CN116973317A - Method for identifying resistance of plant to plutella xylostella based on hyperspectral imaging technology - Google Patents
Method for identifying resistance of plant to plutella xylostella based on hyperspectral imaging technology Download PDFInfo
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- 238000000034 method Methods 0.000 title claims abstract description 58
- 241000500437 Plutella xylostella Species 0.000 title claims abstract description 57
- 238000000701 chemical imaging Methods 0.000 title claims abstract description 32
- 238000005516 engineering process Methods 0.000 title claims abstract description 23
- 241000238631 Hexapoda Species 0.000 claims abstract description 43
- 238000000338 in vitro Methods 0.000 claims abstract description 12
- 238000004458 analytical method Methods 0.000 claims abstract description 10
- 238000003064 k means clustering Methods 0.000 claims abstract description 10
- 238000012216 screening Methods 0.000 claims abstract description 8
- 238000012847 principal component analysis method Methods 0.000 claims abstract description 7
- 238000001228 spectrum Methods 0.000 claims abstract description 7
- 241000196324 Embryophyta Species 0.000 claims description 59
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- 230000003595 spectral effect Effects 0.000 claims description 13
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- 238000001514 detection method Methods 0.000 claims description 6
- 238000000513 principal component analysis Methods 0.000 claims description 6
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- 238000004659 sterilization and disinfection Methods 0.000 claims description 4
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- 230000001488 breeding effect Effects 0.000 abstract description 6
- 238000011835 investigation Methods 0.000 abstract description 3
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- 235000003899 Brassica oleracea var acephala Nutrition 0.000 description 10
- 235000011301 Brassica oleracea var capitata Nutrition 0.000 description 10
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- 235000010149 Brassica rapa subsp chinensis Nutrition 0.000 description 9
- 235000000536 Brassica rapa subsp pekinensis Nutrition 0.000 description 9
- 241000499436 Brassica rapa subsp. pekinensis Species 0.000 description 9
- 241000607479 Yersinia pestis Species 0.000 description 5
- 235000013311 vegetables Nutrition 0.000 description 5
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- 241000219193 Brassicaceae Species 0.000 description 3
- PLUBXMRUUVWRLT-UHFFFAOYSA-N Ethyl methanesulfonate Chemical compound CCOS(C)(=O)=O PLUBXMRUUVWRLT-UHFFFAOYSA-N 0.000 description 3
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/17—Systems in which incident light is modified in accordance with the properties of the material investigated
- G01N21/25—Colour; Spectral properties, i.e. comparison of effect of material on the light at two or more different wavelengths or wavelength bands
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
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Abstract
The application discloses a method for identifying the resistance of a plant to plutella xylostella based on hyperspectral imaging technology, which comprises the following steps: determining screening varieties and raising seedlings; obtaining plutella xylostella larvae and adults; carrying out in-vitro insect grafting and living insect grafting; collecting spectrum data; performing K-means clustering analysis on the data to obtain an insect resistance grade; and verifying the insect resistance grade by adopting a principal component analysis method. According to the method for identifying the resistance of the plant to the plutella xylostella based on the hyperspectral imaging technology, which is provided by the application, the hyperspectral imaging technology is utilized to count the area and the wound of the plutella xylostella biting blade, and the biting data can be furthest represented by a K-means clustering analysis and principal component analysis method, so that the influence of subjective factors in the previous investigation method is avoided, and the identification accuracy is greatly improved; the operation is simple, the workload is saved, and the identification efficiency is higher; is not affected by seasons and climate conditions, is convenient to use, can realize rapid screening of new germplasm resources of the plutella xylostella, and accelerates the breeding process of the plutella xylostella.
Description
Technical Field
The application relates to the technical field of plutella xylostella resistance identification, in particular to a method for identifying plutella xylostella resistance of plants based on hyperspectral imaging technology.
Background
Plutella xylostella (Plutella xylostella) belongs to the order Lepidoptera (Lepidotera) and the family Plutellidae (Plutellidae) is an important pest worldwide. It is classified as one of the most difficult pests to control due to its short life cycle, high mobility, strong tolerance to environmental stress, and enhanced resistance to existing pesticides.
