| Fungal diseases of crops are mainly transmitted by spores.Due to the properties of spores such as large number,small size and lightness,it is easy to spread through air flow,thus forming fungal airborne diseases.The spread speed of fungal air-borne diseases is much faster than that of soil-borne diseases,and it is very easy to re-infect many times.Once the disease occurs,it is difficult to control the disease effectively,which will not only reduce the yield,but even cause crop failure in a large area.Thus,it seriously threatens the agricultural production and food security of our country.The characteristics of fungal airborne diseases determine that the effective control methods should be prevention and control.Compared with the way of diagnosing the disease by observing the appearance of crops,monitoring the number(concentration)of spores in the air can move the prevention and control gate forward,and limit the development of the disease to the initial stage,so it is of great significance to improve the level of disease prevention and control.Studies have shown that the number(concentration)of disease spores in the air is closely related to the degree of disease occurrence.By monitoring the concentration of airborne spores,the disease can be predicted effectively.At present,the conventional airborne spore monitoring method is to capture spores directly in the air by using a spore capture instrument,and then use the methods of culture identification,molecular biology and immunology for detection.This has improved the prevention and control level of agricultural airborne diseases to a certain extent.However,there are still the following problems that need to be solved urgently: 1.Air-borne microorganisms move irregularly and are widely and unevenly distributed in space,so how to avoid missing detection;2.Aerosol components in the air are extremely complex,so how to improve the purity of the target captured spores and realize rapid quantitative detection;3.The airborne microorganisms are invisible and the concentration of specific microorganisms is low.How to improve the detection limit and resolution of spore detection.Traditional macro spore sampler has no effective countermeasures to solve the above three difficult problems.According to the above-mentioned difficult problems,this paper comprehensively comb,conclude and summarize the related research of airborne disease microorganism capture and detection technology at home and abroad,and puts forward a method of airborne disease spore identification and concentration detection based on microfluidic separation technology,and explores the key technical problems of this method theoretically and experimentally.The main work of this paper includes:1.Based on the basic theory of particle-fluid two-phase flow model,the dynamic principle of airborne spore movement in micro-environment is studied by CFD simulation analysis and experimental verification,which proves the theoretical feasibility of the proposed orthogonal coupling separation and gravity sedimentation enrichment method.2.The mechanical analysis of airborne spores in the microchannel with high aspect ratio was carried out,and a microfluidic airborne spore separation method with orthogonal coupling of axial drag and radial drag was proposed.The performance of the separation chip was simulated and analyzed by using COMSOL finite element multi-physical field coupling software,combined with chip size,airflow velocity,spore quality,particle size and other influencing parameters.The results showed that under the determined microchannel structure,the target spores could be accurately extracted by controlling airflow velocity.In view of the non-spherical characteristics of most airborne spores,the fluid-structure coupling interface in COMSOL software was used to study,and the results showed that the spore shape had a great influence on its movement characteristics under micro-scale conditions.3.Based on the dynamic principle of microparticles in still air,the gravity sedimentation behavior of airborne spores was analyzed.The analysis results show that it takes only 0.03 seconds for the particles with a particle size of 10 ?m to settle at a height of 100 ?m in microenvironment.Based on this characteristic,a steep expansion microfluidic enrichment chip was designed,and the gravity deposition enrichment of the target airborne spores was realized by controlling the flow rate.In order to determine the effectiveness of the enrichment chip,the quantitative detection model of optical scattering was adopted,the microfluidic light scattering detection platform was built,and taking the spores of Ustilaginoidea virens as an example,the best light intensity and wavelength suitable for detection were screened out.It was found that there was a good linear correlation between transmitted light intensity and spore concentration,which indicated that light scattering method could be used for spore concentration detection.4.On the basis of separating,capturing,enriching and thickening the target spores,the paper uses the technology of on-chip microscopic image in-situ acquisition and processing combined with artificial intelligence to verify the effect of spore segmentation counting and intelligent recognition.The results show that the recognition accuracy reaches 96.12% by using the autogenous spore data set and using Alex Net and other deep neural networks to carry out recognition training and testing again.The Star Dist deep learning model can better accomplish the segmentation and counting of conglutinated spores,and the relative error of sample detection is less than 10% based on the number of manual detections.5.In order to verify the quantitative detection effect of the microfluidic chip with the functions of dynamic filtration+orthogonal coupling separation+gravity sedimentation enrichment,an integrated airborne spore separation and detection experimental platform was established,and the performance of the platform was tested.The results showed that the isolation rate of mold spores by focusing treatment was 89%,which was much higher than that by unfocused treatment(59%).The purity of mold spores captured by the separation chip is 98%,which is much higher than the traditional sedimentation method of 69%.In the experiment of spore enrichment,when the diameter of enrichment chip is 2mm and the inlet flow rate is 40mm/s,the flow rate in the enrichment center area is reduced to 1.38mm/s,and the enrichment rate of botrytis cinerea spores can reach 95%.Error analysis of the platform shows that the relative error of the detection results is less than 5%under the condition of specific spore flow rate.The results of light scattering detection of enriched spores show that the linear correlation coefficient between microscope detection and light scattering detection is 0.964,which verifies the effectiveness of the proposed light scattering detection method.6.The proposed method and system are applied to typical application scenarios in the field of agricultural engineering.Firstly,the detection of botrytis cinerea disease in facility vegetables is developed,and the performance of the device is tested.The results show that the correlation coefficient between the device and the traditional spore catcher is 0.912,and the relative error is less than 5%.Then,the proposed method was applied to the detection of drug resistance of disease spores.The linear correlation coefficient between microscopic detection and light scattering detection of spore germination inhibition rate was 0.9597,which further verified the effectiveness of light scattering detection method.The research shows that the method of separation and enrichment of airborne spores based on the principle of gas phase hydrodynamics in microenvironment can realize the accurate control of airborne spores.Microfluidic chip technology is applied to the detection of crop pathogens in this paper.It is beneficial to realize the micro-sensitization of detection equipment,and the full coverage monitoring of geographical time and space can be realized by large-scale deployment or airborne cruise.The microfluidic double sheath flow orthogonal coupling method can realize the active separation of airborne spores and realize the accurate capture of target spores;combined with the gravity sedimentation principle of airborne spores and artificial intelligence technology,the enrichment and concentration of airborne spores and rapid detection can be realized.The relevant research ideas and solutions proposed in this study can be applied to the detection of various airborne diseases in agricultural production,and also have a certain reference value for the monitoring of bacteria,viruses and other aerosol microorganisms. |
PDF Full Text Request