Culture device for rapidly identifying thrip resistance of plant germplasm, use method and investigation and evaluation method

Abstract

The invention discloses a culture device for rapidly identifying the resistance of plant germplasm to thrips, a use method and an investigation and evaluation method, and belongs to the technical field of agricultural plant protection. The device comprises a natural environment simulation unit, a culture tube rack arranged in a box body, and a control unit, and the natural environment simulation unit comprises a box body capable of automatically adjusting the internal environment temperature, humidity and illumination and supplementing water; a plurality of placing grooves are formed in the culture tube rack; the insect growth environment simulation unit comprises a plurality of culture tubes positioned in the box body; the culture tube comprises a culture tube body; the culture tube body is arranged in the placing groove; the tube cap is detachably connected with the top opening of the culture tube body; the gauze element covers the top opening of the culture tube body and is pressed and fixed through a tube cap; the centrifugal tube is positioned in the culture tube body; the sponge cushion is arranged at the bottom of the culture tube body. The device is simple, convenient, easy to carry, low in cost and capable of highly simulating the natural environment and avoiding interference of unstable factors.

Description

Technical Field

[0001] This invention belongs to the field of agricultural plant protection technology, and more specifically relates to a culture device and its usage method, as well as a survey and evaluation method for rapidly identifying plant germplasm resistance to thrips. Background Technology

[0002] Thrips belong to the phylum Arthropoda, class Insecta, order Thysanoptera, and are a collective term for insects in this order. Thysanoptera is further divided into two suborders: Terebrantia and Tubulifera. There are numerous species of thrips, with approximately 6,000 known worldwide and over 500 recorded in China. Common harmful species include melon thrips, onion thrips, rice thrips, and western flower thrips.

[0003] Adult thrips are typically 1-3 mm long and highly elusive. Their body color ranges from yellow and brown to black, with some species exhibiting stripes or markings. Their wings are membranous and elongated, with cilia along the edges; some species have vestigial wings. The appendages possess retractable, vesicle-like organs at the ends, facilitating climbing. The female's ovipositor curves ventrally and is often serrated; the male's genitalia can retract into its abdomen. Their rasping-sucking mouthparts pierce and suck sap from new shoots, tender leaves, flowers, and young fruits, causing leaf dieback, fruit deformities, and reduced agricultural product quality. Some species can transmit plant viruses (such as tomato spotted wilt virus and pepper mild mottle virus), exacerbating losses. Thrips have an extremely wide host range, encompassing vegetables, fruit trees, flowers, field crops, and various cash crops, causing serious damage and significant economic losses to agricultural production. In 2023, they were listed as a Class A crop pest by the Ministry of Agriculture and Rural Affairs.

[0004] Currently, screening insect-resistant plant germplasm resources and cultivating new insect-resistant varieties are highly economical and effective strategies for agricultural development. Their core advantages lie in reducing agricultural production costs and improving efficiency across multiple dimensions. Cultivating new insect-resistant varieties can significantly reduce pesticide use, decrease environmental pollution, protect the biodiversity of beneficial insects, directly reduce material and labor costs, delay the development of pesticide resistance in pests, reduce the severity of pest damage, and ensure stable yield and quality of agricultural products. Therefore, establishing a system for identifying and evaluating plant resistance to pests to cultivate new insect-resistant varieties is of great significance for promoting plant variety improvement and ensuring the sustainable development of agricultural production.

[0005] In agricultural production, the prerequisite for breeding varieties is the screening and collection of germplasm resources, which can exhibit characteristics related to insect resistance. Therefore, the rapid and efficient screening of parental materials with insect resistance from plant germplasm resources is a key prerequisite for breeding insect-resistant varieties.

[0006] Currently, the evaluation system for plant resistance to various agricultural pests is still incomplete. Plant resistance assessment is mainly divided into two categories: live inoculation and in vitro inoculation. Live inoculation resistance assessment is mostly conducted in the field, either by weight gain rate (e.g., Chinese patent CN201711417803.7), insect population ratio method (e.g., Chinese patent CN201710058972.X), or by investigating insect population density (e.g., Chinese patent CN201310383520.0). However, the field environment is complex and easily affected by unstable factors, resulting in insufficient accuracy of the evaluation results. Counting insect populations is time-consuming and labor-intensive. Furthermore, using live plants for resistance assessment involves numerous materials and cumbersome procedures, resulting in a large workload, extensive space requirements, and low accuracy. Compared to live inoculation, in vitro inoculation for resistance assessment offers advantages such as controllable conditions, good repeatability, simple operation, and high accuracy. Summary of the Invention

[0007] To solve the above technical problems, the present invention adopts the following technical solution: A culture device for rapidly identifying plant germplasm resistance to thrips, comprising: Natural environment simulation unit, the natural environment simulation unit includes a box with the function of automatically adjusting the internal environment temperature, humidity, light and water replenishment, and a culture tube rack set in the box, the culture tube rack being provided with multiple placement slots; The insect growth environment simulation unit includes multiple culture tubes located within the box and placed in the placement slot; the culture tubes include: The culture tube body is a transparent container with an open top; the culture tube body is placed inside the placement slot; The cap is detachably connected to the top opening of the culture tube body; A mesh screen covers the top opening of the culture tube body and is pressed and fixed by the tube cap; the mesh count of the mesh screen is at least 250. Centrifuge tubes are located inside the culture tube body; A sponge pad is placed at the bottom of the culture tube body; the sponge pad has a hole in the center, and the centrifuge tube is installed in the hole.

