Device and method for evaluating and identifying insect resistance of cruciferous vegetables

Abstract

The invention discloses a device and a method for evaluating and identifying insect resistance of cruciferous vegetables, and relates to the technical field of agricultural vegetable insect resistance identification. The device comprises a breathable net, a cover, agarose, filter paper, moisturizing cotton and a culture dish. A breathable net is arranged on the culture dish cover; the method comprises the following steps: selecting cruciferous vegetable materials which need to be evaluated and identified to resist plutella xylostella; preparing an evaluation and identification device; inoculating third-instar larvae of plutella xylostella; and counting the death number of the plutella xylostella and performing data analysis. According to the device and the method, the blank of the device and the technical method for identifying the insect-resistant germplasm of the major pest plutella xylostella of the cruciferous vegetables is filled, the device and the method for identifying the insect-resistant germplasm of the cruciferous vegetables are creatively created, and the device is easy in material obtaining, low in cost, reusable and simple and convenient to operate; according to the method, the field condition is truly restored, the test result is high in stability, susceptible and high-resistance varieties of the cruciferous vegetables to the plutella xylostella can be accurately and efficiently screened out, and support is provided for insect-resistant breeding of the cruciferous vegetables.

Description

Technical Field

[0001] The present invention relates to the technical field of agricultural vegetable insect resistance identification, and particularly relates to a device and method for evaluating and identifying the insect resistance of cruciferous vegetables. Background Art

[0002] Cruciferae ( Brassicaceae ) plants are widely distributed around the world, with approximately 375 genera and more than 3,200 species. Among them, the plants widely cultivated as vegetables are mainly divided into the genus Brassica ( Brassica ) and the genus Raphanus ( Raphanus L.). The genus Brassica ( Brassica ) vegetables are divided into the cabbage group ( B. oleracea ), the Chinese cabbage group ( B.rapa ), and the mustard group ( B. juncea ). The cabbage group vegetables mainly include cauliflower, broccoli, cabbage, and Chinese kale, etc.; the Chinese cabbage group mainly includes Chinese cabbage, non-heading Chinese cabbage, and turnip, etc.; the mustard group mainly includes leaf mustard, root mustard, stem mustard, and bolt mustard, etc. In the genus Raphanus, it mainly includes wild radish and cultivated radish. In today's diet structure, cruciferous vegetables have attracted much attention due to their rich nutritional components and health benefits, and are also essential foods for three meals a day. Cruciferous vegetables can be used not only as vegetables but also as oilseeds and feeds, so they have a large planting area in China and have extremely important economic value.

[0003] Plutella xylostella Plutella xylostella (L.) belongs to the family Plutellidae of the order Lepidoptera and is a worldwide agricultural pest that damages cruciferous vegetables, with more than 40 host plants. The 1st instar larvae of Plutella xylostella gnaw and bore into the leaves of host plants, and then continuously feed on the mesophyll tissue. When entering the 2nd instar, the larval food intake increases, so that only the remaining leaf epidermis is left on the leaves, which is called "windowpane". When entering the 3rd - 4th instar larvae, it is the gluttonous stage of Plutella xylostella, and the larval food intake increases significantly, gnawing the leaves to form large areas of holes or notches. In severe cases, it can even gnaw the leaf veins, resulting in a decline in the quality of host plants and causing significant economic losses. It is estimated that the global economic losses caused by the damage of Plutella xylostella reach up to $4 - 5 billion annually, and the economic losses caused by its damage in China reach up to $770 million annually.

[0004] The control methods of Plutella xylostella can be divided into agricultural control, biological control, physical control and chemical control. However, chemical control is mainly used in agricultural production. However, the extensive and unreasonable use of chemical insecticides not only causes environmental pollution and varying degrees of damage to the ecosystem, but also leads to the resistance of Plutella xylostella to almost all insecticides, seriously affecting the control of Plutella xylostella and food safety. At present, according to the different insect resistance abilities shown by the differences in genotypes of different vegetable varieties, cultivating new insect-resistant vegetable varieties is the most economical and effective measure to control Plutella xylostella. However, it is not clear which varieties among many vegetable varieties have insect resistance. Therefore, the evaluation and identification of vegetable insect resistance are the premise and basis of vegetable insect-resistant breeding.

