Systems and methods for collecting bioaerosols

Abstract

An apparatus for collecting bioaerosols is provided, the apparatus having a cassette including a mesh made of electrostatically charged fibers held in tension between a pair of fittings that support the collection medium in an extended position to expose a collection surface for collecting bioaerosols, the cassette being replaceably inserted into a wind vane that directs airflow over the collection surface of the cassette to collect bioaerosols.

Description

[Technical field]

[0001] The present disclosure relates generally to the field of agricultural research, including systems and methods for collecting and analyzing bioaerosols. [Background technology]

[0002] Plant diseases are one of the major causes of crop loss, leading to economic losses, food shortages, and loss of viable crops for future reproduction. Pathogens are one of the three drivers of crop disease, the other two being host susceptibility and environmental conditions.

[0003] Pesticides are applied to crops to combat plant diseases caused by pathogens, but application of pesticides is typically based on grower experience and, where available, consideration of local model predictions based on environmental factors such as weather. [Prior art document] [Patent documents] [Patent Document 1] International Publication No. 2013 / 123500 Summary of the Invention [Problem to be solved by the invention]

[0004] Thus, there remains a need for improved systems, devices, and methods for gathering pathogen information to inform pesticide use decisions. [Means for solving the problem]

[0005] In one aspect, a passive particle collection device and system is provided for passively collecting bioaerosols, such as pathogens and spores, without the use of an electrically powered pump.

[0006] In one aspect, an improved passive collection device is provided that is easy to use not only for researchers but also for farmers and growers, and is inexpensive to manufacture, allowing farmers and growers to install the device on their individual fields and collect localized data.

[0007] In another aspect, a replaceable cassette for collecting bioaerosols is provided.

[0008] In another aspect, a pathogen collection system is provided having a cassette and a wind vane apparatus.

[0009] In another aspect, a method is provided for monitoring crops by collecting pathogens using the cassettes and collection systems described herein and detecting the presence and / or absence of target bioaerosols containing the pathogens.

[0010] Many additional features and combinations of the embodiments described herein will be apparent to those of ordinary skill in the art after reading this disclosure.

[0011] In this regard, before describing at least one embodiment in detail, it is to be understood that the embodiments are not limited in their application to the details of construction and the arrangement of components set forth in the following description or illustrated in the drawings, and it is to be understood that the phraseology and terminology used herein is for the purpose of description and should not be regarded as limiting.

[0012] Embodiments of devices, apparatus, and methods are generally described with reference to the drawings. [Brief description of the drawings]

[0013] [Figure 1] FIG. 1 is a perspective view of a cassette for collecting bioaerosols. [Diagram 2] FIG. 2 is a side view of the cassette of FIG. 1; [Diagram 3] FIG. 3 is a front view of the cassette of FIG. 2. [Figure 4] A perspective view of a wind vane. The arrow indicates the direction of airflow (wind direction). [Diagram 5] FIG. 1 is a first perspective view of a wind vane with a cassette attached; [Figure 6] FIG. 6 is a second perspective view of FIG. 5. [Figure 7] FIG. 1 is a flow diagram illustrating the steps involved in providing a pesticide application decision.

[0014] Passive collection of particulates from air streams using passive collectors to capture bioaerosols containing potential pathogens has many advantages over currently existing devices (called volumetric collectors) that actively draw air over a medium using mechanical pumps. Volumetric spore trap collection is used in a wide range of applications for epidemiological, health, and safety settings, but has only been used on a limited scale in agriculture, primarily on a research basis, because commercially available technology is costly and difficult to use. However, volumetric collectors are expensive and require regular maintenance as well as a power supply such as a generator, which is cumbersome when volumetric collectors are installed on agricultural land and vulnerable to weather.

[0015] Passive collection devices do not require expensive components and are easy to install throughout the farm because they do not require a power source. At the same time, passive collection devices draw in less air than those driven by mechanical pumps, allowing less particulate matter, such as pathogens, to pass through. Therefore, improved pathogen collection devices and sensitive sample analysis methods are needed to optimize passive collection devices.

[0016] [Bioaerosol collection] One existing system uses an indoor air sampler and a cassette and includes a slide for identification under a microscope. It provides a short-term "snapshot" of the sample, capturing it for as little as 5-15 minutes, during which spores may not be present in the air. (See Canadian Patent No. 2969282, the entire contents of which are incorporated herein by reference.) Currently, the system uses microscopic ID, which is fairly insensitive and relies on the training and skill of the analyst performing the sampling. The system also lacks robustness and is not designed for other bioaerosols.