[0008] Furthermore, the centrifuge tube includes a centrifuge tube cavity, a centrifuge tube cap, and a connecting component; wherein the centrifuge tube cavity is connected to the centrifuge tube cap through the connecting component.

[0009] Furthermore, the centrifuge tube cap is provided with insertion holes for inserting plant petioles and water injection tubes.

[0010] Furthermore, the culture tube body is a transparent centrifuge tube with a nominal capacity of 50 mL, a height of 11 cm without a cap, and a diameter of 3 cm.

[0011] Furthermore, the centrifuge tubes have a capacity of 1.5 mL, 2 mL, or 5 mL.

[0012] Furthermore, the thickness of the sponge pad is 5mm, and the diameter of the central hole is 1cm.

[0013] Furthermore, an operation hole with a diameter of 5 mm is provided on the upper side wall of the culture tube body.

[0014] A method of using a culture device for rapidly identifying plant germplasm resistance to thrips, comprising the following steps: S10. Material Preparation Plant materials: Plant varieties whose insect resistance was to be evaluated were grown in an artificial climate chamber, including insect-resistant and insect-susceptible control materials; Insect source material: The test insect source was a single natural population of thrips collected from the field and reared in the laboratory for more than 3 generations; S20, Insecticide When the seedlings have grown to the top and the leaves are 3-4 cm wide, retain the leaves and petioles, cut them off at the base of the petioles, and transfer them into the culture tube. Let the petiole pass through the centrifuge tube cap and extend into the centrifuge tube cavity. Inject nutrient solution into the centrifuge tube cavity in advance, and make sure that the cut of the petiole extends below the surface of the nutrient solution. Take one live adult female thrips, place it in the culture tube and place it on a leaf. Cover the opening of the culture tube with a mesh, then press the hollowed-out tube cap onto the mesh and connect it tightly to the threaded opening at the top of the culture tube. Set up sufficient repeat treatments as needed for the experiment. S30. Post-inoculation management After inoculation, the culture tubes are fixed to the culture tube rack through the placement slot. The connecting tubes extending from the outer wall of the culture tubes are connected to the water inlet through the interconnecting hose. The power supply is connected through the power cord interface. The culture environment parameters are set on the digital control display screen. The survey data are observed and recorded at regular intervals according to the inoculation time.

[0015] A method for rapidly identifying and evaluating plant germplasm resistance to thrips includes investigating and evaluating the degree of thrips damage to inoculated leaves cultured using the aforementioned method for rapidly identifying plant germplasm resistance to thrips. The method is as follows: Based on the degree of thrips damage (I) to the inoculated leaves, the leaf damage level is divided into six levels: 0, 1, 3, 5, 7, and 9. Here, I represents the percentage of the leaf area marked by thrips bites. The grading criteria are as follows: The leaf damage level is 0, the leaf is undamaged, I=0; The leaf damage level is 1, with pinhead-sized insect bite marks on the leaf edges, I ≤1%; The leaf damage level is 3, with a small amount of chlorotic spots appearing on the leaves, 1% < I ≤ 5%; The leaf damage level is 5, with slight chlorosis, 5% < I ≤ 15%; The leaf damage level is 7, with obvious chlorosis and leaf tip curling of 15% < I ≤ 30%; The leaf damage level is 9, with leaves turning yellowish-brown, leaf tips curling and drying out (I > 40%). When the leaf damage level of the susceptible control material reaches level 7 or above, an investigation is conducted, and the insect infestation index is calculated: ; Material resistance rating standards: Based on the insect infestation index of the tested plant germplasm materials, the insect resistance of the tested plant germplasm materials is divided into five levels: highly resistant, resistant, moderately resistant, susceptible, and highly susceptible. The specific grading criteria for plant material resistance to thrips are as follows: The resistance level is high resistance, and the insect infestation index If (%) is: 0 < If ≤ 15; The resistance level is resistant, and the insect infestation index If (%) is: 15 < If ≤ 30; The resistance level is moderately resistant, and the insect infestation index If (%) is: 30 < If ≤ 50; The resistance level is susceptible, and the infestation index If (%) is: 50 < If ≤ 70; The resistance level is highly susceptible, and the infestation index If (%) is: If > 70; Among them, when the insect infestation index of the insect-infected control material in the experiment reaches 50% or more, the identification result is considered valid; Record the identification and evaluation results.