[0005] At present, the methods for identifying plant insect resistance include field natural identification method, field inoculation of insect sources to increase the damage pressure method, and net room identification method. However, these methods are usually widely used in field crops (rice, corn and wheat), and they are time-consuming and laborious to use, and the screening cycle is relatively long. In addition, the research on vegetable insect-resistant breeding started relatively late, and the insect resistance identification devices and technologies are relatively scarce. Therefore, it seriously restricts the development of vegetable insect-resistant breeding. At the same time, the traditional methods for identifying crop insect resistance cannot be perfectly matched in vegetable insect resistance identification, and there are problems such as difficult to accurately control experimental conditions and unscientific identification methods, and it is impossible to comprehensively and accurately evaluate the resistance of cruciferous vegetables to Plutella xylostella. Therefore, developing a device and method for evaluating and identifying the insect resistance of cruciferous vegetables is of great significance for the development of vegetable insect-resistant breeding. Summary of the Invention

[0006] The purpose of the present invention is to solve the deficiencies in the existing technologies and methods, and provide a device and method for evaluating and identifying the insect resistance of cruciferous vegetables; the device is easy to operate, low in cost, and can accurately and truly reflect the resistance level of cruciferous vegetables to Plutella xylostella, screen out the susceptible and highly resistant materials of cruciferous vegetables to Plutella xylostella, and solve the problems of lack of devices, time-consuming and laborious statistics and poor result accuracy in the process of evaluating and identifying the resistance of cruciferous vegetables to Plutella xylostella.

[0007] In order to achieve the above purpose, the present invention is realized through the following technical solutions: In the first aspect, the present invention provides a device for evaluating and identifying the insect resistance of cruciferous vegetables, including a breathable net, a lid, 2% agarose, a moistened filter paper, a moisture-retaining cotton and a culture dish; the lid is provided with a breathable net; there is 2% agarose at the bottom of the culture dish, and a moistened filter paper is placed above the agarose. The culture dish, the moistened filter paper, the breathable net and the lid form a closed space, and the 3rd instar larvae of Plutella xylostella and leaves are placed in the culture dish; the petiole of the leaf is wrapped with moisture-retaining cotton.

[0008] Preferably, the mesh number of the breathable net is not less than 100 meshes.

[0009] Preferably, the moisture-retaining cotton is moistened with sterile water.

[0010] In a second aspect, the present invention provides a method for evaluating and identifying the insect resistance of cruciferous vegetables, characterized in that the method is as follows: Step S1: Select cruciferous vegetable materials that need to be evaluated and identified for resistance to Plutella xylostella; Step S2: Prepare the evaluation and identification device; Step S3: Inoculate the 3rd instar larvae of Plutella xylostella on the leaves of the cruciferous vegetables; Step S4: Observe the survival and development of the Plutella xylostella larvae; Step S5: Analyze the data on the resistance of cruciferous vegetables to Plutella xylostella.

[0011] Preferably, for the selection of cruciferous vegetable materials that need to be evaluated and identified for resistance to Plutella xylostella in Step S1, the specific steps are as follows: Sow the seeds of different cruciferous vegetables in culture pots, with 3 seeds sown in each pot, and then place them in a solar greenhouse. When the cruciferous vegetables grow to 4 leaves, remove the weak seedlings and retain the healthy and strong ones. Then, when the seedlings grow to 8 leaves, take the 4th - 5th leaves for insect resistance identification, and repeat 3 times for each variety.

[0012] Preferably, for the preparation of the evaluation and identification device in Step S2, the specific steps are as follows: Select a 90 mm plastic petri dish, place 2% agarose at the bottom of the petri dish, place a filter paper with a diameter of 86 mm above the agarose, moisten the filter paper with a writing brush dipped in sterile water, cut out a 40 - 60 mm area in the center of the petri dish lid, and stick a 100 - mesh breathable net to the vacant area with hot melt glue.

[0013] Preferably, for the inoculation of the 3rd instar larvae of Plutella xylostella in Step S3, the specific steps are as follows: Select the 3rd instar larvae of Plutella xylostella, inoculate 10 3rd instar larvae of Plutella xylostella in each petri dish, cover the top of the petri dish with absorbent paper, and then cover the petri dish lid.

[0014] Preferably, the culture conditions after inoculating the 3rd instar larvae of Plutella xylostella in Step S3 are: in an artificial climate insect - rearing room with an environmental temperature of 25 ± 1°C and a relative humidity of 65%.