The plutella xylostella mainly damages cruciferae vegetables such as cabbages, cabbages (green cabbages, purple cabbages and collards), broccoli, rapes, radishes, leaf mustard, cauliflower and the like, and when the damage is serious, the cruciferae vegetables can be reduced by more than 90 percent, and even are out of harvest. The germplasm resources of the diamondback moth resistant in the cruciferous vegetables are very deficient, and the wide screening of the resistant germplasm resources is an important content in vegetable breeding work.
Practice proves that the cultivation of the insect-resistant variety is the most effective method in the comprehensive pest control, can reduce the dependence of crops on chemical pesticides, and provides guarantee for sustainable development of agriculture. Therefore, the method has the advantages of wide collection, systematic research and reasonable utilization of the insect-resistant germplasm resources, simple, convenient, accurate and efficient development of the resistance identification technology, germplasm resource assessment and mutant screening, cultivation of insect-resistant varieties and utilization, and great significance for preventing and controlling the damage of plutella xylostella.
The insect resistance identification is a precondition and basis for breeding and utilizing plant insect resistance varieties, and the main task is to identify the type of resistance of plant genetic resources to insect pests and evaluate the degree of resistance under the condition of natural or artificial inoculation, so as to screen out excellent resistant materials with strong resistance or special characteristics. At present, the traditional insect resistance identification method comprises 3 kinds of field resistance identification, net indoor insect-grafting resistance identification and indoor insect-grafting identification which take naturally occurring insect pests as insect sources. However, the traditional insect-resistant identification method is long in time consumption, easy to be influenced by seasons and climate conditions, high in difficulty and large in workload, the insect-biting area cannot be accurately detected, and the detection result is also easy to be influenced by artificial subjective factors, so that the accuracy and objectivity of identification are influenced.
Therefore, how to develop a plant seedling-stage diamondback moth-resistant identification method with high identification efficiency, good accuracy and good repeatability is a technical problem which needs to be solved by the technicians in the field.
Disclosure of Invention
The application aims to provide a method for identifying the resistance of a plant to plutella xylostella based on a hyperspectral imaging technology, and provides a method which is rapid, accurate, efficient, simple to operate, convenient to use, and more suitable for accurately detecting the biting area and wounds of the plutella xylostella in cabbages, so that the plant is rapidly and accurately identified, the germplasm resources of the plutella xylostella are screened, and a new process of breeding the plutella xylostella resistant to the cabbages is accelerated.
In order to achieve the above purpose, the application provides a method for identifying the resistance of a plant to plutella xylostella based on a hyperspectral imaging technology, which utilizes the hyperspectral imaging technology to count the area and wound of the plutella xylostella biting leaves, and screens the germplasm resources of the plutella xylostella, and specifically comprises the following steps:
(1) Determining plant material varieties to be screened, and carrying out seedling culture;
(2) Obtaining a sufficient quantity of 2-year larvae and adults of plutella xylostella;
(3) Respectively carrying out in-vitro insect grafting and living insect grafting on the same variety;
(4) Collecting spectral data of biting leaves and plants;
(5) Performing K-means clustering analysis on the collected spectrum data to obtain an insect resistance grade of a detection sample;
(6) And verifying the obtained insect resistance grade by adopting a principal component analysis method.
Preferably, the seeds and the culture medium are effectively sterilized before seedling raising in the step (1), and the sterilization method is a sterilization method which does not influence the germination rate of the seeds and furthest maintains the nutrition components and the structural morphology of the culture medium; the seedling is carried out in a container which is convenient to move, such as a nutrition pot, a seedling tray and the like, and the seedling distance is not less than 7cm.
Preferably, in the step (2), before hatching eggs, other non-target insects in the incubator and the feeding box are removed, and the incubator, the feeding box and the plutella xylostella eggs are sterilized; the method for sterilizing the ova comprises the steps of mixing 8% formaldehyde solution: soaking in water solution with volume ratio of 1:4 for 15-20min, washing with clear water for 3-4 times, and air drying in shade and ventilated place.