[0017] Other existing collection devices include the Roto® rods, which have a sticky adhesive on the rod and are messy, difficult to use and not very robust, and the Burkhard®, which is a very expensive and difficult to use device.

[0018] The inventors have discovered that the use of mesh material allows optimal collection of bioaerosols containing crop pathogens while allowing airflow and molecular analysis with minimal sample preparation. In some embodiments, a cassette containing mesh material for bioaerosol collection is left in the field for several days (typically 3-7 days) to provide long-term collection. Long-term collection provides more comprehensive data compared to snapshot techniques. Spores in the air depend on various factors (e.g., wind speed, weather conditions such as rain, time of day, season). Snapshot techniques are hit-or-miss, but comprehensive long-term collection has the opportunity to collect under various conditions, increasing the probability of collecting targets. Therefore, a cassette is provided for long-term collection. In one embodiment, a pathogen collection device is provided having fibers, preferably electrostatically charged fibers.

[0019] As used herein, "bioaerosols" refers to biological aerosols, which are small airborne particles that are biological in nature. Bioaerosols may be derived from living organisms (such as house pet dander or tree pollen) or may be living organisms (such as bacteria or viruses). As used herein, "pathogen" refers to any substance that can cause disease. Airborne pathogens include plant pathogens. In one embodiment, the pathogen collection device collects spores, spore fragments, and / or hyphae.

[0020] In some embodiments of the device for collecting bioaerosols or pathogens, the bioaerosols or pathogens include powdery mildew, downy mildew, botytris, fusarium, early blight, and apple scab.

[0021] In some embodiments of the device for collecting spores, the spores are from the plant pathogen "Phytophthora." As used herein, the term "Phytophthora" includes all species of the genus Phytophthora. Species of Phytophthora collected are 'Phytophthora taxon Agathis', 'Phytophthora alni', 'Phytophthora boehmeriae', 'Phytophthora botryose', 'ibrassicae', 'Phytophthora cactorum', 'Phytophthora cajani', 'Phytophthora cambivora', 'Phytophthora capsici”, “Phytophthora cinnamomi”, “Phytophthora citricola”, “Phytophthora citrophthora”, “Phytophthora clandestine”, “Phytophthora colocasiae”, “Phytophthora cryptogea”, “Phytophthora drechsleri”, “Phytophthora diwan ackerman”, “Phytophthora erythroseptica', 'Phytophthora fragariae', 'Phytophthora fragariae var. rubi', 'Phytophthora Gemini', 'Phytophthora glovera', 'Phytophthora ``Phytophthora gonapodyides'', ``Phytophthora heveae'', ``Phytophthora hibemalis'', ``Phytophthora humicola'', ``Phytophthora hydropathical'', ``Phytophthora irrigate'', ``Phytophthora idaei'', ``Phytophthora ilicis'', ``Phytophthora infestans'', ``Phytophthora inflate'', ``Phytophthora ipomoeae”, “Phytophthoraincluding any of "iranica", "Phytophthora katsurae", "Phytophthora kemoviae", "Phytophthora lateralis", "Phytophthora medicaginis", "Phytophthora megakarya", "Phytophthora megasperma", "Phytophthora melonis", "Phytophthora mirabilis", "Phytophthora multivesiculata", "Phytophthora nemorosa", "Phytophthora nicotianae", "Phytophthora PaniaKara", "Phytophthora palmivora", "Phytophthora phaseoli", "Phytophthora pini", "Phytophthora porri", "Phytophthora plurivora", "Phytophthora primulae", "Phytophthora pseudosyringae", "Phytophthora pseudotsugae", "Phytophthora quercina", "Phytophthora ramorum", "Phytophthora sinensis", "Phytophthora sojae", "Phytophthora syringae", "Phytophthora tentaculata", "Phytophthora trifolii", "Phytophthora vignae".