[0015] Preferably, for the observation of the survival and development of the Plutella xylostella larvae in Step S4, the specific steps are as follows: 24 h, 48 h, and 72 h after completing Step S3, observe and record the number of dead Plutella xylostella larvae on the leaves of the cruciferous vegetables.

[0016] Preferably, the data analysis of the resistance of cruciferous vegetables to Plutella xylostella in step S5 is carried out according to the following specific steps: statistical analysis and graphing are carried out based on the number of dead Plutella xylostella larvae counted in step S4, and cruciferous vegetable varieties susceptible and highly resistant to Plutella xylostella are screened out.

[0017] The present invention has the following beneficial effects: (1) The present invention provides a device for evaluating and identifying the insect resistance of cruciferous vegetables. The materials of this device are easily obtained, the assembly is convenient, the operation is easy, and the cost is low.

[0018] (2) The present invention provides a device for evaluating and identifying the insect resistance of cruciferous vegetables. This device provides 2% agarose that can maintain moisture continuously, overcoming the defect of short moisture retention time of single-layer filter paper in the past.

[0019] (3) The present invention provides a device for evaluating and identifying the insect resistance of cruciferous vegetables. An area of 40 - 60 mm is dug out in the center of the lid of this device and a 100-mesh breathable net is adhered, increasing the air permeability of the device and overcoming the defect that inaccurate detection data was caused by a large number of dead insects due to excessive humidity in the past.

[0020] (4) The present invention provides a device for evaluating and identifying the insect resistance of cruciferous vegetables. The culture dish selected for this device has a diameter of 90 mm, which can hold the entire leaf to be tested, avoiding the defect that inaccurate detection data was caused by severe water loss and withered yellow leaves due to leaf cutting in the past.

[0021] (5) When the present invention uses this device to evaluate and identify the insect resistance of cruciferous vegetables, this method breaks the long-term dominant position of European and American countries in the method of identifying crop insect-resistant germplasms, and creatively provides a method for identifying vegetable insect-resistant germplasms belonging to China.

[0022] (6) When the present invention uses this device to evaluate and identify the insect resistance of cruciferous vegetables, this method selects the 3rd instar larvae of Plutella xylostella, a specialized pest of cruciferous vegetables. The larvae at this instar are in the period of voracious eating of field pests, which can represent the period when field pests cause the most serious damage and has obvious representativeness.

[0023] (7) The present invention uses this device to evaluate and identify the insect resistance of cruciferous vegetables, filling the gap in the device and technical method for identifying insect-resistant germplasms of the diamondback moth, a major pest of cruciferous vegetables. It has pioneered the creation of a device and method for identifying the insect resistance of cruciferous vegetables. This method is simple to operate and low in cost, and can truly and accurately detect the susceptible and highly susceptible materials of cruciferous vegetables to the diamondback moth. During the test process, interference from external factors to the test results is avoided, and the real situation in the field is largely restored, improving the authenticity and accuracy of the test results. It saves time and effort, has good repeatability, further ensures the reliability and stability of the test data, and fills the gap in the lack of devices and methods for evaluating and identifying the insect resistance of cruciferous vegetables in current insect-resistant breeding. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 It is a schematic diagram of the device for evaluating and identifying the insect resistance of cruciferous vegetables in the present invention; Figure 2 It is a physical diagram of the device for evaluating and identifying the insect resistance of cruciferous vegetables in the present invention; Figure 3 It is a physical diagram of the culture dish and the breathable net in the present invention; Figure 4 It is a first perspective view of the usage state of the device for evaluating and identifying the insect resistance of cruciferous vegetables in the present invention; Figure 5 It is a second perspective view of the usage state of the device for evaluating and identifying the insect resistance of cruciferous vegetables in the present invention; Figure 6 It is a schematic diagram of the test implementation of the device for evaluating and identifying the insect resistance of cruciferous vegetables in the present invention; Figure 7 It is the evaluation and identification results of the insect resistance of cabbage and broccoli in cruciferous vegetables in the embodiment, where A is the resistance level of 10 cabbage varieties to diamondback moth larvae, and B is the resistance level of 10 broccoli varieties to diamondback moth larvae.

[0025] In the figure: 1, breathable net; 2, lid; 3, 3rd instar larvae of diamondback moth; 4, leaf; 5, moistened filter paper; 6, moisture-retaining cotton; 7, plastic culture dish; 8, 2% agarose. DETAILED DESCRIPTION OF THE EMBODIMENTS

[0026] The following further illustrates the present invention in conjunction with embodiments, but it shall not be used as a basis for limiting the present invention.

