algae, insect and / or parasitic plants. The term plant pathogen should be understood to mean an individual organism or several organisms of the plant pathogen.

[0047] “Location” as used herein may refer to a specific position, location, area or region.

[0048] “Species” used herein is to be understood as a group of organisms that can reproduce with one another in nature and produce fertile offspring. The use of “species” herein is to be understood as both a single group of organisms and a plurality of groups of organisms. Furthermore, “species” may include pathogenic, neutral and / or beneficial species to a crop.

[0049] “Species community” used herein is to be understood as a population or group of species.

[0050] “Resistance”, “fungicide resistance”, or “pesticide resistance”, refers to the ability of a plant pathogen to survive the exposure to pesticides designed to control it. In other words, any decreased, partial or reduced sensitivity of a species to a disease control agent is considered as “resistance”.

[0051] As used herein, “resistance profile” refers to the ability of a plant pathogen or a population of a plant pathogen to overcome and survive the exposure to a pesticide, fungicide, a pesticide class or fungicide class as defined by FRAC, or other chemical or biological agent used for controlling development, viability and reproduction of the plant pathogen.

[0052] “Resistance management” as used herein refers to strategies to delay resistance of plant pathogens to plant control products.

[0053] As used herein, “virulence profile” refers to the ability of a plant pathogen or a population of a plant pathogen to infect and cause damage to a host such as a plant. The host may have a type of built in resistance to the pathogen (resistant traits), for example it may be a cultivar having host resistance.

[0054] Referring now to the figures, figure 1 illustrates an example method 100 for supporting crop disease management by profiling a species community of a location. The example method 100 may comprise collecting an environmental sample 101 of a location, profiling a species community of the environmental sample by DNA sequencing 110 and determining an effective crop disease management strategy105. The example method 100 operations will now be described in more detail.

[0055] As mentioned above, the example method 100 may comprise collecting an environmental sample 101. The environmental sample may be air, soil, water, plant tissue and / or seed. In cases where the environmental sample is air, spore traps may be used for detecting fungal spores, as well as airborne bacteria, in the air. The spore traps may be stationary or mobile, and they may be located in, near fields or in non-agronomic locations (e.g. a city) to sample local air, or high up above ground to sample regional airflow. One example of a spore trap that may be used is a high volume spore trap that actively pumps a large volume of air each minute into a device and deposits spores / bacteria into collection tubes. This type of sampler may be located above ground, for example, 10 to 15 meters above ground rather than directly in a field in order to sample regional airflow. Another example of a spore trap that may be used is a passive or light active pumping spore trap that processes a much smaller volume of air compared to the high volume spore trap. Also the light active pumping spore trap collects and deposits spores into tubes, filters or cassettes. This type of spore trap may be located on ground level, for example 1 to 2 meters above ground, in or near a field, so as to sample local air. Other spore traps may be used to collect environmental samples such as mobile spore traps located on a vehicle, such as a car, tractor or drone. The spore traps can be configured to collect samples at specific times and days. Furthermore, the spore traps described herein are not limited to collecting only fungal spores but can be used for collecting other airborne microorganisms in the environment including bacteria.

[0056] Soil and water samples may be collected at a location of interest such as a field of crops. In some examples, the soil and / or water samples may be collected far away from a location of interest so as to get an understanding of the species community across a wider location, area or region.

[0057] Other types of environmental samples may be used too, and in some examples, plant material including plant tissue and / or seed material may be collected. In some examples, just a single type of sample is used, e.g. aerial, and in other examples different types of samples are used (e.g. aerial and soil) to obtain a more informative understanding of the species community in a location.

[0058] In some examples, a network of sample collecting nodes or sensors may be used, for example, a network of spore traps as described above. These nodes can be configured to collect aerial samples at particular time intervals, for example, the spore trap may be sampling throughout the year with defined collection windows (e.g. 1 day, 2, days, 3 days, 1 week...) for the full day or defined collection intervals (e.g. from 6 AM to 6 PM). The network of nodes may be spread out across a field, larger area or region, including across a country or several countries. In some examples, the network of sample collecting nodes are not limited to spore traps but can be of different types of nodes for collecting different types of samples, e.g. aerial, soil and water.