[0016] Compared with the prior art, the present invention has the following beneficial effects: The culture device provided by this invention for rapidly identifying plant germplasm resistance to thrips is simple, portable, and low in cost. It can highly simulate the natural environment, avoid interference from unstable factors, conduct multiple reproducible tests simultaneously, and extend the leaf preservation time by supplementing nutrient solution, making it convenient to accurately observe the complete life cycle of thrips.

[0017] The present invention provides a method for using and evaluating a culture device for rapidly identifying plant germplasm resistance to thrips, which involves inoculating plant leaves with a single female adult thrips. Its advantages are: ① Compared to inoculation with larvae, inoculation with adults overcomes the limitations of larvae's low activity level and single feeding site. ② Compared with inoculating with larvae of the same age, the method of inoculating with female adults overcomes the disadvantages of difficulty in distinguishing between male and female thrips larvae in their early stages, significant differences in feeding habits, and easy impact on evaluation results. ③ The survey method uses the ratio of the area of ​​thrips scabs on the leaves to the total leaf area as the grading standard. Based on this, the degree of damage caused by thrips to plant leaves can be accurately distinguished. At the same time, multiple replicates can be set up according to the experimental needs to scientifically reflect the resistance trend of plant germplasm to thrips and increase the accuracy of identification. Attached Figure Description

[0018] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on the provided drawings without creative effort.

[0019] Figure 1 This is a three-dimensional structural schematic diagram of a culture device for rapidly identifying plant germplasm resistance to thrips according to the present invention; Figure 2 This is a schematic diagram of the internal structure of a culture device for rapidly identifying plant germplasm resistance to thrips according to the present invention; Figure 3 This is a front view of the culture tube; Figure 4 This is a front view of a centrifuge tube; Figure 5 This is a top view of a centrifuge tube.

[0020] Figure 6 This invention relates to a culture device for rapidly identifying plant germplasm resistance to thrips. The device is used to identify adult female thrips (taking *Thrips serratus*, *Thrips melonica*, *Thrips serratus*, and *Thrips spp.* as examples).

[0021] Figure 7 The study investigated the leaf damage of 12 tomato varieties 3 days after inoculation with western flower thrips.

[0022] Figure 8 To investigate the insect infestation index of 12 tomato varieties 3 days after inoculating detached leaves with western flower thrips.

[0023] Figure 9 The results of the survey and evaluation of the resistance of 12 tomato varieties to western flower thrips.

[0024] The components include: 1. Box body; 2. Door; 3. Door handle; 4. CNC display screen; 5. Ventilation vent; 6. Water inlet; 7. Power cord interface; 8. Door hinge; 9. Heating wire; 10. White light tube; 11. Fan; 12. Metal cover; 13. Temperature and humidity probe; 14. Culture tube rack; 15. Placement slot; 16. Controller; 17. Culture tube; 171. Culture tube body; 172. Tube cap; 173. Mesh; 174. Operating hole; 175. Substrate filling layer; 176. Sponge pad; 177. Centrifuge tube; 1771. Centrifuge tube cavity; 1772. Centrifuge tube cover; 1773. Connecting parts; 1774. Insertion hole; 178. Water inlet pipe. Detailed Implementation

[0025] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0026] Example 1

[0027] refer to Figure 1 - Figure 5 A culture device for rapidly identifying plant germplasm resistance to thrips, comprising: The natural environment simulation unit includes a chamber that can automatically adjust the internal temperature, humidity, light and water replenishment, a culture tube rack set inside the chamber, and multiple placement slots on the culture tube rack. The insect growth environment simulation unit includes multiple culture tubes 17 located inside a box; each culture tube 17 is a small culture tube, comprising: The culture tube body 171 is a transparent container with an open top; the culture tube body 171 is placed in the placement slot 15; The cap 172 is detachably connected to the top opening of the culture tube body 171; A mesh 173 covers the top opening of the culture tube body 171 and is pressed and fixed by a tube cap 172; the mesh count of the mesh 173 is at least 250 mesh. Centrifuge tube 177 is located inside culture tube body 171; A sponge pad 176 is placed at the bottom of the culture tube body 171; the sponge pad 176 has a hole in the center, and the centrifuge tube 177 is installed in the hole.

[0028] In this embodiment, the enclosure includes an enclosure body 1, a door 2, a door handle 3, a digital control display screen 4, a ventilation opening 5, a water inlet 6, a power cord interface 7, a door hinge 8, a heating wire 9, a white light tube 10, a fan 11, a metal cover 12, a temperature and humidity probe 13, and a culture tube rack 14, a placement slot 15, a controller 16, and a culture tube 17 disposed inside the enclosure body 1.