[0027] As Figures 1 - 3Shown: The device for evaluating and identifying the insect resistance of cruciferous vegetables according to the present invention includes a plastic petri dish 7, 2% agarose 8, a moistened filter paper 5, a moisture-retaining cotton 6, a lid 2 and a ventilation net 1; there is 2% agarose 8 at the bottom of the plastic petri dish 7; the moistened filter paper 5 is placed on the 2% agarose 8; the lid 2 is provided with a ventilation net 1, and the plastic petri dish 7, 2% agarose 8, moistened filter paper 5, ventilation net 1 and lid 2 form a closed space, and the 3rd instar larvae 3 of Plutella xylostella and leaves 4 are placed in the plastic petri dish 7; the petiole of the leaf 4 is wrapped with a moisture-retaining cotton 6.

[0028] In this embodiment, the plastic petri dish 7 used has a diameter of 90 mm and a height of 20 mm, the ventilation net 1 has a mesh number of 100 meshes, the filter paper 5 has a diameter of 86 mm, and the concentration of agarose is 2%.

[0029] Please refer to Figures 4 - 6 , the method for evaluating and identifying the insect resistance of cruciferous vegetables according to the present invention includes the following: Step S1: Select cruciferous vegetable materials that need to be evaluated and identified for resistance to Plutella xylostella; Select the cruciferous vegetable varieties to be identified, sow different seeds in the culture pots to cultivate seedlings. In this embodiment, the culture pot has a specification of an upper diameter of 11 cm, a bottom diameter of 7 cm, and a height of 8 cm; 3 seeds are sown in each culture pot to ensure that there are seedlings in each culture pot, and then placed in a solar greenhouse. When the cruciferous vegetables grow to 4 leaves, remove the weak seedlings and retain the healthy and strong seedlings; then when the seedlings grow to 8 leaves, take the 4th - 5th leaves for insect resistance identification, and repeat each variety 3 times.

[0030] Step S2: Prepare the evaluation and identification device; Select a plastic petri dish with a diameter of 90 mm, place 2% agarose at the bottom of the petri dish, place a filter paper with a diameter of 86 mm above the agarose, moisten the filter paper with a writing brush dipped in sterile water, dig out the area of 40 - 60 mm in the center of the petri dish lid, and stick a 100 - mesh ventilation net to the vacant area with hot melt adhesive.

[0031] Step S3: Inoculate the 3rd instar larvae of Plutella xylostella on the cruciferous vegetable leaves; Select the 3rd instar larvae of Plutella xylostella with stronger vitality, pick up the larvae with a soft writing brush, and then gently place them on the leaves to be tested in the petri dish. The petri dish is covered with absorbent paper above, and then the petri dish lid is covered. In this embodiment, 10 3rd instar larvae of Plutella xylostella are inoculated in each petri dish. Then the petri dish is placed in an artificial climate insect - rearing room with an environmental temperature of 25 ± 1 °C and a relative humidity of 65% for cultivation.

[0032] Step S4: Observe the survival and development of Plutella xylostella larvae; At 24 h, 48 h, and 72 h after insect inoculation, observe and record the number of dead Plutella xylostella larvae on cruciferous vegetable leaves.

[0033] Step S5: Data analysis of the resistance of cruciferous vegetables to Plutella xylostella; Based on the statistically recorded number of dead Plutella xylostella larvae, use GraphPad Prism 8.3 graphing software to create graphs to visually reflect the insect resistance of different cruciferous vegetable varieties, and screen out the susceptible and highly resistant varieties of cruciferous vegetables to Plutella xylostella.

[0034] In this example, 10 cabbage varieties and 10 broccoli varieties were selected as materials. Among them, for cabbage, the main cultivated variety JingFeng No.1 in the Chinese fields was used as the control, and for broccoli, the main cultivated variety ZhongQing No.15 in the Chinese fields was used as the control. They were divided into two groups, A and B, for evaluation and identification. Graphs were drawn based on the statistical data as Figure 7 shown, and the data is as shown in the following table.

[0035] Table 1 Statistics of the number of dead Plutella xylostella larvae after feeding on cabbage in Group A

[0036] Table 2 Statistics of the number of dead Plutella xylostella larvae after feeding on broccoli in Group B

[0037] The above has shown and described the basic principles, main features, and advantages of the present invention. However, the above are only specific embodiments of the present invention, and the technical features of the present invention are not limited thereto. Any other embodiments obtained by those skilled in the art without departing from the technical solution of the present invention should be covered within the patent scope of the present invention.

