Thrip resistance identification device and application

Abstract

The invention discloses a thrip resistance identification device and application. The device comprises an inoculation barrel, a preservation substrate is laid at the bottom in the inoculation barrel, an isolation structure with one or more through holes covers the preservation substrate, and a sealing structure with one or more air holes covers an opening in the top of the inoculation barrel. When the device is used, the whole plant does not need to be cultivated, in-vitro flowering branches can be picked for identification, rapid screening of large-scale germplasm resources can be achieved, and repeatability and consistency of experimental results are good. Meanwhile, when the device is used, buds and functional leaves of in-vitro flowering branches are reserved, the actual feeding part of the Frankliniella occidentalis is simulated, and compared with in-vitro leaf inoculation, the real resistance level can be better reflected. The device has the advantages of easily available materials, low cost and strong operability, can be used for identifying the resistance of the Frankliniella occidentalis of various materials such as chrysanthemum and related wild species, cultivated species, breeding offspring and the like, provides a high-quality parent material for insect-resistant breeding, can be used for researching an insect-resistant mechanism, and is suitable for popularization and application in basic breeding units.

Description

Technical Field

[0001] This invention belongs to the field of plant resistance breeding technology, and particularly relates to a thrips resistance identification device and its application. Background Technology

[0002] chrysanthemum( Chrysanthemum × morifolium Chrysanthemums are one of the world's four major cut flowers and hold an important position in the global flower industry. However, they are susceptible to various pests during their growth, among which thrips, especially the western flower thrips (Thrips spp.), are the dominant pests affecting chrysanthemums. The western flower thrips not only feed directly on plant sap with their rasping-sucking mouthparts, leaving silvery-white spots on leaves and flowers, leading to plant stunting and deformities, but they are also highly efficient vectors for various plant viruses. For example, infection with tomato wilt virus and impatiens necrotic spot virus causes ring spots, necrosis, stunted growth, and even death on leaves, and once infected, it is incurable, resulting in continuous losses to the ornamental quality and yield of chrysanthemums. Studies have shown that there are significant differences in resistance to western flower thrips among chrysanthemum varieties, and traits such as flower color and shape affect the thrips' feeding preferences. However, the resistance mechanism is complex and not determined by a single factor, but involves the combined effects of various phenolic compounds and other secondary metabolites.

[0003] Currently, the identification of chrysanthemum resistance to western flower thrips mainly relies on two types of live inoculation methods. Whole-plant inoculation requires culturing plants to a specific growth stage, typically taking 30-60 days. This method is time-consuming and requires a large area, making it unsuitable for large-scale germplasm resource screening. Furthermore, environmental conditions are difficult to control, affecting experimental consistency. Additionally, western flower thrips are small, highly mobile, and easily escape, leading to inaccurate inoculation amounts. While detached leaf inoculation shortens the time, it primarily reflects leaf resistance, neglecting the thrips' objective feeding preferences and failing to simulate the overall resistance response of the flower stalk. Moreover, detached leaves are prone to water loss and wilting, requiring frequent replacement and making the process cumbersome. Summary of the Invention

[0004] Purpose of the invention: The purpose of this invention is to provide a thrips resistance identification device that has good moisturizing effect, reliable resistance evaluation results, and can prevent thrips from escaping.

[0005] Technical Solution: The thrips resistance identification device of the present invention includes an inoculation tank. A preservative substrate is laid at the bottom of the inoculation tank, and an isolation structure with one or more through holes is placed on top of it. The top opening of the inoculation tank is covered with a sealing structure with one or more vent holes. In this device, the preservative substrate fully fills the inoculation tank, the isolation structure completely covers the preservative substrate, separating the preservative substrate from the upper space inside the inoculation tank, and the sealing structure ensures the inoculation tank is sealed, preventing thrips from escaping.

[0006] Preferably, the inoculation barrel is a transparent cylindrical container, and the material thereof includes but is not limited to glass, polyethylene terephthalate, polycarbonate resin, polymethyl methacrylate, polyethylene, and polypropylene; further preferably, the bottom diameter of the inoculation barrel is 15-25 cm, and the height is 5-15 cm.

[0007] Preferably, the fresh-keeping substrate is water agar; further preferably, the concentration of the water agar is 1.5-2.5%, and the thickness is 1-1.5 cm.

[0008] Preferably, the isolation structure and the sealing structure are both organic polymer material films; further preferably, the organic polymer material films include but are not limited to polyurethane, polyester, cellulose derivative, and polyolefin; more preferably, the thickness of the isolation structure and the sealing structure is both 0.1-0.3 mm.