[0059] In cases where environmental samples are collected from a plurality of locations, and profiled as described in more detail below, a more comprehensive understanding of the species communities associated with these locations can be obtained. For example, it can be determined how species communities differ between locations and also how they change over time. This understanding can further support crop disease management in that information from the profiled species community of the plurality of locations can be interpolated in order to estimate a profile of a species community in a new location. In other examples, the profiled species communities associated with the plurality of locations can be used for forecasting a change in a species community of one of the plurality of locations or a new location.

[0060] For the sake of clarity, where the following description of example method 100 refers to an “environmental sample”, it should be understood to also apply to a plurality of environmental samples that may be from a plurality of locations as described above.

[0061] Returning now to figure 1 , after the environmental sample has been collected, the species community in that environmental sample is profiled 110. This is achieved by subjecting the sample to DNA sequencing 102, and based on the DNA sequencing, determining and quantifying at least one fungal and / or bacterial species of the species community, and also identifying and quantifying the presence of any or at least one genetic variance of the identified species 103.

[0062] It should be understood that the profiled species community may include beneficial and / or pathogenic organisms, both of which can contribute to improved crop disease management. In particular, identifying beneficial species can be indicative of soil health and can be effective as biological control agents, and by identifying pathogenic species and their variances crop disease management can be improved by identifying appropriate disease control agents, both chemical and biological, whilst considering resistance risks.

[0063] In some examples, a plurality, or even all, of the species in a species community of an environmental sample(s) are profiled which provides a comprehensive or holistic understanding of the species community. This provides many advantages including the possibility of managing multiple pathogens or pests simultaneously rather than an individual pathogen or pest, contributing to effective integrated crop management.

[0064] In one example, collection of environmental samples 101 at a single location or a plurality of locations and profiling of the species community by DNA sequencing as described herein may be carried out at time intervals. For example, operations 101 , 102 and 103 of example method 100 may be repeated at various time intervals and / or at various locations. This enables changes in the species community in terms of species, genetic variation and / or quantity to be monitored over time and / or across a plurality of locations. Based on this, various aspects can be determined including spatio-temporal quantification of a disease, spatiotemporal quantification of a species community’s sensitivity to mode of action of pesticides, and / or spatio-temporal quantification of a species community’s virulence. Furthermore, monitoring changes in species communities over time and / or location enable agronomic decisions to be optimised, the agronomic decisions relating to disease control strategies including type of application of a plant protection product such as a pesticide, applying biological control agents, timing or intervals of application of a plant protection product, biological control agents and / or selection of an optimal or particular resistant crop.

[0065] To further profile the species community, example method 100 may further comprise determining 104 a resistance profile to a disease control strategy and / or virulence profile of the identified species and associated genetic variances as determined in operations 102 and 103. This is indicated in figure 1 with dashed lines to illustrate that this operation is optional. Generating a resistance profile may comprise associating the at least one genetic variance and corresponding quantity with a level of resistance to a pesticide and / or a group of pesticides, and / or generating a virulence profile may comprise associating the at least one genetic variance and corresponding quantity with a level of virulence. Using the resistance profile and / or virulence profile for further profiling a species community of an environmental sample enables tailored disease control strategies to be made to manage the development, reproduction and / or viability of the species whilst minimizing resistance development.

[0066] As described above, based on the profiled species community, the example method 100 may further comprise determining an effective crop disease management strategy 105, see figure 1. The effective crop disease management strategy is for promoting plant health and may be considered to include any method or process for managing crop diseases, for example, disease control strategies including determining adaptation to a disease control strategy. Disease control strategies may comprise management of a species or species community of fungi and / or bacteria, for example, by application of a plant protection product such as a pesticide, using biological control such as biological control agents, timing or intervals of application of a plant protection product / biological control, and / or selection of an optimal or particular resistant crop in order to control the development, reproduction and / or viability of at least one species in the species community whilst minimizing resistance development. Advantageously, by using the example methods described herein, disease control strategies can be tailored to manage the identified species taking into account any resistance and / or genetic variance(s) and quantities rather than employing disease control strategies based on historical data of previous seasons. Moreover, in one example, the adapted disease control strategies can be sent or recommended to a user, authority, farmer, service provider or the like, who can then implement the disease control strategies accordingly.