Preferably, the in-vitro insect-receiving method in the step (3) comprises the following steps: when the plant seedlings grow to 6-8 true leaves, the newly grown true leaves are cut, placed in a culture dish, inoculated with 10 heads of 2-year-old larvae in each culture dish with the diameter of 6cm, and inoculated in vitro with the setting of male and female larvae in each half, and each plant material is provided with 3 biological repeats.
Preferably, the living body insect-receiving method in the step (3) is as follows: after the seedlings grow to four leaves and one heart, the seedlings are transferred into an insect-catching net room, the plant spacing is not less than 7cm, 10 plants are used for each part of material, all plants to be detected are arranged in a random group mode, and the plant spacing is 5.6m 2 Inoculating 30 adult plutella xylostella with basically consistent eclosion degree in the net room, and arranging male and female halves to carry out living body insect grafting; the insect-receiving net chamber is made of a 40-mesh net gauze cover.
Preferably, the spectrum data acquisition in the step (4) is completed through a hyperspectral imaging system; the hyperspectral imaging system consists of a computer, a light shield, a light source, a sample placement table to be detected and a portable ground object spectrometer, wherein the light source is a halogen lamp, the working voltage is 230V, and the power is 35W; the spectrum data acquisition method comprises the following steps: firstly, calibrating a portable ground object spectrometer, then placing the biting leaves and/or plants in a hyperspectral imaging system, scanning by using the portable ground object spectrometer, collecting the reflectivity of 320-800nm wave band leaves and/or plants, and then extracting the reflectivity of 450nm, 475nm, 550nm and 650nm characteristic wave bands by using an SPA method to obtain spectral data of the biting leaves and/or plants; the in vitro insect-grafting experiment and the living insect-grafting experiment respectively collect spectral data of biting leaves and plants on the 3 rd day and the 10 th day of insect grafting.
Preferably, in the K-Means clustering analysis in the step (5), the number of K-Means clusters is set to be 5 and the maximum iteration number is set to be 10 according to the international grading standard of insect resistance.
Preferably, the principal component analysis in the step (6) is performed on spectral data in characteristic bands of 450nm, 475nm, 550nm, and 650 nm.
The application of the method for identifying the resistance of the plant to the plutella xylostella based on the hyperspectral imaging technology in identifying the insect resistance of the plant is provided.
Therefore, the method for identifying the resistance of the plant to the plutella xylostella based on the hyperspectral imaging technology has the following specific technical effects:
(1) The identification method provided by the application utilizes the hyperspectral imaging technology to count the biting blade area and the wound of the plutella xylostella, and can furthest characterize biting data through a K-means clustering analysis and principal component analysis method, so that the influence of subjective factors in the previous investigation method is avoided, and the identification accuracy is greatly improved;
(2) The identification method provided by the application only needs to place the sample to be identified in the detection system, and then uniformly analyzes and processes the collected data, so that the operation is simple, the workload is saved, and the identification efficiency is higher;
(3) The identification method provided by the application is not influenced by seasons and climate conditions, is convenient to use, can realize rapid screening of new germplasm resources of the plutella xylostella, and accelerates the breeding process of the plutella xylostella.
The technical scheme of the application is further described in detail through the drawings and the embodiments.
Drawings
In order to more clearly illustrate the technical solutions of the embodiments of the present application, the drawings that are needed in the description of the embodiments of the present application will be briefly described below, it being obvious that the drawings in the following description are only some embodiments of the present application, and that other drawings may be obtained according to these drawings without inventive effort for a person skilled in the art.
FIG. 1 is a block diagram of a hyperspectral imaging system provided by the present application;
FIG. 2 is a graph showing reflectance of a leaf of a non-biting cabbage as measured in accordance with an embodiment of the present application;
FIG. 3 is a graph of the reflectance of insect resistant levels of different mutant cabbages obtained in an embodiment of the application;
FIG. 4 is a graph showing the results of principal component analysis in the example of the present application.
