A method for increasing the incidence of southern rice black streaked dwarf disease

Abstract

The present application relates to the technical field of agricultural science, and particularly relates to a method for improving the incidence of southern rice black-streaked dwarf disease, comprising the following steps: (1) breeding of virus-carrying white-backed planthoppers; (2) micro-wound pretreatment of rice seedling sheaths; (3) virus transmission of white-backed planthoppers; (4) inoculation of rice materials; and (5) rice transplanting and incidence phenotype statistics; from the overall synergistic effect, the technical scheme of the present application provides a basis for sufficient virus input through the screening of virus-carrying white-backed planthoppers, guarantees efficient virus migration and tissue protection through micro-wound pretreatment of sheaths and composite modified microneedles, creates conditions for stable virus transmission process through optimized virus transmission environment, and finally stably improves the incidence of rice through mutual cooperation and progressive steps of each step; meanwhile, the identification period is shortened to 35 days or less, effectively solves the problems of low incidence, long period and poor accuracy of traditional methods, and efficiently meets the demand of rapid screening of rice disease-resistant varieties.

Description

Technical Field

[0001] This invention relates to the field of agricultural science and technology, specifically to a method for identifying the incidence of rice black-streaked dwarf disease in southern China. Background Technology

[0002] Southern rice black-streaked dwarf virus (SRBSDV) is a major viral disease of rice caused by SRBSDV and primarily transmitted by the white-backed planthopper. It occurs annually in rice-growing areas of southern my country, and in severe cases can lead to yield reductions of 30%-50%, or even total crop failure. Accurately identifying the resistance of rice varieties to this disease is crucial for screening and breeding resistant varieties and developing disease control strategies. Therefore, establishing efficient and stable methods for disease incidence assessment is of significant practical importance in agricultural production.

[0003] Currently, commonly used methods for identifying the incidence of Southern Rice Black-Streaked Dwarf Virus (SRBSDV) in the industry generally suffer from low identification efficiency and poor result stability. These problems are mainly reflected in the following aspects: The screening of infected white-backed planthoppers lacks standards. Most methods simply select infected individuals for transmission without considering the correlation between nymph weight and virus load (the viral load in underweight nymphs is often below 0.5 μg / mL), and also fail to control nymph feeding frequency (when feeding frequency is <5 times / h, virus transmission efficiency drops significantly), resulting in insufficient virus input during transmission and a rice incidence rate generally below 30%. SRBSDV mainly spreads systemically through the rice phloem. Under natural conditions, the mouthparts of the white-backed planthopper have limited piercing depth, making it difficult to break through the leaf sheath tissue barrier. The virus mostly remains on the surface of the leaf sheath, failing to induce typical disease phenotypes throughout the plant, further reducing the incidence rate. These problems collectively lead to traditional methods having an identification cycle of over 40 days, low incidence rates, and unstable results, failing to meet the need for rapid screening of disease-resistant rice varieties. Summary of the Invention

[0004] The purpose of this invention is to overcome the shortcomings of the prior art and propose a method for identifying the incidence of rice black-streaked dwarf disease in southern China.

[0005] The specific technical solution is as follows: A method for identifying the incidence of rice black-streaked dwarf disease in southern rice, comprising the following steps:

[0006] (1) Cultivation of white-backed planthoppers carrying the Southern Rice Black-streaked Dwarf Virus: Cultivate white-backed planthopper populations carrying the Southern Rice Black-streaked Dwarf Virus, and screen for individuals carrying the virus. The weight of the screened individuals carrying the virus is 0.3-0.5 mg / person, and the number of feeding times per hour is ≥5 times.