[0067] Another example of determining an effective crop disease management strategy may comprise determining an indication of disease risk to a plant. To explain further, by determining an indication or level of disease risks to a plant at a location, an outbreak of the disease can be controlled or even prevented. For example, an indication of disease risk to a plant can be alerted to a user, authority, farmer, service provider or the like, who can then make an optimal agronomic decision on how to manage the disease risk.

[0068] In yet another example, determining an effective crop disease management strategy may be in association with supporting seed certification. In this example, the environmental sample may be a seed material, for example, a whole seed(s) and / or part of a seed(s). By profiling the species community associated with the seed material, the process of seed certification can be supported. In another example, the environmental sample is from a crop production field for producing seeds, and the profiled species community is used for certifying seeds produced from the crop production field.

[0069] In a further example, the example method 100 may further comprise identifying allergens and / or toxin producing species based on the profiled species community. For example, the profiled species community can be used for identifying allergens such as sooty mould wheat, other fungal allergens, bacteria, and / or the profiled species community can be used for identifying species that produce toxins. These allergens and / or toxins can then be managed through crop disease management as described herein, for example, crops can be treated and / or a user can be alerted and / or provided with recommendations on how to manage the identified allergen and / or toxin producing species.

[0070] In addition to the aforementioned advantages, example method 100 provides the advantage of significant reduction in time for producing and analysing the DNA sequencing data compared to other known methods where identifying and analysing species and variants typically takes weeks at which point the data is no longer a representation of the disease progression in real-time. The example method 100 enables the quantitative and qualitative profiling of a species community in a location to be determined in real-time, close to real-time, within 48 hours or a few days such that the profiled species community is a representation of the current species community present in the location of the environmental sample. This provides opportunities to have a clear and more detailed understanding of the possible disease risks, tailored disease control strategies whilst also considering resistance management that are relevant to the current profiled species community at a specific location. As such, one example of use of example method 100 is to identify the most effective type of pesticide(s) for controlling the current, profiled species. In contrast, crop disease management strategies known from the prior art are based on previous seasons and experience, theoretical mathematical model without considering scouting and profiling the complexity of factors challenging a crop, and so are less effective as they estimate the current species community.

[0071] As described herein, an environmental sample is subjected to DNA sequencing in order to profile a species community associated with a location, see for example methods 100, 300 and 400. The DNA sequencing of the present disclosure may be a single DNA sequencing process or it may be two separate DNA sequencing processes. In the case of two separate DNA sequencing process, a first DNA sequencing process is for identifying and quantifying at least one fungal and / or bacterial species of the species community present in the environmental sample. Once the species have been identified and quantified a second DNA sequencing process may be performed for identifying and / or quantifying the presence of at least one genetic variance of the identified species. An example of preparation and DNA sequencing method 200 that may be used in relation to methods 100, 300 and 400 will now be described with reference to figure 2.

[0072] The example method 200 may comprise using a sequencer capable of high throughput sequencing of minimum of at least 200, 300, 400 or 500 base pairs. For example, Oxford Nanopore sequencing Technologies such as MinlON, GridlON or PromethlON or PacBio Sequel Systems implementing single-molecule real-time sequencing as provided by PACBIO may offer the required capability. These technologies may be referred to as third generation sequencer and they provide a high throughput combined with larger sequenced genetic regions from a few hundred base pairs up to 10,000 base pairs or more. The technology provided by Oxford Nanopore Technologies (Oxford, United Kingdom) comprises flow cells which contain an array of tiny holes referred to as nanopores that are embedded in an electroresistant membrane. Each nanopore corresponds to its own electrode connected to a channel and sensor chip, which measures the electric current that flows through the nanopore. When a molecule passes through a nanopore, the current is disrupted to produce a characteristic ‘squiggle’ or a current intensity value. The squiggle is then decoded using base calling algorithms to determine the DNA. Base calling is a computational process of translating the squiggle into DNA sequences. Specific bioinformatics pipelines may be combined to enable quantification of genetic variants in pathogen populations.