[0022] In one embodiment, the device collects spores from the plant pathogen Sclerotinia. As used herein, the term Sclerotinia includes all species of the genus Sclerotinia. Sclerotinia species that may be collected include Sclerotinia borealis, Sclerotinia bulborum, Sclerotinia homoeocarpa, Sclerotinia minor, Sclerotinia ricini, Sclerotinia sclerotiorum, Sclerotinia spermophila, Sclerotinia sulcata, Sclerotinia trifoliorum, and Sclerotinia veratri.

[0023] In some embodiments, the device collects pathogens from one or more of those listed in Table 1.

[0024] [Table 1] TIFF0007636800000002.tif192161

[0025] 1 and 3, one embodiment of a pathogen collection device is shown in the form of a cassette 100. The cassette includes a collection medium 110 for passively collecting airborne pathogens, and a support frame 120. The support frame 120 supports and holds the collection medium 110 in tension, thereby exposing a collection medium surface 130 to the airflow. As the air flows through the collection medium surface 130, it collects airborne pathogens.

[0026] In one embodiment, the collection medium is comprised of electrostatically charged fibers. Preferably, the collection medium is a polymer mesh comprised of electrostatically charged fibers. In some embodiments, the polymer mesh is woven from monofilament fibers. In other embodiments, the polymer mesh is woven from multifilament fibers.

[0027] In some embodiments, the polymer mesh is made of a polyester material. In one embodiment, the polymer mesh is made of polyamide, polyethylene, polypropylene, ethylene tetrafluoroethylene, or polyether ether ketone fibers, or a combination of these fibers. In one embodiment, the polymer mesh is made of polyamide.

[0028] In some embodiments, the polymer mesh has a mesh size of 1 μm to 200 μm, preferably 10 μm to 150 μm. In one embodiment, the mesh size is 10 μm, 15 μm, 20 μm, 25 μm, 30 μm, 50 μm, 100 μm, or 150 μm. In some embodiments, the mesh size is selected based on the target pathogen.

[0029] 2, the support frame 120 includes a pair of interlocking members 130a, 130b that fit together under compression to hold the collection media 110 therebetween and maintain the collection media surface 120 in taut condition over the entire area enclosed by the pair of interlocking members. In one embodiment, the pair of interlocking members are two interlocking rings having an inner diameter of 0.5 to 3 inches, preferably 1 to 2 inches, and more preferably about 1.5 inches. In other embodiments, the pair of interlocking members are square, polygonal, or other shapes.

[0030] In some embodiments, the support frame is also electrostatically charged, hi one embodiment, the support frame is constructed of plastic, such as a styrene or polystyrene plastic.

[0031] The cassette 100 is disposable. Pathogens are captured in the cassette by capture, diffusion, impaction, electrostatic attraction, and / or sedimentation. Some filtering occurs, but this is not the primary cause of particle / pathogen capture. Additionally, the capture media 110 of the cassette 100 can be easily removed from the cassette by unlocking the pair of mating members 130a, 130b. The capture media 110 can then be further analyzed using molecular analysis to identify the captured pathogens.

[0032] In some embodiments, the collection medium 110 is a mesh that is removed from the cassette and placed directly into a vial for DNA extraction. Bioaerosols such as spores that are mostly bound to the mesh by static attraction are easily released from the mesh when a liquid solution is applied. Therefore, bioaerosols do not act as inhibitors of PCR because they are not bound to the mesh by any sticky properties. Also, the mesh is compatible with standard PCR analysis procedures.

[0033] [Pathogen collection and analysis] In use, the cassettes are replaceably inserted into a rotatable wind vane apparatus 200 to direct air into the cassettes. As shown in FIG. 4, the wind vane apparatus 200 has a funnel 210, vanes 220, and a post adapter 230. The funnel 210 focuses the incoming air and the vanes 220 orient the funnel based on the wind direction. The post adapter 230 allows the wind vane 200 to be attached to the end of a post. When attached, the wind vane 200 rotates about the post based on the wind direction.

[0034] In some embodiments, the wind apparatus does not have blades and the funnel is positioned based on the desired direction. In some implementations, the wind apparatus does not have a funnel and has blades. In other embodiments, the wind apparatus does not have blades or a funnel.

[0035] In some embodiments, the wind device is a drone, hi other embodiments, the cassette is placed on a drone or other vehicle used in agriculture, such as a tractor or truck.

[0036] In some embodiments, the wind device has vanes and can rotate around a stake. For example, the wind vane can be attached to a ½ inch MIP fitting or standard plumbing threads with integral thread pipe. This allows the end user to get the desired length of pipe for the desired placement.