[0009] Preferably, the pore diameter of the through hole on the isolation structure is 0.8-1.2 cm, and the distance between the through holes is 2-5 cm.

[0010] Preferably, the pore diameter of the air-permeable hole on the sealing structure is 0.1-0.3 cm.

[0011] The thrips resistance identification device can be applied to the resistance identification of the western flower thrips in the Asteraceae plant.

[0012] Preferably, the application steps include: S1, picking an in-vitro Asteraceae plant flower branch with a length of 10-15 cm, and uniformly reserving at least one full flower bud and at least one healthy functional leaf; S2, inserting the flower branch obtained in S1 into the fresh-keeping substrate through the through hole on the isolation structure, and the insertion depth is 1-1.5 cm; S3, inoculating 40-60 starved western flower thrips adults in the inoculation barrel, sealing the sealing structure, and culturing for 6-8 days, counting the number of western flower thrips, and completing the resistance level identification of the horticultural plant.

[0013] Beneficial Effects: Compared with existing technologies, this invention has the following significant advantages: 1. This thrips resistance identification device does not require the cultivation of whole plants; detached flower branches can be used for identification, with an experimental cycle of only 7 days, which is significantly shorter than whole-plant inoculation identification, enabling rapid screening of large-scale germplasm resources; 2. The device's sealed design prevents thrips from escaping, and combined with artificial climate chamber cultivation, it reduces interference from environmental and human factors, resulting in good repeatability and consistency of experimental results; 3. This device is specifically designed for detached chrysanthemum flower branches, preserving flower buds and functional leaves to simulate the actual feeding sites of western flower thrips on chrysanthemum plants, reflecting the true resistance level of chrysanthemums better than detached leaf inoculation; 4. The device uses readily available and inexpensive materials, has simple experimental operation steps, requires no complex equipment, and is highly operable. It can be used for the identification of western flower thrips resistance in various materials such as wild species, cultivated varieties, and breeding progeny of the Chrysanthemum genus and its closely related genera, providing high-quality parental materials for insect-resistant breeding. It can also be used for research on insect resistance mechanisms and is suitable for promotion and application in grassroots breeding units. Attached Figure Description

[0014] Figure 1 This is a schematic diagram of the thrips resistance identification device in Example 1; Figure 2 This is a schematic diagram of the isolation structure with through holes in the thrips resistance identification device of Example 1; Figure 3 Example 1 shows the thrips resistance identification device used for identification of western thrips resistance in Chrysanthemum plants. In the diagram, a is a schematic diagram of the flower branch layout, b is a schematic diagram of inoculation of western thrips after covering the preservation substrate with an isolation structure, c is a schematic diagram of the preservation substrate, d is a schematic diagram of western thrips resistance identification of wild Chrysanthemum resources in the flowering period, and e is a schematic diagram of western thrips resistance identification of cut Chrysanthemum in the bud stage. Figure 4 This is a schematic diagram of counting western flower thrips after soaking in alcohol, where a - schematic diagram of western flower thrips after soaking in alcohol; b - enlarged view of a part; c - inoculated western flower thrips adults. Detailed Implementation

[0015] The technical solution of the present invention will be further described below with reference to the accompanying drawings.

[0016] Figure 1 The attached diagrams are labeled as follows: Inoculation bucket-1, Preservation substrate-2, Isolation structure-3, Through hole-4, Sealing structure-5, Ventilation hole-6.

[0017] Example 1: Establishment of a Thrips Resistance Identification Device A thrips resistance identification device, such as Figure 1 As shown, the inoculation container 1 is a transparent cylindrical polypropylene (PP) container with a diameter of 20 cm and a height of 15 cm, which is convenient for observing the condition of the flower branches and the activity of thrips after inoculation.

[0018] The bottom of the inoculation bucket 1 is lined with a preservation substrate 2. In this embodiment, a 2.0% concentration of water agar is used as the preservation substrate. After the inoculation bucket 1 is disinfected, sterilized water agar is poured in. After solidification, the thickness is 1 cm. Water agar can provide a stable water supply for the detached flower branches, prolonging the survival period of the flower branches to more than 7 days, and can also prevent the flower branches from rotting due to excessive water.