Reference numerals
1. A halogen lamp; 2. a portable ground object spectrometer; 3. a computer; 4. a sample to be detected; 5. a sample placement stage to be tested; 6. a light shield.
Detailed Description
The technical scheme of the application is further described below through the attached drawings and the embodiments.
In order to make the objects, technical solutions and advantages of the present application more clear, thorough and complete, the technical solutions of the present application will be clearly and completely described below through the accompanying drawings and examples. The following detailed description is of embodiments, and is intended to provide further details of the application. Unless defined otherwise, all technical terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs.
The special seedling raising matrix for the celery cabbage is produced by baoding Shuofeng gardening limited company; the plutella xylostella larva feed and plutella xylostella egg are purchased from Henan province source white cloud industry limited company; the portable ground object spectrometer is manufactured by Beijing European Purpura technology Co., ltd, and the model is PSR-1100.
Example 1
A method for identifying the resistance of a plant to plutella xylostella based on hyperspectral imaging technology comprises the following steps:
(1) Determining plant materials to be screened and carrying out seedling raising
By taking Chinese cabbage of Brassica of Brassicaceae as a material, taking a high-generation homozygous inbred line A03 of Chinese cabbage from a key laboratory and originating from vegetable germplasm innovation of Hebei province as a reference, randomly selecting 300 parts of mutagenic progeny as plutella xylostella resistance identification materials from a mutant library (Lu Yin. Creation and screening identification of Chinese cabbage EMS mutant library [ D ] Hebei university of agriculture, 2014;LuY,Dai S,Gu A,et al.Microspore induced doubled haploids production from Ethyl Methanesulfonate (EMS) soaked flower buds is an efficient strategy for mutagenesis in Chinese cabbage [ J ] Frontiers in Plant Science,2016,7 (e 41570): 1780 ]), and carrying out resistance experiments for 10 times. Selecting 10 full Chinese cabbage seeds per material, and soaking in 50deg.C warm water for 30min. Sterilizing special seedling medium for Chinese cabbage in autoclave at 121deg.C for 30min, cooling to room temperature, loading into 7cm nutrition pot, sowing the soaked seeds into the medium, and culturing at 23deg.C under 70% humidity with photoperiod of 16 hr light-8 hr darkness;
(2) Obtaining enough quantity of 2-year larva and adult plutella xylostella
And (5) removing other non-target insects in the incubator and the feeding box, and sterilizing the incubator and the feeding box by using 75% alcohol.
The eggs of plutella xylostella are put into formaldehyde solution with concentration of 8 percent: soaking in formaldehyde solution diluted with water at a volume ratio of 1:4 for 20min, washing with clear water for 4 times, air drying at shady and ventilated place, and incubating the sterilized plutella xylostella eggs in dark environment with a temperature of 25deg.C, a relative humidity of 60% and a photoperiod of 16-8 h.
The volume of the blocks is 1cm 3 Placing the feed in sterilized feeding boxes, placing 50-100 eggs hatched for 1 day in each feeding box, starting in-vitro insect-grafting experiment when the diamondback moth larvae grow to 2 years old, and performing in-vivo insect-grafting experiment when the larvae grow to adults with basically consistent eclosion degree.
(3) The same part of the mutagenesis material is respectively subjected to in vitro insect grafting and living insect grafting
When the plant seedlings grow to 6-8 true leaves, each plant shears 3 newly grown true leaves, the plant seedlings are placed in a culture dish with the diameter of 6cm, 10 head 2-year-old plutella xylostella larvae, male and female larvae are half, and three biological repeats are arranged on each plant material.
After the Chinese cabbage seedlings grow to four leaves and one core, the Chinese cabbage seedlings are transferred into an insect-receiving net room made of a 40-mesh net gauze cover, 10 plants of each material are arranged in a random group, and each net room is inoculated with 30 larvae of 2-3 years old, wherein the male and female parts are half.