[0037] In some embodiments, the vane has a receptacle for receiving the cassette, and the funnel directs the air flow to the collection surface of the cassette. In one embodiment, the cassette is located adjacent to the funnel and downstream of the neck 214 of the funnel. As used herein, the terms "upstream" and "downstream" refer to the direction of air flow. In one embodiment, the cassette is located inside the funnel, such as near the upstream end of the funnel, in the middle of the funnel, or near the downstream end of the funnel, to capture particulates and pathogens as air flows through the funnel. In one embodiment, as shown in FIG. 4, the neck 214 of the funnel 210 has an opening 212 sized to receive the cassette.

[0038] 5 and 6 show a pathogen collection system 300 having an anemometer 200 with a cassette 100 inserted therein. The cassette is inserted into the neck 214 of the funnel 210 through an opening 212. In one embodiment, the diameter of the cassette corresponds to the inner diameter of the neck such that the collection media surface 120 spans substantially the entire circular cross section of the neck perpendicular to the direction of airflow. In another embodiment, the diameter of the cassette is smaller than the inner diameter of the neck such that the collection media surface 120 spans only part of the circular cross section of the neck perpendicular to the direction of airflow.

[0039] The cassettes are replaced every day, every two days, every three days, or more. After use, the cassettes are retrieved for molecular analysis. As used herein, "molecular analysis" refers to analytical techniques such as, but not limited to, real-time PCR, conventional PCR, quantitative PCR, multiplex PCR, nested PCR, community sequencing, high-throughput sequencing, recombinase polymerase amplification (RPA), loop-mediated isothermal amplification (LAMP), antibody / antigen assays, colorimetric assays, and ELISA. Molecular analysis is used to determine the presence or absence of pathogen-containing bioaerosols on the cassette. Molecular analysis is used to quantify pathogen-containing bioaerosols on the cassette.

[0040] The pathogen collection system is not limited to a specific type of pathogen or pathogenic particle (spores, spore / hypha fragments) and can passively collect any wind-dispersed pathogenic particle. The system can also collect multiple types of spores simultaneously, and molecular testing for multiple types of spores is possible by changing the standard PCR cycle to a multiplex PCR cycle.

[0041] [Spreading decision] Currently, decisions to spray pesticides are often made by growers and agricultural specialists as a preventative measure based on host susceptibility and environmental factors. Information about the pathogen causing the disease is often only available post-infection, either through visual inspection of the grower's field or through information disseminated from similar measures in neighboring fields or regions.

[0042] The present disclosure also provides monitoring systems and methods that allow pre-infection pathogen information to be collected and made available to growers and agricultural professionals. Pathogenic particles can be detected in the air before they cause infection. This allows more information to be considered when deciding when to spray, what to spray, and whether to spray.

[0043] Referring to Figure 7, the monitoring system and method includes first identifying a crop and a target pathogen 701. The weather vanes described herein are installed and positioned at various heights depending on the crop and bioaerosol / pathogen, often depending on the height of the canopy of the field. The weather vanes are left installed in the field for all or part of the growing season depending on the crop. Each crop has a window of susceptibility to various pathogens, and preferably the pathogen collection system described herein is installed at least partially during this window of susceptibility.

[0044] When collection of airborne pathogens is desired, a cassette as described herein is attached to the wind vane (702). Single or multiple cassettes can be used to collect pathogens. For example, cassettes can be optionally replaced after a predetermined period of time to maximize collection of pathogens (703). Multiple wind vanes can be placed in the field to collect pathogens at different locations.

[0045] After pathogen collection, the cassettes are retrieved for molecular analysis (704). Optionally, weather data associated with the time the pathogen collection occurred is obtained (706).

[0046] The target spores captured by the cassette are differentiated or identified (707) by a number of methods that determine the presence of the target organism and provide a value, for example, that is numerical, specifically quantitative, specifically qualitative, or semi-quantitative, or semi-qualitative.

[0047] This value is then used to determine a spray decision, including spraying based on the presence of the organism, not spraying based on the presence of the organism, not spraying based on the absence of the organism, or spraying based on the absence of the organism.

