A staining test method for screening Riptortus pedestris sucking soybean pods

Abstract

A staining inspection method for screening the puncture of Riptortus pedestris on soybean pods according to the present invention includes five steps: cleaning, staining, elution, observation, and result determination. The phenol-fuchsin staining test paper detection method is adopted, which is easier to carry and store, can quickly and intuitively obtain the puncture situation of soybean pods, and can avoid cross-contamination to a greater extent. The detection process of the present invention is short, the operation is simple, and the detection cost is low. It is applicable to screening the puncture sites of Riptortus pedestris on soybean pods and grains, can improve the detection efficiency of the puncture damage of Riptortus pedestris, and thus helps to realize the dynamic monitoring of the early occurrence of Riptortus pedestris.

Description

Technical Field

[0001] The present invention relates to the technical field of plant damage detection, and specifically to a staining test method for screening the sucking damage of soybean pods by Riptortus pedestris. Background Art

[0002] Monitoring the occurrence dynamics of field pests is an important task in plant protection and is of great significance for scientifically controlling agricultural and forestry pests. Riptortus pedestris is a sucking pest that mainly damages leguminous plants. In the late growth stage of soybeans, Riptortus pedestris causes sparse or normal pod formation, and the soybean pods of the whole plant or some branches cannot normally swell or do not swell at all. Before the present invention, generally, based on experience, the symptoms of the above-mentioned whole soybean plant, branches or pods were observed by visual inspection in the field to infer whether there was damage by Riptortus pedestris or other pests. When symptoms were visually observed, pests such as Riptortus pedestris had damaged soybean pods and seeds for some time. This method could not determine whether there was sucking damage caused by Riptortus pedestris, nor could it determine the sucked parts and damage symptoms on soybean pods. This method was only applicable to the inference after symptoms appeared, and there was no room for recovering the resulting economic losses. Therefore, there was an urgent need for a method with a short detection process, simple operation, low detection cost and wide applicability to provide a convenient way for on-site detection in the fields, improve the detection efficiency of the sucking damage of soybean pods by Riptortus pedestris, and thus contribute to the effective monitoring of the early occurrence dynamics of Riptortus pedestris. Summary of the Invention

[0003] The purpose of the present invention is to provide a staining test method for screening the sucking damage of soybean pods by Riptortus pedestris, which can be used for the qualitative and preliminary quantitative analysis of the damaged parts and damage symptoms of soybean pods after being sucked by Riptortus pedestris.

[0004] To achieve the above purpose, the present invention provides the following technical solution: A staining test method for screening the sucking damage of soybean pods by Riptortus pedestris, comprising the following steps:

[0005] S1, Prepare a phenol-fuchsin staining test paper;

[0006] S2, Cleaning: Clean the collected soybean pods or seeds to be tested with a cleaning solution for 0.5 min to 1 min;

[0007] S3, Staining: Put 1 to 2 pieces of the phenol-fuchsin staining test paper prepared in step S1 into a suitable container, pour an appropriate amount of water until the phenol-fuchsin staining test paper is fully stirred and dissolved, then add a mordant, and then put the soybean pods or seeds to be tested into the container and soak for 10 min to 30 min until fully stained;

[0008] S4, Elution: Subsequently, use forceps to pick up the dyed soybean pods or seeds and place them in the eluent to wash the solution on the surface of the soybean pods or seeds. The washing time shall not exceed 1 minute.

[0009] S5, Observation: Carefully observe the soybean pods or seeds damaged by sucking of Riptortus pedestris through the above steps with the naked eye or visual optical instruments.

[0010] S6, Result determination: The main characteristics of the damage caused by sucking of Riptortus pedestris are as follows: Wherever there are small red dots with a diameter of about 0.3 mm on the soybean pods or seeds, they are the sucking points of Riptortus pedestris; If there are no small red dots on the soybean pods or seeds, they are neither the sucking points of Riptortus pedestris nor the mechanical damage points.

[0011] Preferably, for step S1, the specific operation of preparing the phenol-fuchsin staining test paper is as follows:

[0012] S11, Take a blank neutral filter paper and cut it into a specific size of 1 - 5 cm × 1 - 5 cm.

[0013] S12, Prepare the phenol-fuchsin staining solution, which is composed of phenol, basic fuchsin, citric acid and water.

[0014] In the phenol-fuchsin staining solution, the mass concentration of basic fuchsin is 1% - 5%, the mass concentration of citric acid is 10% - 20%, and the mass concentration of phenol is 1% - 5%.

[0015] S13, Immerse the neutral filter paper of the specific size in the phenol-fuchsin staining solution at 0 - 35°C until the neutral filter paper is saturated.