[0029] Specifically, the front of the cabinet body 1 is provided with a door 2, which is connected to the cabinet body by a door hinge 8. A door handle 3 is provided on the side of the door 2 to facilitate opening the cabinet. A digital control display screen 4 is provided in the upper middle part of the door 2, and a ventilation opening 5 is provided in the middle of the door 2. A water inlet 6 and a power cord interface 7 are provided on the bottom side of one side of the cabinet body 1.

[0030] The bottom of the main body 1 of the box is equipped with a culture tube rack 14. The culture tube 17 is fixed on the culture tube rack 14 through the placement groove 15. The water injection pipe 178 extending from the outer wall of the culture tube 17 is connected to the water injection port 6 through the interconnecting hose. After the nutrient solution is injected from the water injection port 6, it can directly reach the centrifuge chamber 1771.

[0031] At the bottom of the main body 1, below the culture tube rack 14, a controller 16 is installed. The controller 16 obtains power through the power cord interface 7.

[0032] A temperature and humidity probe 13 is installed on the inner wall of the cabinet body 1. The temperature and humidity probe 13 senses the temperature and humidity of the space inside the cabinet and transmits the real-time temperature and humidity data to the digital control display screen 4 through the controller 16 connected by electrical connection. According to the temperature and humidity range set by the user on the digital control display screen 4, the probe 13 automatically controls the start and stop of the fan 11 and the heating wire 9, thereby adjusting the temperature and humidity inside the cabinet body 1 to a constant and controllable range.

[0033] A pair of white light tubes 10 are installed on the inner wall of the box body 1 and are electrically connected to the controller 16. The daily light cycle time program is set through the digital control display screen 4 according to the needs of the cultivated plants to control the switching of the light tubes.

[0034] In this embodiment, the centrifuge tube 177 includes a centrifuge tube cavity 1771, a centrifuge tube cap 1772, and a connecting component 1773; wherein, the centrifuge tube cavity 1771 is connected to the centrifuge tube cap 1772 through the connecting component 1773.

[0035] In this embodiment, the centrifuge tube cap 1772 is provided with an insertion hole 1774 for inserting plant petioles and water injection pipes.

[0036] In this embodiment, the culture tube body 171 is a transparent centrifuge tube with a nominal capacity of 50 mL, a height of 11 cm without a cap, and a diameter of 3 cm.

[0037] In this embodiment, the centrifuge tube 177 has a capacity of 1.5 mL, 2 mL, or 5 mL.

[0038] In this embodiment, the thickness of the sponge pad 176 is 5mm, and the diameter of the central hole is 1cm.

[0039] In this embodiment, an operation hole 174 is provided on the upper side wall of the culture tube body 171. The diameter of the operation hole 174 is 5mm, and a sterile cotton plug is provided on the operation hole 174.

[0040] Specifically, the culture tube body 171 is a cylindrical structure with an open top, made of transparent plastic to facilitate observation of the thrips' activity and leaf condition inside the device. The culture tube body 171 is equipped with a cap 172 for sealing the opening, the size of which can be selected according to the size of the detached plant leaf. In this embodiment, a transparent centrifuge tube with a nominal capacity of 50mL is used as the culture tube body 171. The length of the culture tube body 171 with the cap 172 is 12cm, the diameter is 3cm, and the height without the cap 172 is 11cm.

[0041] Cap 172 and mesh 173: Cap 172 is connected to the top opening of culture tube body 171 by a screw thread; mesh 173 has a diameter larger than the opening diameter at the top of culture tube body 171, and is fixed by screwing on cap 172 after covering the opening; the mesh 173 used in this embodiment has a mesh count of 250 to ensure air circulation inside and outside the device, while effectively preventing thrips from escaping.

[0042] Sponge pad 176: Located in the lower part of the culture tube body 171, the sponge pad 176 is 5mm thick and has a hole in the center. The diameter of the hole is adapted to the outer diameter of the centrifuge tube 177. It is used to fix the centrifuge tube 177 and also plays a role in buffering and isolation to prevent the substrate filling layer 175 from directly contacting the bottom of the centrifuge tube 177.

[0043] Centrifuge tube 177: Includes centrifuge tube cavity 1771, centrifuge tube cap 1772, connecting component 1773, and water inlet pipe 178. The centrifuge tube cavity 1771 and centrifuge tube cap 1772 are connected by the connecting component 1773. The centrifuge tube cap 1772 has two insertion holes 1774 in its center. One insertion hole is connected to the water inlet pipe 178 and extends into the centrifuge tube cavity 1771 for delivering nutrient solution to provide water and nutrients to the detached plant leaves. The other insertion hole is used to insert the petiole of the detached plant leaf to be tested. The diameter of the insertion hole is preferably such that the petiole of the detached plant leaf to be tested can be inserted, and the insertion depth of the petiole is preferably such that the cut surface of the petiole can be immersed in the nutrient solution. Its front view is shown below. Figure 4 Top view Figure 5Centrifuge tube 177 passes through the holes in the sponge pad 176. Centrifuge tube cap 1772 and part of centrifuge tube lumen 1771 are located above the sponge pad 176. The capacity of centrifuge tube 177 can be selected as 1.5ml, 2ml or 5ml according to the size of the culture tube body 171 and the water requirement of the leaves. The leaf culture time can be further extended by artificially supplementing nutrient solution.