[0007] (2) Pretreatment of rice seedling leaf sheaths with micro-trauma: The rice seedlings to be tested in the early tillering stage were subjected to pretreatment of leaf sheaths with micro-trauma. The seedlings in the early tillering stage were 15-20 cm tall and had 4-5 leaves on the main stem. During the pretreatment, a microneedle array modified with composite nano-silver-chitosan was used to puncture the base of the leaf sheath of the main stem of the rice at a distance of 2-3 cm from the ground. The microneedles of the array were conical with a tip diameter of 5-8 μm, a needle height of 200-250 μm, a puncture depth of 50-80 μm, and a puncture density of 2-3 per mm. 2 A micrometer-scale wound channel is created through puncture;

[0008] (3) Inoculation and virus transmission of white-backed planthoppers in rice materials: The white-backed planthoppers obtained in step (1) were inoculated on rice seedlings treated in step (2) at a density of 1-2 plants per plant. Virus transmission was carried out in a virus transmission environment for 48 hours. The temperature of the virus transmission environment was 25-28℃, the relative humidity was 70-80%, the photoperiod was 14h / 10h (light / dark), and the carbon dioxide concentration was 500-600ppm.

[0009] (4) Statistics on rice transplanting and disease incidence phenotype: After removing white-backed planthoppers, rice seedlings were transplanted to the identification nursery and cultured under conventional field management conditions. Disease incidence was counted 30-35 days after transplanting.

[0010] The composite nano-silver-chitosan modified microneedle array is used to promote the directional migration of viruses from the vascular bundles to the phloem, while inhibiting bacterial growth at the wound site.

[0011] Preferably, the preparation method of the composite nano-silver-chitosan modified microneedle array in step (2) includes:

[0012] Preparation of microneedle substrate: Medical-grade polylactic acid-glycolic acid copolymer (PLA-glycolic acid copolymer) with a lactic acid to glycolic acid molar ratio of 7:3 and an intrinsic viscosity of 0.6-0.8 dL / g was prepared. The PLA-glycolic acid copolymer was then formulated into a 15-18 wt% dichloromethane-N,N-dimethylformamide mixed solution, with a dichloromethane to N,N-dimethylformamide volume ratio of 4:1. The mixed solution was cast into a silicon mold and vacuum dried at 35-38℃ for 8-10 h to obtain the microneedle substrate.

[0013] Preparation of composite coating solution: Take chitosan with a degree of deacetylation ≥90% and a viscosity of 100-200 mPa·s; dissolve chitosan in 1% acetic acid aqueous solution to prepare a 1.5-2.0 wt% chitosan solution; take 0.3-0.6 wt% silver nanoparticle sol, and mix the chitosan solution and silver nanoparticle sol at a volume ratio of 1:1 to obtain the composite coating solution;

[0014] Coating and drying: A composite coating liquid was coated on the surface of the microneedle substrate using a spin coating method. The spin coating speed was 800 r / min and the coating time was 30 s, forming a composite coating with a thickness of 8-12 μm. The microneedle array was obtained by drying in a forced air at 40℃ for 2 h.

[0015] Preferably, the nano-silver sol is prepared by ascorbic acid reduction-gelatin stabilization method, the specific steps of which are as follows: take 0.02-0.04 mol / L silver nitrate aqueous solution and 1.5-1.8 wt% food-grade pigskin gelatin solution, mix them at a mass ratio of 1:2; under the condition of keeping warm at 60℃, add 0.1 mol / L ascorbic acid solution dropwise at a rate of 1 mL / min until the solution turns brownish-red, and continue stirring for 1 h; dialyze and purify using a dialysis bag with a molecular weight cutoff of 3500 Da for 48 h, changing the deionized water every 12 h during the dialysis process, and finally obtain the nano-silver sol.

[0016] Preferably, in step (2), the rice seedlings need to be sprayed with a salicylic acid-melatonin composite inducer 24 hours before the micro-trauma treatment. The specific spraying method is as follows: spray with a manual sprayer with an atomization particle size of 50-80μm; after spraying, the rice leaves are tested, and the test results must meet the requirements of no obvious yellowing and chlorophyll content ≥2.0mg / g (FW).

[0017] Preferably, the preparation method of the salicylic acid-melatonin complex inducer is as follows: take salicylic acid and melatonin, mix them at a mass ratio of 3:1; dissolve them in a 5% (v / v) ethanol aqueous solution, and then dilute with deionized water to a concentration of 20 mg / L, and use immediately after preparation.