Claims

1. An apparatus for evaluating and identifying the insect resistance of cruciferous vegetables, characterized in that, It includes a breathable net (1), a lid (2), a moistened filter paper (5), a moisturizing cotton (6), a petri dish (7), and 2% agarose (8); the lid (2) is provided with a breathable net (1); 2% agarose (8) is placed at the bottom of the petri dish (7), a moisturizing filter paper (5) is placed on the 2% agarose (8), and the petri dish (7), 2% agarose (8), the moistened filter paper (5), the breathable net (1), and the lid (2) form a closed space, and the 3rd instar larvae (3) and leaves (4) of Plutella xylostella are placed in the petri dish (7); the petiole of the leaf (4) is wrapped with a moisturizing cotton (6).

2. The device for evaluating and identifying the insect resistance of cruciferous vegetables according to claim 1, characterized in that The mesh number of the breathable net (1) is not less than 100 meshes.

3. The device for evaluating and identifying the insect resistance of cruciferous vegetables according to claim 1, wherein, The moisturizing cotton is moistened with sterile water.

4. A method for evaluating and identifying the insect resistance of cruciferous vegetables using the device according to any one of claims 1-3, characterized in that, The method is as follows: Step S1: Select cruciferous vegetable materials that need to be evaluated and identified for resistance to Plutella xylostella. Step S2: Inoculate the 3rd instar larvae of Plutella xylostella on the leaves of the cruciferous vegetables. Step S3: Observe the survival and development of the Plutella xylostella larvae. Step S4: Analyze the data on the resistance of cruciferous vegetables to Plutella xylostella.

5. The method for evaluating and identifying the insect resistance of cruciferous vegetables according to claim 4, wherein For the selection of cruciferous vegetable materials that need to be evaluated and identified for resistance to Plutella xylostella in Step S1, the specific steps are as follows: Sow the seeds of different cruciferous vegetables in a culture pot, sow 3 seeds in each culture pot, and then place them in a solar greenhouse. When the cruciferous vegetables grow to 4 leaves, remove the weak seedlings and retain the healthy and strong seedlings. Then, when the seedlings grow to 8 leaves, take the 4th - 5th leaves for insect resistance identification, and repeat each variety 3 times.

6. The method for evaluating and identifying the insect resistance of cruciferous vegetables according to claim 4, wherein, For the preparation of the evaluation and identification device in Step S2, the specific steps are as follows: Select a plastic petri dish with a diameter of 90 mm, place 2% agarose at the bottom of the petri dish, place a filter paper with a diameter of 86 mm above the agarose, moisten the filter paper with a writing brush dipped in sterile water, dig out the area with a diameter of 40 - 60 mm in the center of the petri dish lid, and use hot melt adhesive to stick a 100 - mesh breathable net on the vacant area.

7. The method for evaluating and identifying the insect resistance of cruciferous vegetables according to claim 4, characterized in that, For the inoculation of the 3rd instar larvae of Plutella xylostella in Step S3, the specific steps are as follows: Select healthy and lively 3rd instar larvae of Plutella xylostella, inoculate 10 larvae in each petri dish, cover the top of the petri dish with absorbent paper, and then cover the petri dish lid.

8. The method for evaluating and identifying the insect resistance of cruciferous vegetables according to claim 4, wherein The culture conditions after inoculating the 3rd instar larvae of Plutella xylostella in Step S3: In an artificial climate insect - rearing room with an environmental temperature of 25 ± 1 °C and a relative humidity of 65%.

9. The method for evaluating and identifying the insect resistance of cruciferous vegetables according to claim 4, characterized in that, For the observation of the survival and development of the Plutella xylostella larvae in Step S4, the specific steps are as follows: 24 h, 48 h, and 72 h after completing Step S3, observe and record the number of dead Plutella xylostella larvae on the leaves of the cruciferous vegetables.

10. The method for evaluating and identifying the insect resistance of cruciferous vegetables according to claim 4, characterized in that, For the data analysis of the resistance of cruciferous vegetables to Plutella xylostella in Step S5, the specific steps are as follows: Conduct statistical analysis and graphing based on the number of dead Plutella xylostella larvae counted in Step S4, and screen out the cruciferous vegetable germplasms that are susceptible and highly resistant to Plutella xylostella.

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