[0044] Matrix filling layer 175: Filled inside the culture tube body 171, surrounding the centrifuge tube cavity 1771, with a filling height up to the centrifuge tube cap 1772; the matrix filling layer 175 is made of materials that can simulate the field soil environment to meet the soil requirements of different morphologies in the life history of thrips (such as the 3rd and 4th instar pseudopupa stages of thrips).

[0045] Sterile cotton plug and operation hole 174: At least one operation hole 174 with a diameter of 5 mm is opened on the side wall of the culture tube body 171. A sterile cotton plug is inserted into the operation hole 174. The operation hole 174 is used to use a suction device to remove thrips of different ages, male and female, or eggs, for the purpose of statistical analysis of egg production, damage level, and other measurement indicators. The sterile cotton plug ensures the sealing of the operation hole, preventing thrips from escaping during suction, and also makes it easy to remove and operate at any time.

[0046] In this embodiment, as a preferred technical solution, the components of the cultivation device are selected and assembled as follows: ① Selection of device components.

[0047] The white light tube 10 uses a continuous plant spectrum of 400nm to 780nm, which is an LED plant light that meets the needs of plant cultivation. The power is ≤24W and the light intensity is about 5000Lux to 10000Lux. The heating temperature of the metal wire is usually 30℃ to 100℃. The temperature measurement range of the temperature and humidity probe 13 (sensor) is 0℃ to 50℃, and the humidity measurement range is usually 0%RH to 100%RH. The wind speed of the fan 11 is dynamically adjustable between 0 and 3m / s.

[0048] The culture tube body 171 is a transparent centrifuge tube with a nominal capacity of 50ml. The centrifuge tube cap 1772 is 12cm long, 3cm in diameter, and 11cm high without the cap. The centrifuge tube 177 is 1.5ml. The gauze 173 is 250 mesh. The sponge pad 176 is 5mm thick with a 1cm hole in the center. The substrate filling layer 175 is a material made of sterile soil and vermiculite mixed in a volume ratio of 1:1. The sterile cotton plug operation hole 174 is 5mm in diameter.

[0049] ② Device assembly steps.

[0050] A controller 16 is installed at the bottom of the enclosure. The controller 16 obtains power through the power cord interface 7 and electrically starts the fan 11, temperature and humidity probe 13, heating wire 9 and white light tube 10. The above electrical structures are electrically connected to the CNC display screen 4 to display the readings. A culture tube rack 14 is installed above the controller 16, and a culture tube 17 is placed in the placement trough 15. The outer wall of the culture tube 17 is connected to the water inlet 6 through an interconnecting hose. An appropriate amount of nutrient solution is injected into the water inlet 6, and the nutrient solution reaches the centrifuge chamber 1771 through the hose and the water inlet pipe 178. Take a 1.5ml centrifuge tube 177 containing nutrient solution, tighten the cap 1772, select fresh tomato leaves detached from the body, trim the petiole to a length of 2cm to 3cm, insert it into the insertion hole 1774, so that the end of the petiole is immersed in the nutrient solution, which is Hoagland's nutrient solution diluted 10 times. Centrifuge tube 177 is inserted through the central hole of sponge pad 176, so that sponge pad 176 is located in the lower middle part of centrifuge tube 177, ensuring that centrifuge tube cavity 1771 is stably fixed, and then placed into culture tube body 171. Fill the culture tube body 171 with the substrate filling layer 175 until the height of the substrate filling layer 175 is almost flush with the centrifuge tube cap 1772. Gently compact the substrate filling layer to avoid damaging the leaves. The right-angled glass water injection tube 178 is inserted through the small hot-melt hole on the outer wall of the culture tube body 171 and then inserted into the centrifuge tube 177 along the insertion hole 1774 on the tube cap 1772. Cover the top opening of the culture tube body 171 with a 250-mesh gauze 173, with the edge of the gauze 173 extending 10mm to 20mm beyond the opening. Tighten the tube cap 172 to fix the gauze 173. Insert a sterile cotton plug into the 5mm operation hole 174 pre-set on the side wall of the culture tube body 171 to complete the assembly of the culture tube 17. Place the culture tube 17 into the placement tank 15, connect one end of the transparent flexible tube to the water injection tube 178, and the other end to the water injection port 6 to complete the device assembly.