[0073] Before subjecting an environmental sample to DNA sequence, the example method 200 may comprise sample information collection 202 which identifies where and when the sample was collected, meteorological and / or crop parameters. Thereafter, the sample processing 203 may be performed on the sample which comprises bulking the collected specimens into a single sample such that it represents of the species community of the location. Thereafter, DNA may be extracted 204 to create a DNA sample. Genes of interest may then be amplified from the DNA sample by single step or multiplex PCR using specific primers 205. Thereafter, DNA barcoding 206 may be performed to prepare a library of samples (e.g. multiple sites, collection windows or collection intervals) so that species and / or genetic variants can be identified after the samples have been sequenced. The next operation may be sequencing preparation 207 followed by the operation of DNA sequencing 208 using a third-generation sequencer as described herein. The DNA sequencing 208 determines the nucleic acid sequence and this data is then analysed 209 by comparing the determined DNA sequencing with a reference DNA sequence (or a database) from the prepared library. By doing so, the species community is profiled including identifying and quantifying at least one fungal and / or bacterial species of the species community present in the environmental sample, and identifying and / or quantifying the presence of at least one genetic variance of the identified species. As described herein, in some examples method 200 can be performed twice, once for identifying and quantifying at least one fungal and / or bacterial species of the species community present in the environmental sample, and a second time for identifying and / or quantifying the presence of at least one genetic variance of the identified species.

[0074] Furthermore, based on the results of the profiling, the environmental sample can be further profiled by generating a resistance profile and / or virulence profile as described herein.

[0075] Quantification of species and the presence of genetic variants can be expressed as a frequency and this may be determined as a percentage of the whole population of a species or the whole species community present in the environmental sample. The frequency can also be expressed in alternative ways, for example, heatmaps, boxplots, or pie charts associated with or without maps. The performance of a pesticide is expected to be more affected in situations of high frequency of specific resistant individuals within a pathogen population. The adaptation of a population to a fungicide and / or a fungicide class is the result of the population composition, in which each variant might be associated to different resistance factors. An index of adaptation can be obtained integrating the percentage of the single genotypes to their resistance factors. Similarly, populations with high frequency of individuals with high virulence determinates are expected to infect resistant crops more efficiently. As for the adaptation index, an index of virulence for a population can be inferred from the frequency of the single genotypes and their associated phenotype.

[0076] Another example method 300 for supporting crop disease management will now be described with reference to figure 3. This example method 300 may comprise any of the features mentioned in relation to example method 100 and associated advantages. The example method 300 comprises profiling a species community associated with a location 310 by subjecting an environmental sample of the location to DNA sequencing 302, identifying and quantifying at least one fungal and / or bacterial species of the species community present in the environmental sample based on the DNA sequencing, and identifying and / or quantifying the presence of at least one genetic variance of the identified species based on the DNA sequencing 303. The example method 300 further comprises, based on the profiled species community, determining an effective crop disease management strategy 305. By profiling a species community in an environmental sample, part or even the whole fungal and / or bacterial community can be understood. The profiled species community may beneficial and / or pathogenic organisms. Example method 300 enables a crop disease management strategy to be improved or optimised as explained in more detail in relation to figure 1 , however as an example, disease control strategies can be tailored to manage the identified species taking into account any genetic variance(s) and quantities, and / or resistance to pesticides. Furthermore, beneficial organisms in terms of soil health and / or as biological control agents can also be profiled from example method 300 and be used for supporting a crop disease management strategy.

[0077] In one example, a plurality of species in a species community associated with a location is profiled such that a plurality of fungal and / or bacterial species of the species community are identified and quantified including any genetic variance of the identified and quantified species. By profiling a plurality, or even all, of the species in a species community of an environmental sample, a comprehensive understanding of the species community can be obtained. This provides many advantages including the possibility of managing multiple pathogens simultaneously rather than an individual pathogen thereby providing an integrated crop management solution.