[0016] S14, Loosely place the saturated neutral filter paper in a vacuum drying oven. After evacuating the vacuum drying oven, let it stand still, keep the temperature at 30°C - 40°C, and let it stand still for 1 h - 3 h after evacuating the vacuum to obtain the phenol-fuchsin staining test paper.

[0017] Preferably, in step S2, the cleaning solution is selected as pure water.

[0018] Preferably, seal the phenol-fuchsin staining test paper in a sealed bag added with an antioxidant and a desiccant.

[0019] Preferably, the preparation method of the mordant in step S3 is as follows:

[0020] The mordant is made from an aqueous solution of sorbitol, and the mass concentration is 1% - 5%.

[0021] Preferably, the preparation method of the eluent in step S4 is as follows:

[0022] The mass concentration of Na2HPO4 is 0.1% - 0.5%, the mass concentration of KH2PO4 is 0.01% - 0.05%, the mass concentration of NaCl is 0.1% - 5%, and the mass concentration of KCl is 0.01% - 0.1%. Stir well to dissolve, and then add concentrated hydrochloric acid to adjust the pH of the mixed solution to 7 - 8.5.

[0023] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0024] (1) The staining inspection method for screening the puncture of soybean pods by Riptortus pedestris described in the present invention is simple, can ensure the stability of the concentration, is easier to carry and store, can quickly and intuitively obtain the puncture situation of soybean pods, and can avoid cross - contamination to a greater extent.

[0025] (2) The detection process of the present invention is short, the operation is simple, and the detection cost is low. It is suitable for screening the puncture sites of Riptortus pedestris on soybean pods and seeds, can improve the detection efficiency of the puncture damage of Riptortus pedestris, and thus helps to realize the dynamic monitoring of the early occurrence of Riptortus pedestris. It is suitable for use by departments such as agricultural production, plant protection, inspection and quarantine.

[0026] Description of the Drawings

[0027] Figure 1 It is the daily feeding rhythm diagram of adult Riptortus pedestris;

[0028] Figure 2 It is the daily feeding rhythm diagram of the 5th instar nymph of Riptortus pedestris;

[0029] Figure 3 It is the daily feeding rhythm diagram of the 3rd instar nymph of Riptortus pedestris;

[0030] Figure 4 It is a schematic diagram of the puncture sites of the 3rd instar nymph of Riptortus pedestris on 15 - day - old soybean seeds;

[0031] Figure 5 It is a schematic diagram of the puncture sites of adult Riptortus pedestris on 15 - day - old soybean seeds;

[0032] Figure 6 It is a schematic diagram of the puncture sites of adult Riptortus pedestris on 15 - day - old soybean pods. Detailed Embodiments

[0033] Next, please clearly and completely describe the technical solutions in the embodiments of the present invention according to the drawings. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative efforts shall fall within the protection scope of the present invention.

[0034] Example 1

[0035] Refer to the attached specification Figures 1-6 , the present invention provides a technical solution: a staining inspection method for screening the soybean pods punctured by Riptortus pedestris, comprising the following steps:

[0036] S1, Prepare phenol-fuchsin staining test paper;

[0037] S2, Cleaning: Clean the collected soybean pods or seeds to be tested with a cleaning solution for 0.5 min to 1 min;

[0038] S3, Staining: Put 1 to 2 pieces of the phenol-fuchsin staining test paper prepared in step S1 into a suitable container, pour an appropriate amount of water until the phenol-fuchsin staining test paper is fully stirred and dissolved, then add a mordant solution, and then put the soybean pods or seeds to be tested into the container and soak for 10 min to 30 min until fully stained;

[0039] S4, Elution: Then use tweezers to pick up the stained soybean pods or seeds and place them in the eluent to clean the solution on the surface of the soybean pods or seeds, and the cleaning time does not exceed 1 min;

[0040] S5, Observation: Carefully observe the soybean pods or seeds damaged by the puncture of Riptortus pedestris obtained through the above steps by the naked eye or visual optical instruments;

[0041] S6, Result determination: The main characteristics of being punctured by Riptortus pedestris are: wherever there are small red dots with a diameter of about 0.3 mm on the soybean pods or seeds, they are the puncture points of Riptortus pedestris; if the soybean pods or seeds do not show small red dots, they are neither the puncture points of Riptortus pedestris nor the mechanical damage points.

[0042] Specifically, for step S1, the specific operation of preparing the phenol-fuchsin staining test paper is as follows:

[0043] S11, Take a blank neutral filter paper and cut it into a specific size of 1 - 5 cm × 1 - 5 cm;

[0044] The specific size of the cutting is carried out according to actual needs, and the side length of each neutral filter paper is not less than 1 cm and not more than 5 cm.