[0051] Example 2

[0052] A method for using a culture device for rapidly identifying plant germplasm resistance to thrips, using the culture device for rapidly identifying plant germplasm resistance to thrips provided in Example 1, the method includes the following steps: S10. Material Preparation Plant materials: Plant varieties to be evaluated for insect resistance were grown in an artificial climate chamber, including insect-resistant and insect-susceptible control materials; seeds of the test plant varieties were sown in seedling trays and cultured at a temperature of 25℃~28℃, humidity of 60%~80%, and a photoperiod of 16h:8h (L:D), with other conventional culture methods, and ensuring that they were not affected by any diseases or pests. Insect source material: The test insect source was a single natural thrips population collected from the field and raised indoors in the laboratory for more than 3 generations, without any contact with chemical pesticides during the rearing and breeding period; Specifically, let's take the test tomato variety as an example.

[0053] Seeds of the tomato varieties to be tested (including insect-resistant and insect-susceptible control materials) were soaked in water and germinated. After germination, they were sown in seedling trays containing substrate and cultured at a temperature of 25℃–28℃, humidity of 60%–80%, and a photoperiod of 16h:8h (L:D). Other culture methods were standard. Twenty seedlings of each variety were sown, and the seedlings were ready for use when the top leaves reached a width of 3–4 cm.

[0054] The test insects were a single population of western flower thrips collected from the field and reared indoors in the laboratory for more than three generations, without exposure to any chemical pesticides during the rearing and breeding period. Fully emerged female thrips were then used for testing.

[0055] S20, Insecticide

[0056] When the seedlings grow to the top and the leaves are 3-4 cm wide, retain the leaves and petioles, cut them off at the base of the petioles, and place them in the culture tube 17. Let the petiole pass through the centrifuge tube cap 1772 and extend into the centrifuge tube cavity 1771. Nutrient solution is injected into the centrifuge tube cavity 1771 in advance, and the cut of the petiole extends below the surface of the nutrient solution.

[0057] One live adult female thrips was placed in the culture tube body 171 and positioned on a leaf. A mesh screen 173 was placed over the opening of the culture tube body 171, and then the hollowed-out tube cap 172 was pressed onto the mesh screen 173, ensuring a tight connection with the threaded opening at the top of the culture tube body 171. Twenty replicates were set up for each tomato variety, as needed for the experiment. S30. Post-inoculation management After inoculation, the culture tube 17 is fixed to the culture tube rack 14 through the placement groove 15. The water injection tube 178 extending from the outer wall of the culture tube 17 is connected to the water injection port 6 through the interconnecting hose. The power supply is connected through the power cord interface 7. The culture environment parameters are set on the CNC display screen 4, and the survey data is observed and recorded at regular intervals according to the inoculation time.

[0058] Specifically, the culture tube 17 after inoculation is fixed on the culture tube rack 14 through the placement groove 15 (multiple replicates can be set according to the needs of the experiment). The water injection tube 178 extending from the outer wall of the culture tube 17 is connected to the water injection port 6 through the interconnecting hose. The power supply is connected through the power cord interface 7. The culture environment parameters are set on the digital control display screen 4: culture temperature 25℃~28℃, humidity 60%~80%, photoperiod 16h:8h (L:D), light intensity around 5000Lux~10000Lux. The inoculation time is recorded and the activity and feeding status of thrips are observed regularly. The damage to the leaves and the survey data are recorded. During this process, the nutrient solution level in the centrifuge chamber 1771 is observed. Nutrient solution is added to the centrifuge chamber 1771 in a timely manner through the water injection port 6 so that the petiole cut of the tomato leaf being tested is always immersed in the nutrient solution.

[0059] Example 3

[0060] A method for rapidly identifying and evaluating plant germplasm resistance to thrips includes investigating and evaluating the degree of thrips damage to inoculated leaves cultured using the cultivation device for rapidly identifying plant germplasm resistance to thrips provided in Example 2. The investigation and evaluation method is as follows: Based on the degree of thrips damage (I) to the inoculated leaves, the leaf damage level is divided into six levels: 0, 1, 3, 5, 7, and 9. Here, I represents the percentage of the leaf area marked by thrips bites. The grading criteria are as follows: The leaf damage level is 0, the leaf is undamaged, I=0; The leaf damage level is 1, with pinhead-sized insect bite marks on the leaf edges, I ≤1%; The leaf damage level is 3, with a small amount of chlorotic spots appearing on the leaves, 1% < I ≤ 5%; The leaf damage level is 5, with slight chlorosis, 5% < I ≤ 15%; The leaf damage level is 7, with obvious chlorosis and leaf tip curling of 15% < I ≤ 30%; The leaf damage level is 9, with leaves turning yellowish-brown, leaf tips curling and drying out (I > 40%). Please see Table 1 below for specific grading standards: Table 1. Grading criteria for thrips damage to plant leaves

[0061] The algorithm for the leaf damage area ratio (I) is as follows: Open the image of the leaf sample on your computer, use ImageJ software to count the feeding area of ​​thrips on the leaf, and use ImageJ to calculate the percentage of the area. This can generally be done by following these steps: Open the image: Launch ImageJ software, click the "File" menu, select "Open", and open the image you want to analyze.