[0078] In one example, the example method 300 may comprise profiling the species community at time intervals and / or at a plurality of locations, similar to example method 100.

[0079] Example method 300 may further comprise determining a resistance profile to a disease control strategy and / or virulence profile of the identified species based on the identified and / or quantified presence of at least one genetic variance of the identified species, and profiling a species community based on the resistance profile and / or virulence profile.

[0080] The DNA sequencing may comprise the method described with reference to figure 2.

[0081] In one example, the operation of subjecting an environmental sample of the location to DNA sequencing 302 comprises a first DNA sequencing process for identifying and quantifying at least one fungal and / or bacterial species of the 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 variance of the identified species based on the DNA sequencing. In another example, there is only a single DNA sequencing process for operation 302.

[0082] The DNA sequencing in operation 302 may be configured to provide a plurality of genetic variances and associated quantity of the identified species in a single read or experiment. Furthermore, subjecting the sample to DNA sequencing may comprise using a sequencer capable of sequencing at least 200 base pairs in a single read or experiment. In one example, subjecting the sample to DNA sequencing comprises using a sequencer configured to process at least 100 reads in a single read or experiment. Furthermore, subjecting the sample to DNA sequencing may comprise using nanopore sequencing technology or single-molecule real-time sequencing, examples of which are described herein.

[0083] The environmental sample may be obtained from air, soil, plant material and / or water, wherein plant material may be plant tissue and / or seed material (including a whole seed(s) and / or parts of a seed (s)).

[0084] In one example, a plurality of environmental samples are obtained from a plurality of locations, and each environmental sample is subjected to the DNA sequencing so as to profile the species community across the plurality of locations. The profiled species can be used for interpolating information to estimate a profile of a species community in a new location and / or to forecasting a change in the profiled species community.

[0085] Based on the profiled species community, an indication of disease risk to a plant can be determined and the example method 300 may further comprise alerting a user, such as an authority, farmer, service provider or the like, of said indication. In another example, an adaptation to a disease control strategy in a location may be determined and example method 300 may further comprise recommending the adaptation to a user such as an authority, farmer, service provider or the like.

[0086] In yet another example, determining an effective crop disease management strategy may be in association with supporting seed certification. In this example, the environmental sample may be a seed material, for example, a whole seed(s) and / or part of a seed(s). By profiling the species community associated with the seed material, the process of seed certification can be supported. In another example, the environmental sample is from a crop production field for producing seeds, and the profiled species community is used for certifying seeds produced from the crop production field.

[0087] In a further example, the example method 100 may further comprise identifying allergens and / or toxin producing species based on the profiled species community. For example, the profiled species community can be used for identifying allergens such as sooty mould wheat, other fungal allergens, bacteria, and / or the profiled species community can be used for identifying species that produce toxins. These allergens and / or toxins can then be managed through crop disease management as described herein, for example, crops can be treated and / or a user can be alerted and / or provided with recommendations on how to manage the identified allergen and / or toxin producing species.

[0088] As explained above, example method 300 may comprise any of the features of example method 100 and associated advantages.

[0089] Another example method 400 for supporting seed certification will now be described with reference to figure 4. This example method 400 may comprise any of the features mentioned in relation to example methods 100 and 300 and associated advantages.

[0090] Figure 4 illustrates the example method 400 for supporting seed certification and the method comprises profiling a species community associated with a sample 410 by subjecting a sample to DNA sequencing 402, and identifying and quantifying at least one fungal and / or bacterial species of the species community present in the sample based on the DNA sequencing, and identifying and / or quantifying the presence of at least one genetic variance of the identified species based on the DNA sequencing 403. Thereafter, the example method comprises using the profiled species community to certify seeds associated with the sample 405. In example method 400, the sample may be a seed sample relating to at least one seed, and the profiled species community is used for certifying seeds associated with the seed sample. In another example, the sample is from a crop production field for producing seeds, and the profiled species community is used for certifying seeds produced from the crop production field.