[0045] S12, Prepare a phenol-fuchsin staining solution, which is composed of phenol, basic fuchsin, citric acid and water;

[0046] In the phenol-fuchsin staining solution, the mass concentration of basic fuchsin is 1% - 5%, the mass concentration of citric acid is 10% - 20%, and the mass concentration of phenol is 1% - 5%;

[0047] S13, Immerse the neutral filter paper of the specific size in the phenol-fuchsin staining solution at 0 - 35 °C until the neutral filter paper is saturated;

[0048] S14. Loosely place the saturated neutral filter paper in a vacuum drying oven. After evacuating the vacuum drying oven, let it stand still, keep the temperature at 30°C to 40°C, and let it stand still for 1 h to 3 h after evacuation to obtain the phenol-fuchsin staining test paper.

[0049] An appropriate number of phenol-fuchsin staining test papers can be prepared for standby according to the actual situation. When detection is needed, directly conduct the detection. The phenol-fuchsin staining test paper remains stable for at least 3 months in an environment of 0°C to 35°C.

[0050] Specifically, in step S2, the cleaning solution is pure water.

[0051] Specifically, seal the phenol-fuchsin staining test paper in a sealed bag added with an antioxidant and a desiccant.

[0052] Specifically, the preparation method of the mordant in step S3 is as follows:

[0053] The mordant is made from an aqueous solution of sorbitol with a mass concentration of 1% to 5%.

[0054] Specifically, the preparation method of the eluent in step S4 is as follows:

[0055] The mass concentration of Na2HPO4 is 0.1% to 0.5%, the mass concentration of KH2PO4 is 0.01% to 0.05%, the mass concentration of NaCl is 0.1% to 5%, and the mass concentration of KCl is 0.01% to 0.1%. Stir well to dissolve, and then add concentrated hydrochloric acid to adjust the pH of the mixed solution to 7 to 8.5.

[0056] To ensure the accuracy of the detection, 30 pods or seeds are randomly collected and observed from each soybean plot.

[0057] Specifically, when detecting each soybean plot, a total of 30 pods or seeds are randomly selected and observed, and at least 10 of either pods or seeds.

[0058] In the present invention, before staining the soybean pods or seeds, an operation of cleaning is carried out to remove impurities, thereby reducing the background interference of staining observation.

[0059] In the present invention, a small amount of carbon citric acid is added as a stabilizer to the phenol-fuchsin solution, and a small amount of sorbitol is added as a mordant to play a role in assisting osmosis.

[0060] The staining effect of the present invention can be maintained for more than 24 h until the soybean pods or seeds wilt and deteriorate.

[0061] The phenol-fuchsin staining test paper involved in the present invention remains stable for at least 3 months in an environment of 0 to 35°C and can be stored for 3 years or more in a sealed bag with an antioxidant and a desiccant added.

[0062] Example 2

[0063] Experimental treatment: Put potted soybeans in an insect-rearing cage and introduce 10 unpaired and unmated adult Riptortus pedestris, 10 fifth-instar nymphs and 10 third-instar nymphs respectively. Observe each adult and nymph of Riptortus pedestris for 24 hours, and observe and record whether they pierce and suck the soybean pods and the number of piercing and sucking times. The insect-rearing cage is placed under natural light conditions and is consistent with the external natural environment at night. According to the described test kit for screening Riptortus pedestris piercing and sucking soybean pods and its detection steps, observe and record the piercing and sucking points of the pods and seeds.

[0064] Main results: Adult Riptortus pedestris have feeding behaviors throughout the day. The feeding on soybean pods mainly concentrates in the evening and early morning, and the feeding time is 1 to 2 hours. Fifth-instar nymphs of Riptortus pedestris mainly feed on soybean pods, feeding on soybean pods multiple times a day with a relatively long feeding time on soybean pods. There is a feeding peak from 9 to 11 am; from 7 to 9 pm, the feeding frequency is also relatively high. The feeding pattern of third-instar nymphs of Riptortus pedestris is similar to that of fifth-instar nymphs, mainly piercing and sucking soybean pods. Third-instar nymphs also feed on soybean pods multiple times, and the feeding frequency on soybean pods is lower than that of fifth-instar nymphs.

[0065] Example 3

[0066] Experimental treatment: Adopt the method of covering potted plants with nets to catch insects. Prepare 40 soybean varieties and cover the potted soybean plants with gauze nets. Retain 10 pods with consistent growth and development on the plants at the pod-setting and grain-filling stage for each variety. Introduce adult Riptortus pedestris into the net covers at a density of 2 pairs per plant. Pick the pods 2 days after introducing the insects, and according to the described test kit for screening Riptortus pedestris piercing and sucking soybean pods and its detection steps, observe and record the piercing and sucking points of the pods and seeds.