(4) Collecting spectral data of biting leaves and plants
Placing the biting leaves and/or plants on a sample placing table 5 to be detected of a hyperspectral imaging system shown in figure 1 on the 3 rd day and the 10 th day of an in-vitro insect grafting experiment and a living body insect grafting experiment respectively, opening a halogen lamp 1, covering a light shield 6, calibrating a portable ground object spectrometer 2, scanning the biting leaves and/or plants (sample 4 to be detected) by the portable ground object spectrometer 2, collecting the reflectivity of the 320-800nm wave band leaves and/or plants, transferring the reflectivity into a computer 3 connected with the portable ground object spectrometer 2, and extracting the reflectivity of the 450nm, 475nm, 550nm and 650nm characteristic wave bands by using an SPA method, namely the spectral data of the biting leaves and/or plants.
(5) K-means clustering analysis is carried out on the collected spectrum data to obtain the insect resistance grade of the detection sample
And (3) inputting the spectral data acquired in the step (4) into SPSS software for K-Means clustering analysis, setting the clustering number in a K-Means data space to be 5 according to the insect resistance international grading standard (see table 1), setting the maximum iteration number to be 10, and carrying out measurement and classification by taking the square of Euclidean distance as the standard, so as to obtain the insect resistance grade of a detection sample, wherein the reflectance curve of the leaf of the non-biting cabbage is shown in fig. 2, and the reflectance curves of the insect resistance grades of different mutant cabbages are shown in fig. 3.
TABLE 1
(6) And verifying the obtained insect resistance grade by adopting a principal component analysis method.
The principal component analysis was performed on all spectral data of bite leaves and plants obtained at characteristic bands of 450nm, 475nm, 550nm, 650nm using Origin software, and the results are shown in FIG. 4.
The results show that 1 principal component is finally obtained, and the contribution rate is as high as more than 90%, so that the test of the embodiment can represent the information of the original data by the principal component. And (3) carrying out cluster analysis on the new data recoded by the principal component analysis so as to effectively monitor, wherein the principal component analysis verification result shows that the grading result in the step (5) is accurate.
Therefore, the method for identifying the resistance of the plant to the plutella xylostella based on the hyperspectral imaging technology provided by the application utilizes the hyperspectral imaging technology to count the area and the wound of the plutella xylostella biting blade, and the biting data can be furthest represented by a K-means clustering analysis and principal component analysis method, so that the influence of subjective factors in the previous investigation method is avoided, and the identification accuracy is greatly improved; the operation is simple, the workload is saved, and the identification efficiency is higher; is not affected by seasons and climate conditions, is convenient to use, can realize rapid screening of new germplasm resources of the plutella xylostella, and accelerates the breeding process of the plutella xylostella.
Finally, it should be noted that: the above embodiments are only for illustrating the technical solution of the present application and not for limiting it, and although the present application has been described in detail with reference to the preferred embodiments, it will be understood by those skilled in the art that: the technical scheme of the application can be modified or replaced by the same, and the modified technical scheme cannot deviate from the spirit and scope of the technical scheme of the application.
Claims (9)
1. The method for identifying the resistance of the plant to the plutella xylostella based on the hyperspectral imaging technology is characterized by counting the area and wound of the plutella xylostella biting leaves by using the hyperspectral imaging technology, and screening the germplasm resources for resisting the plutella xylostella, and the specific method is as follows:
(1) Determining plant material varieties to be screened, and carrying out seedling culture;
(2) Obtaining a sufficient quantity of 2-year larvae and adults of plutella xylostella;
(3) Respectively carrying out in-vitro insect grafting and living insect grafting on the same variety;
(4) Collecting spectral data of biting leaves and plants;
(5) Performing K-means clustering analysis on the collected spectrum data to obtain an insect resistance grade of a detection sample;
(6) And verifying the obtained insect resistance grade by adopting a principal component analysis method.