[0062] Convert image type: Click the "Image" menu, select "Type" to convert the image to an 8-bit grayscale image for subsequent processing.

[0063] To set the threshold: Click the "Image" menu, select "Adjust," and then click "Threshold" to open the threshold adjustment dialog box. Adjust the threshold by dragging the slider or entering a value so that the area to be calculated is highlighted in red (default). If you need to measure a bright area, check the "Dark background" option. After setting, click the "Set" and "OK" buttons.

[0064] To set measurement parameters: Click the "Analyze" menu, select "Set Measurements," and in the pop-up dialog box, check the "Area Fraction" option. You can also check other measurement parameters as needed, and then click "OK." To measure area percentage: Click the "Analyze" menu, select "Measure," or use the shortcut "M." ImageJ will automatically calculate and display the area percentage of the selected region in the results window.

[0065] When the leaf damage level of the susceptible control material reaches level 7 or above, an investigation is conducted, and the insect infestation index is calculated: ; Material resistance rating standards: Based on the insect infestation index of the tested plant germplasm materials, the insect resistance of the tested plant germplasm materials is divided into five levels: highly resistant, resistant, moderately resistant, susceptible, and highly susceptible. The specific grading criteria for plant material resistance to thrips are as follows: The resistance level is high resistance, and the insect infestation index If (%) is: 0 < If ≤ 15; The resistance level is resistant, and the insect infestation index If (%) is: 15 < If ≤ 30; The resistance level is moderately resistant, and the insect infestation index If (%) is: 30 < If ≤ 50; The resistance level is susceptible, and the infestation index If (%) is: 50 < If ≤ 70; The resistance level is highly susceptible, and the infestation index If (%) is: If > 70; The specific rating criteria are shown in Table 2: Table 2. Grading Criteria for Thrips Resistance Identification in Plant Materials

[0066] Among them, when the insect infestation index of the insect-infected control material in the experiment reaches 50% or more, the identification result is considered valid; Record the identification and evaluation results Record detailed data including identification location (latitude and longitude), date of insect inoculation, date of investigation, investigator, test materials and their sources, insect infestation index, and insect resistance level.

[0067] Using a simple culture apparatus made from the small culture tube 17, and simulating the natural environment under artificial culture chamber conditions, the resistance of tomato varieties to thrips was identified. The results are shown in the appendix to the instruction manual. Figures 7-9 .

[0068] Device testing: 1. Environmental adaptability test.

[0069] The assembled device was placed in environments with temperatures of -20℃, 25℃, and 50℃ and relative humidity of 10%, 60%, and 90% for 7 days under each environmental condition. The simulated environmental conditions such as temperature, humidity, and light in the device all met the set conditions and the device operated stably. The structural integrity of the device and the freshness of the leaves were observed: the results showed that the device had no cracks or deformation, the cultivated tomato leaves could remain fresh under each environment, and the thrips samples were active normally, proving that the device met the requirements of the working range.

[0070] 2. Repeated testing.

[0071] After use, the device was cleaned. The culture tube body 171, centrifuge tube 177, and tube cap 172 were wiped and disinfected with 75% alcohol. The sponge pad 176 and gauze 173 were sterilized by high-pressure steam. The device was reassembled and used for three culture experiments. The results showed that the structural stability of the device did not decrease, and the leaf preservation time and thrips culture effect were consistent with the first use, proving that the device can be reused and reducing experimental costs.

[0072] The technical solutions of the present invention have been fully described above. It should be noted that the specific embodiments of the present invention are not limited to the above description. All technical solutions formed by those skilled in the art based on the spirit and essence of the present invention by adopting equivalent transformations or equivalent transformations in terms of structure, method or function fall within the protection scope of the present invention.

Claims

1. A culture device for rapid identification of plant germplasm resistance to thrips, characterized by, It comprises: a natural environment simulation unit, which comprises a box body with the functions of automatically adjusting the internal environment temperature, humidity, illumination and water replenishment, and a culture tube rack arranged in the box body, and a plurality of placing slots are arranged on the culture tube rack; a worm body growth environment simulation unit, which comprises a plurality of culture tubes arranged in the box body; the culture tube comprises: a culture tube body, which is a transparent container with an open top; the culture tube body is arranged in the placing slot; a tube cap, which is detachably connected with the top opening of the culture tube body; a gauze, which is covered at the top opening of the culture tube body and is tightly fixed by the tube cap; the gauze has a mesh number of at least 250 meshes; a centrifugal tube, which is located in the interior of the culture tube body; a sponge pad, which is arranged at the bottom of the culture tube body; the sponge pad is provided with a hole in the center, and the centrifugal tube is installed in the hole.