[0019] A 12-hole isolation structure 3 is provided on the preservation substrate 2. In this embodiment, a 0.15 mm thick polyvinyl chloride (PVC) film is used as the isolation structure 3. The holes 4 are spaced 2 cm apart and evenly arranged along the edge of the PVC film. The distance between the holes 4 and the edge of the film is 0.8 cm. The isolation structure 3 with holes 4 (e.g. Figure 2 (As shown) It can fix the position of the flower branches, ensure the even distribution of the flower branches, and isolate thrips from water agar. Moreover, the PVC material has high transparency and strong observation, which can prevent thrips from hiding in the moisturizing layer and being difficult to count.

[0020] The top opening of the inoculation container 1 is covered by a sealing structure 5 with multiple ventilation holes 6. In this embodiment, commercially available polyethylene (PE) plastic wrap is used as the sealing structure 5, and holes are evenly punched in the plastic wrap with a needle with a specification of 0.6×25 mm TWLB as ventilation holes 6. The sealing structure 5 with ventilation holes 6 not only ensures air circulation inside the inoculation container 1 to meet the breathing needs of thrips and flower branches, but also effectively prevents thrips from escaping and ensures the accuracy of the inoculation amount during resistance identification.

[0021] Experimental Example 1: Thrips Resistance Identification Device Used for Identification of Thrips Resistance in Western Flower 1. Identification of western flower thrips resistance in germplasm resources of Chrysanthemum and its closely related genera. In this experiment, the following species were provided by the National Southern Herbaceous Flower Germplasm Resource Nursery established by Nanjing Agricultural University: Hibiscus Chrysanthemum, Daruma Chrysanthemum, Nanjing Wild Chrysanthemum, Huaji Chrysanthemum, Tangshan Wild Chrysanthemum, Zijinshan Wild Chrysanthemum, Maohua Chrysanthemum, Satsuma Wild Chrysanthemum, and Chrysanthemum Brain.

[0022] 1.1 Identification of resistance to western flower thrips based on detached flower branches Healthy detached flower branches from each material were selected and uniformly treated to a length of 14 cm, retaining 4 flower buds and 2 functional leaves. For *Chrysanthemum indicum* and *Chrysanthemum morifolium*, 1 flower bud was retained; for *Chrysanthemum indicum*, which had smaller leaves, 4 leaflets were retained. The thrips resistance identification device described in Example 1 was used, with two test chambers per chamber, containing 5 species and 3 flower branches of each species. Figure 3d) Two sets of experiments were conducted, ensuring that at least 6 stems of each Chrysanthemum genus and its closely related genera were inoculated. Both sets included Chrysanthemum satsumae. The flower stems were inserted into water agar through the holes in the PVC film to a depth of 1 cm. Subsequently, 50 adult western flower thrips (from the College of Plant Protection of Nanjing Agricultural University, provided by the College of Horticulture for propagation and subculture) were inoculated into the inoculation tank after being starved for 10 h. The tank was sealed with PE plastic wrap. The experiment was conducted in an artificial climate chamber at a temperature of 25℃, a relative humidity of 65%, and a photoperiod of 16L:8D. Seven days after inoculation, the detached flower branches were completely immersed in 75% ethanol for 14 hours to ensure that all thrips had detached from the flower branches before counting (e.g. Figure 4 (as shown in a).

[0023] 1.2 Identification of resistance to western flower thrips based on detached leaves Select the third round of leaves from the terminal bud of each material for in vitro preservation; arrange the leaf tips evenly in a circular pattern around the center of a 150mm diameter culture dish, with 8 leaves of each material per dish, and repeat 5 times per treatment, ensuring that at least 5 leaves are inoculated for each genus of Chrysanthemum and its closely related genera. Use petiole water agar to keep the leaves moist, and carefully inoculate 50 adult western flower thrips that have been starved for 10 hours in the center area of ​​the culture dish with a brush; immediately cover the dish with commercially available polyethylene (PE) plastic wrap, and use a 0.6×25 mm TWLB needle to make evenly puncture holes in the plastic wrap as ventilation holes; The number of thrips on the leaves was counted 24 hours after inoculation using a grading method, and the leaves were graded according to the number of thrips.

[0024] The results of resistance identification of western flower thrips based on detached flower branches and leaves are shown in Table 1. Based on the detached flower branch resistance identification, two highly resistant materials (Damo Chrysanthemum and Hibiscus Chrysanthemum) and two highly susceptible materials (Chrysanthemum bud and Nanjing wild Chrysanthemum) were selected. These results showed some differences compared to the results of detached leaf inoculation. Specifically, in the detached flower branch experiment, western flower thrips reproduction could be observed (e.g., Figure 4 (b, 4c) indicates that the experiment not only reflects the feeding tendency of western flower thrips, but also its reproductive and settlement tendency. Combined with the ornamental characteristics of chrysanthemum, it fully proves that flower branch inoculation can better simulate the actual situation and more accurately reflect the resistance of chrysanthemum materials.