2. The method for identifying the resistance of plants to plutella xylostella based on hyperspectral imaging technology as claimed in claim 1, wherein the method comprises the following steps: the seeds and the culture medium are effectively disinfected before seedling raising in the step (1), and the disinfection method is a disinfection method which does not influence the germination rate of the seeds and furthest maintains the nutrition components and the structural morphology of the culture medium; the seedling is carried out in a container which is convenient to move, such as a nutrition pot, a seedling tray and the like, and the seedling distance is not less than 7cm.
3. The method for identifying the resistance of plants to plutella xylostella based on hyperspectral imaging technology as claimed in claim 1, wherein the method comprises the following steps: in the step (2), other non-target insects in the incubator and the feeding box are removed before hatching eggs, and the incubator, the feeding box and the plutella xylostella eggs are disinfected; the method for sterilizing the ova comprises the steps of mixing 8% formaldehyde solution: soaking in water solution with volume ratio of 1:4 for 15-20min, washing with clear water for 3-4 times, and air drying in shade and ventilated place.
4. The method for identifying the resistance of the plant to the plutella xylostella based on the hyperspectral imaging technology as claimed in claim 1, wherein the in-vitro insect-grafting method in the step (3) is as follows: when the plant seedlings grow to 6-8 true leaves, the newly grown true leaves are cut, placed in a culture dish, inoculated with 10 heads of 2-year-old larvae in each culture dish with the diameter of 6cm, and inoculated in vitro with the setting of male and female larvae in each half, and each plant material is provided with 3 biological repeats.
5. The method for identifying the resistance of plants to plutella xylostella based on hyperspectral imaging technology as claimed in claim 1, wherein the method comprises the following steps: the living body insect-grafting method in the step (3) comprises the following steps: after the seedlings grow to four leaves and one heart, the seedlings are transferred into an insect-catching net room, the plant spacing is not less than 7cm, 10 plants are used for each part of material, all plants to be detected are arranged in a random group mode, and the plant spacing is 5.6m 2 Inoculating 30 adult plutella xylostella with basically consistent eclosion degree in the net room, and arranging male and female halves to carry out living body insect grafting; the insect-receiving net chamber is made of a 40-mesh net gauze cover.
6. The method for identifying the resistance of a plant to plutella xylostella based on the hyperspectral imaging technique according to claim 1, wherein the acquisition of the spectral data in the step (4) is completed by a hyperspectral imaging system; the hyperspectral imaging system consists of a computer, a light shield, a light source, a sample placement table to be detected and a portable ground object spectrometer, wherein the light source is a halogen lamp, the working voltage is 230V, and the power is 35W; the spectrum data acquisition method comprises the following steps: firstly, calibrating a portable ground object spectrometer, then placing the biting leaves and/or plants in a hyperspectral imaging system, scanning by using the portable ground object spectrometer, collecting the reflectivity of 320-800nm wave band leaves and/or plants, and then extracting the reflectivity of 450nm, 475nm, 550nm and 650nm characteristic wave bands by using an SPA method to obtain spectral data of the biting leaves and/or plants; the in vitro insect-grafting experiment and the living insect-grafting experiment respectively collect spectral data of biting leaves and plants on the 3 rd day and the 10 th day of insect grafting.
7. The method for identifying the resistance of the plant to the plutella xylostella based on the hyperspectral imaging technology as claimed in claim 1, wherein in the K-Means clustering analysis in the step (5), the number of K-Means clusters is set to be 5 and the maximum iteration number is set to be 10 according to the international grading standard of the resistance to insects.
8. The method for identifying the resistance of plants to plutella xylostella according to claim 1, wherein the principal component analysis in the step (6) is performed on spectral data in characteristic bands of 450nm, 475nm, 550nm and 650 nm.
9. Use of the hyperspectral imaging technique-based plant-to-plutella xylostella resistance identification method as claimed in any one of claims 1 to 8 in plant insect resistance identification.
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| Application Number | Priority Date | Filing Date | Title |
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| CN202310694808.3A CN116973317A (en) | 2023-06-13 | 2023-06-13 | Method for identifying resistance of plant to plutella xylostella based on hyperspectral imaging technology |
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