[0048] Numerous details are set forth to provide an understanding of the embodiments described herein. Embodiments may be practiced without these details. This description should not be construed as limiting the scope of the embodiments described herein. EXAMPLES

[0049] Example 1: Phytophthora infestans Searching for Phytophthora infestans (Late blight of Potato) in a potato field. Potatoes are susceptible to this disease at any point in their life cycle. Therefore, the pathogen collection system described above can be left in place in the field for the entire growing season. The cassettes are changed every 3-4 days and sent to a lab for analysis.

[0050] Example 2: Sclerotinia sclerotiorum Searching for Sclerotinia sclerotiomm (Stem rot of Canola) in a canola field. Canola is susceptible to this disease only during the flowering stage. Therefore, the pathogen collection system described above can be installed in the field during this period and removed after flowering. The cassettes are changed every two days during flowering only and sent to the laboratory for analysis.

[0051] Although the embodiments have been described in detail, it should be understood that various changes, substitutions, and alterations are possible herein. In addition, the scope of the present application is not intended to be limited to the specific embodiments or examples described herein. As can be understood, the examples described and shown above are intended to be illustrative only.

[0052] For example, it is contemplated that the present invention may incorporate any of the features shown in any of the embodiments described herein with any of the features shown in any of the other embodiments described herein and still be within the scope of the present invention. [Explanation of symbols]

[0053] 100 cassettes 110 Collection media 120 Support Frame 130 Collection medium surface 130a, 130b Fitting members 200 Wind vane 210 Funnel 220 Feather 230 Post Adapter 300 Pathogen Collection System

Claims

1. 1. A cassette for capturing a fungal pathogen, comprising: a collection medium comprising a single layer of electrostatically charged fibers; a support frame supporting the collection media in an extended position to expose a collection surface for passively collecting the fungal pathogens; A cassette comprising:

2. 10. The cassette of claim 1, wherein the collection medium comprises a polymer mesh, optionally wherein the polymer mesh is constructed from woven monofilament fibers.

3. 3. The cassette of claim 2, wherein the polymer mesh is comprised of polyamide, polyethylene, polypropylene, ethylene tetrafluoroethylene, polyether ether ketone, or combinations thereof.

4. The cassette of claim 2 or 3, wherein the polymer mesh has a mesh size of from 1 μm to 200 μm.

5. 5. The cassette of claim 1, wherein the support frame is electrostatically charged styrene or the support frame has a pair of mating members configured to secure the collection media therebetween when the pair of mating members are coupled together, and optionally the support frame has a pair of mating rings.

6. 6. The cassette of claim 5, wherein the collection media spans an open area defined by the pair of mating members.

7. A cassette according to any one of claims 1 to 6 for capturing a plant pathogen.

8. 1. A fungal pathogen collection system comprising: The cassette according to any one of claims 1 to 7; a wind device including a receptacle for receiving the cassette and a funnel for directing a flow of air onto the collection surface of the cassette; A system comprising:

9. 9. The system of claim 8, wherein the receptacle has an opening in a neck portion of the funnel for inserting the cassette, or the collection surface of the cassette extends at least partially across a cross section of the neck portion of the funnel.

10. 10. The system of claim 8 or 9, wherein the wind device has blades for directing the funnel based on wind direction, or the system comprises a stake and the wind device is rotatably mounted to the stake.

11. 1. A method for monitoring a crop, comprising: Identifying a target fungal pathogen; - placing the cassette according to any one of claims 1 to 8 in a wind device to direct an air flow onto the collection surface of the cassette; retrieving the cassette; and analyzing the cassette for the presence of the target fungal pathogen; 13. The method of claim 12, further comprising:

12. 12. The method of claim 11, wherein the cassette is replaced after a predetermined time and multiple cassettes are collected and analyzed.

13. 13. The method of claim 11 or 12, wherein analyzing the cassette comprises molecular analysis of the microparticles captured by the cassette by real-time PCR, conventional PCR, quantitative PCR, multiplex PCR, nested PCR, community sequencing, high-throughput sequencing, recombinase polymerase amplification (RPA), loop-mediated isothermal amplification (LAMP), antibody / antigen assay, colorimetric assay, and / or ELISA.

14. 14. The method of any one of claims 11 to 13, comprising providing a decision based on the presence of a target pathogen, the decision being one of a decision to spray when the presence of the target pathogen is detected and a decision to spray in the absence of the presence of the target pathogen, or the decision being further based on weather data.

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