[0025] Table 1. Identification results of the genus *Chrysanthemum* and its closely related genera.

[0026] Among them, HS stands for High susceptible; S stands for Susceptible; MS stands for Moderates susceptible; R stands for Resistant; and HR stands for High resistant.

[0027] 2. Identification of western flower thrips resistance in cut chrysanthemums The cut chrysanthemums used in this experiment were all provided by the Baguazhou Chrysanthemum Garden in Qixia District, Nanjing.

[0028] Healthy detached flower branches from each material were selected and uniformly treated to a length of 13 cm, retaining 4 flower buds and 2 functional leaves; using the thrips resistance identification device described in Example 1, one experiment consisted of two tanks, with 5 species in each tank and 3 flower branches of each species. Figure 3 d) Seven sets of experiments were conducted, ensuring that at least 6 stems of each cut chrysanthemum material were inoculated. The flower stems were inserted into the preservation substrate through the through-holes in the isolation structure to a depth of 1-1.5 cm. Subsequently, 50 adult western flower thrips (from the College of Plant Protection of Nanjing Agricultural University, which was provided by the College of Horticulture for propagation and subculture) were inoculated into the inoculation bucket after being starved for 10 h. The bucket was sealed using a sealed structure. The experiment was conducted in an artificial climate chamber at a temperature of 25℃, a relative humidity of 65%, and a photoperiod of 16L:8D. Seven days after inoculation, the detached flower branches were completely immersed in 75% ethanol for 14 hours to ensure that all thrips had detached from the flower branches before counting.

[0029] The results are shown in Table 2. The identification results show that the number of insects in the highly resistant materials is ≤1, while the number of insects in the highly susceptible materials is >12. The materials with different resistance levels show significant differences, which verifies the accuracy and reliability of this method.

[0030] Table 2. Identification results of cut chrysanthemums

[0031] Among them, HS stands for High susceptible; S stands for Susceptible; MS stands for Moderates susceptible; R stands for Resistant; and HR stands for High resistant.

Claims

1. A thrips resistance identification device, characterized in that, The application relates to a thrips resistance identification device, which comprises a inoculation barrel (1), the bottom of the inoculation barrel (1) is paved with a preservation substrate (2), the preservation substrate (2) is covered with an isolation structure (3) provided with one or more through holes (4), and the top of the inoculation barrel (1) is covered with a sealing structure (5) provided with one or more air permeation holes (6).

2. The device for identifying resistance to a plant bug according to claim 1, wherein The inoculation barrel (1) is a transparent cylindrical container.

3. The device for identifying resistance to a plant bug according to claim 1, wherein The preservation substrate (2) is water agar.

4. The device for identifying resistance to a plant bug according to claim 3, wherein The water agar has a concentration of 1.5-2.5% and a thickness of 1-1.5 cm.

5. The device for identifying resistance to a plant bug according to claim 1, wherein The isolation structure (3) and the sealing structure (5) are both organic high-molecular material films.

6. The device for identifying resistance to a thrips according to claim 5, wherein The thickness of the isolation structure (3) and the sealing structure (5) is 0.1-0.3 mm.

7. The device for identifying resistance to a plant bug according to claim 1, wherein The through hole (4) has a hole diameter of 0.8-1.2 cm, and the distance between the through holes (4) is 2-5 cm.

8. The device for identifying resistance to a thrips according to claim 7, wherein The air permeation hole (6) has a hole diameter of 0.1-0.3 cm.

9. The application of the thrips resistance identification device according to any one of claims 1-8 in the resistance identification of the western flower thrips of the asteraceae plant.

10. Use according to claim 9, characterized in that, The application steps comprise: S1, picking off the flower branches of the asteraceae plant with a length of 10-15 cm, and uniformly reserving at least one full flower bud and at least one healthy functional leaf; S2, inserting the flower branches obtained in S1 into the preservation substrate (2) through the through holes (4) on the isolation structure (3) with an insertion depth of 1-1.5 cm; S3, inoculating 40-60 starved western flower thrips adults in the inoculation barrel (1), sealing the sealing structure (5) and culturing for 6-8 days, counting the number of western flower thrips, and completing the resistance level identification of the horticultural plant.

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