2. The culture device for rapid identification of plant germplasm resistance to plant thrips according to claim 1, characterized in that, The centrifugal tube comprises a centrifugal tube cavity, a centrifugal tube cover and a connecting component; wherein the centrifugal tube cavity is connected with the centrifugal tube cover through the connecting component.

3. The culture device for rapid identification of plant germplasm resistance to plant thrips according to claim 2, characterized in that, The centrifugal tube cover is provided with a insertion hole for inserting a plant petiole and a water injection tube.

4. The culture device for rapid identification of plant germplasm resistance to plant thrips according to claim 1, wherein, The culture tube body is a transparent centrifugal tube with a nominal capacity of 50 mL, and the height without cover is 11 cm and the diameter is 3 cm.

5. The culture device for rapid identification of plant germplasm resistance to thrips according to claim 1, wherein, The capacity of the centrifugal tube is 1.5 mL, 2 mL or 5 mL.

6. The culture device for rapid identification of plant germplasm resistance to plant thrips according to claim 1, wherein, The thickness of the sponge pad is 5 mm, and the diameter of the center hole is 1 cm.

7. The culture device for rapid identification of plant germplasm resistance to thrips according to claim 1, wherein, An operation hole is arranged on the upper end side wall of the culture tube body, and the diameter of the operation hole is 5 mm.

8. A method of using a culture device for rapid identification of plant germplasm resistance to thrips, characterized by, The use method of the culture device for rapidly identifying the resistance of plant germplasm to thrips according to claims 1 to 7 comprises the following steps: S10, material preparation Plant material: plant variety materials to be evaluated for insect resistance are planted in a phytotron, which includes insect-resistant and insect-susceptible material controls; Worm source material: the test worm source is a natural thrips single population collected from the field and has been bred in the laboratory for more than 3 generations; S20, insect inoculation When the seedlings grow to a width of 3-4 cm at the top of the leaves, the leaves and petioles are retained, cut at the root of the petiole, and moved to the culture tube, with the petiole passing through the centrifugal tube cover and extending into the centrifugal tube cavity, the centrifugal tube cavity being pre-injected with nutrient solution, and the cut of the petiole extending below the liquid level of the nutrient solution; Take 1 head of thrips adult female live worm body, move it to the culture tube body and place it on the leaf, cover the culture tube body with a gauze, press the center hollow tube cap on the gauze, and tightly connect it with the top threaded opening of the culture tube body; according to the test requirements, set enough repeated treatments; S30, management after insect inoculation After inoculation, the culture tube is fixed on the culture tube rack through the placing slot, the connecting tube protruding from the outer wall of the culture tube is connected to the water injection port through the intercommunication hose, the power supply is connected through the power line interface, the culture environment parameters are set on the numerical control display screen, and the data are observed, recorded and investigated at regular intervals according to the inoculation time.

9. A method for the rapid identification of plant germplasm for thrips resistance investigation and evaluation, characterized by, The investigation and evaluation of the damage degree of the inoculated leaves cultured by the use method of the culture device for rapidly identifying the resistance of plant germplasm to thrips according to claim 8 are carried out by thrips, and the investigation and evaluation method is: According to the degree of leaf blade damage by thrips, the leaf blade damage level is divided into 0, 1, 3, 5, 7, 9, wherein I is the ratio of the area of leaf blade damaged by thrips to the whole leaf blade area, and the grading standard is as follows: The leaf blade damage level is 0, the leaf blade is not damaged, and I = 0; The leaf blade damage level is 1, there are pinhead-sized insect bite marks on the edge of the leaf blade, and I ≤ 1%; The leaf blade damage level is 3, a small amount of green fading marks appear on the leaf blade, and 1% < I ≤ 5%; The leaf blade damage level is 5, the leaf blade is slightly yellow, and 5% < I ≤ 15%; The leaf blade damage level is 7, the leaf blade is obviously yellow, and the leaf tip is rolled and shriveled, and 15% < I ≤ 30%; The leaf blade damage level is 9, the leaf blade shows yellowish brown, and the leaf tip is rolled and dried, and I > 40%; When the leaf blade damage level of the susceptible control material reaches level 7 or above, the investigation is carried out, and the insect condition index is calculated: ； The resistance rating standard of the material: According to the insect condition index of the identified material, the resistance of the test plant germplasm material is divided into high resistance, resistance, medium resistance, susceptibility and high susceptibility, and the resistance rating standard of the plant material to thrips is as follows: The resistance level is high resistance, the insect condition index If (%) is: 0 < If ≤ 15; The resistance level is resistance, the insect condition index If (%) is: 15 < If ≤ 30; The resistance level is medium resistance, the insect condition index If (%) is: 30 < If ≤ 50; The resistance level is susceptible, the insect condition index If (%) is: 50 < If ≤ 70; The resistance level is high susceptibility, the insect condition index If (%) is: If > 70; Among them, when the insect condition index of the susceptible control material in the test reaches 50% or more, it is considered that the identification result is valid; Record the identification and evaluation results.

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