[0067] Main results: The results of the piercing and sucking points of Riptortus pedestris on the pods of different soybean varieties are shown in Table 1. There are significant differences in the piercing and sucking points of the pods of 40 soybean varieties (F = 3.61, P < 0.05). The highest piercing and sucking points on the pods of Suinong 14 are 5.50 ± 1.31, which is significantly higher than those of 28 varieties such as Kenfeng 13; the lowest piercing and sucking points on the pods of Longhuang No. 1 are 0.43 ± 0.09, which is significantly lower than those of 5 varieties such as Suinong 28.

[0068] Table 1 Piercing and sucking points of Riptortus pedestris on the pods of 40 soybean varieties

[0069]

[0070]

[0071] The puncture points of the seeds of 40 soybean varieties by *Riptortus pedestris* are shown in Table 2. There were significant differences in the puncture points of the pods and seeds of 40 soybean varieties by *Riptortus pedestris* (F = 4.63, P < 0.05). The highest puncture point of the pods and seeds was found in Tongnong 943, which was 1.93 ± 0.04, significantly higher than 32 varieties such as Suinong 29; the lowest puncture point of the seeds was found in Longhuang 1, which was 0.16 ± 0.04, significantly lower than 5 varieties such as Changnong 34.

[0072] Table 2 Puncture points of *Riptortus pedestris* on the seeds of 40 soybean varieties

[0073]

[0074]

[0075] Although the embodiments of the present invention have been shown and described, it will be understood by those of ordinary skill in the art that various changes, modifications, substitutions and variations can be made to these embodiments without departing from the principles and spirit of the present invention, and the scope of the present invention is defined by the appended claims and their equivalents.

Claims

1. A staining test method for screening Riptortus pedestris sucking soybean pods, characterized in that It includes the following steps: S1. Prepare a phenol-fuchsin staining test paper; S2. Cleaning: Clean the collected soybean pods or grains to be tested with a cleaning solution for 0.5 min to 1 min; S3. Staining: Place 1 to 2 pieces of the phenol-fuchsin staining test paper prepared in step S1 in a suitable container, pour an appropriate amount of water until the phenol-fuchsin staining test paper is fully stirred and dissolved, then add a mordant solution, and then place the soybean pods or grains to be tested in the container and soak for 10 min to 30 min until fully stained; S4. Elution: Then use tweezers to pick up the stained soybean pods or grains and place them in the eluent to clean the solution on the surface of the soybean pods or grains, and the cleaning time does not exceed 1 min; S5. Observation: Carefully observe the soybean pods or grains damaged by sucking of Riptortus pedestris obtained through the above steps by relying on the naked eye or a visual optical instrument; S6. Result determination: The characteristics of being damaged by sucking of Riptortus pedestris are as follows: Whenever there are small red dots with a diameter of about 0.3 mm on the soybean pods or grains, they are the sucking points of Riptortus pedestris; if the soybean pods or grains do not show small red dots, they are neither the sucking points of Riptortus pedestris nor the mechanical damage points.

2. The staining inspection method for screening the puncture and sucking of soybean pods by Riptortus pedestris according to claim 1, characterized in that: In step S1, the specific operation of preparing the phenol-fuchsin staining test paper is as follows: S11. Take a blank neutral filter paper and cut it into a specific size of 1 - 5 cm × 1 - 5 cm; S12. Prepare a phenol-fuchsin staining solution, which is composed of phenol, basic fuchsin, citric acid and water; In the phenol-fuchsin staining solution, the mass concentration of basic fuchsin is 1% - 5%, the mass concentration of citric acid is 10% - 20%, and the mass concentration of phenol is 1% - 5%; S13. Immerse the neutral filter paper of the specific size in the phenol-fuchsin staining solution at an environment of 0 - 35 °C until the neutral filter paper is saturated; S14. Loosely place the saturated neutral filter paper in a vacuum drying oven, evacuate the vacuum drying oven and then let it stand still, keep the temperature at 30 °C - 40 °C, and let it stand still for 1 h to 3 h after evacuation to obtain the phenol-fuchsin staining test paper.

3. The staining inspection method for screening Riptortus pedestris sucking soybean pods according to claim 1, characterized in that: In step S2, the cleaning solution is selected as pure water; the phenol-fuchsin staining test paper is sealed in a sealed bag added with an antioxidant and a desiccant; the mordant solution in step S3 is made from an aqueous solution of sorbitol with a mass concentration of 1% - 5%; the preparation method of the eluent in step S4 is as follows: the mass concentration of Na2HPO4 is 0.1% - 0.5%, the mass concentration of KH2PO4 is 0.01% - 0.05%, the mass concentration of NaCl is 0.1% - 5%, the mass concentration of KCl is 0.01% - 0.1%, fully stir and dissolve, and then add concentrated hydrochloric acid to adjust the pH of the mixed solution to 7 - 8.5.

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