[0091] The methods described herein are not limited to a particular plant or crop, growth condition, geographical region, plant pathogen or plant protection product such as a pesticide. The methods can be applied to any cultivated or wild crop where resistance development needs to be managed or monitored. For example, the methods can be applied to other patho- systems such as Phakopsora pachyrhizi, Corynespora cassiicola, Diaporthe and Colletotrichum spp as example of soybean pathogenic species, Pyrenophora teres, Rhynchosporium commune and Ramularia collo-cygni in barley, Alternaria solani and Phytophthora infestans in potato and tomatoes, Pseudoperonospora cubensis in cucurbits, Plasmopara viticola, Uncinula necatorand Botrytis in grapes, Peronospora destructor'm onion, Plasmopara halstedii in sunflowers.

[0092] It should be noted that the above-mentioned examples illustrate rather than limit the invention, and that those skilled in the art will be able to design 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 a claim, “a” or “an” does not exclude a plurality. Any reference signs in the claims shall not be construed so as to limit their scope.

Claims

CLAIMS1 . A method for supporting crop disease management, the method comprising; profiling a species community associated with a location by; subjecting an environmental sample of the location to DNA sequencing, identifying and quantifying at least one fungal and / or bacterial species of the species community present in the environmental sample based on the DNA sequencing, identifying and / or quantifying the presence of at least one genetic variance of the identified species based on the DNA sequencing, and based on the profiled species community, determining an effective crop disease management strategy.

2. A method according to claim 1 , wherein a plurality of species in a species community associated with a location is profiled such that a plurality of fungal and / or bacterial species of the species community are identified and quantified including any genetic variance of the identified and quantified species.

3. A method according to any preceding claim, comprising profiling the species community at time intervals and / or at a plurality of locations.

4. A method according to any preceding claim, further comprising determining a resistance profile to a disease control strategy and / or virulence profile of the identified species based on the identified and / or quantified presence of at least one genetic variance of the identified species, and profiling a species community based on the resistance profile and / or virulence profile.

5. A method according to any preceding claim, wherein subjecting an environmental sample of the location to DNA sequencing comprises a first DNA sequencing process for identifying and quantifying at least one fungal and / or bacterial species of the 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 variance of the identified species based on the DNA sequencing.

6. A method according to any preceding claim, wherein the DNA sequencing is configured to provide a plurality of genetic variances and associated quantity of the identified species in a single read.

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

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

9. A method according to any preceding claim, wherein subjecting the environmental sample to DNA sequencing comprises using nanopore sequencing technology or singlemolecule real-time sequencing.

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

11. A method according to any preceding claim, wherein environmental samples are obtained from a plurality of locations, and each environmental sample is subjected to the DNA sequencing so as to profile the species community across the plurality of locations.

12. A method according to claim 11 , further comprising interpolating information from the profiled species community of the plurality of locations in order to estimate a profile of a species community in a new location.

13. A method according to claim 11 , further comprising forecasting a change in the profiled species community.

14. A method according to any preceding claim, further comprises alerting a user of a determined indication of disease risk to a plant.

15. A method according to any preceding claim, further comprising recommending a user of a determined adaptation to a disease control strategy in the location.

16. A method according to any preceding claim, wherein the environmental sample comprises at least seed material and the method comprises profiling the species community present in the seed material so as to support seed certification.

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

18. A method for supporting seed certification, the method comprising; profiling a species community associated with a sample by; subjecting a sample to DNA sequencing, identifying and quantifying at least one fungal and / or bacterial species of the species community present in the sample based on the DNA sequencing, identifying and / or quantifying the presence of at least one genetic variance of the identified species based on the DNA sequencing, and using the profiled species community to certify seeds associated with the sample.

19. A method according to claim 18, wherein the sample is a seed sample relating to at least one seed, and the profiled species community is used for certifying seeds associated with the seed sample.

20. A method according to claim 18, wherein the sample is from a crop production field for producing seeds, and the profiled species community is used for certifying seeds produced from the crop production field.

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