[0018] Preferably, the concentration of the compound inducer applied is 20-25 mg / L, and the application rate is 10 mL per rice plant.

[0019] Preferably, after step (3) of virus transmission is completed, the effectiveness of virus transmission needs to be verified. The verification method includes the following steps:

[0020] One hundred white-backed planthoppers were randomly selected, and their SRBSDV load was detected by qRT-PCR. The detection result must meet the requirement that the load is ≥0.8μg / mL, which can ensure that the insect carries sufficient virus.

[0021] Preferably, the statistical method for the incidence rate in step (4) is as follows: observe the phenotype of rice plants. Diseased plants must meet at least one of the following characteristics: the rice plants are severely stunted and the plant height is ≤1 / 3 of the normal plant height; the heart leaf breaks through the leaf sheath of the lower leaf and the breakthrough length is ≥5mm; some plants extend spirally from the lower leaf occipital opening; and some leaf tips show spiral curling. Healthy rice plants are characterized by normal plant height and vigorous tillering. When making statistics, the average incidence rate of two replicates at each test site is used as the result of the incidence rate.

[0022] Preferably, the incidence rate should be counted 30-35 days after the rice is transplanted to the test nursery, and the incidence rate count should also meet the following requirements:

[0023] To determine whether a diseased plant is "severely stunted and its height is ≤1 / 3 of the normal height," the plant height must be measured with a ruler with an accuracy of 0.1 cm.

[0024] The determination of "the heart leaf breaking through the lower leaf sheath with a breakthrough length ≥ 5 mm" requires measuring the length of the heart leaf extending beyond the sheath of the lower leaf, ensuring that the length is ≥ 5 mm.

[0025] Preferably, two replicate nurseries are set up at each test site, with an area of ​​10m2 for each replicate nursery, and the number of rice seedlings to be tested planted in each replicate nursery is ≥50.

[0026] Compared with the prior art, the present invention has the following beneficial effects:

[0027] This invention uses selectively infected insects weighing 0.3-0.5 mg / head and feeding ≥5 times per hour as the virus transmission vector, ensuring the virus-carrying capacity and transmission efficiency of the white-backed planthopper at the microscopic level. Nymphs within this weight range can maintain a stable SRBSDV load above 0.8 μg / mL, significantly higher than the 0.5 μg / mL level of unselected nymphs in traditional methods, providing sufficient virus input to rice. The feeding frequency of ≥5 times per hour ensures continuous virus injection into rice within the 48-hour transmission cycle, avoiding insufficient virus transmission due to feeding interruptions. This effectively solves the problem of "inconsistent quality of virus transmission vectors" in traditional methods, significantly increasing the basic probability of rice infection with the virus and laying the foundation for subsequent high incidence rates.

[0028] The composite nano-silver-chitosan modified microneedle array in this invention, combined with a salicylic acid-melatonin composite inducer, forms a synergistic system of wound guidance, antibacterial protection, and physiological maintenance. From a microstructural perspective, it can form micron-level wound channels at the base of the leaf sheath. These channels directly connect the vascular bundles to the outside environment, guiding the virus to migrate directionally from the outside of the vascular bundles to the phloem, overcoming the physical barrier that makes it difficult for viruses to enter the phloem in traditional methods. Furthermore, the salicylic acid-melatonin inducer sprayed before pretreatment can maintain the chlorophyll content of rice at ≥2.0 mg / g (FW), ensuring that rice maintains normal physiological activity after wounding and avoiding interference with disease incidence statistics due to abnormal growth. This invention solves the problem of low virus migration efficiency and eliminates interference from bacterial infection, further increasing the probability of systemic disease in rice.

[0029] From the perspective of overall synergistic effect, the technical solution of this invention provides a foundation for sufficient virus input through screening of infected white-backed planthoppers, ensures efficient virus migration and tissue protection through leaf sheath micro-trauma pretreatment and composite modified microneedles, and creates conditions for stable virus transmission through an optimized transmission environment. The steps cooperate with each other and progress step by step, ultimately leading to a stable increase in rice disease incidence, which is significantly better than traditional methods. At the same time, the identification cycle is shortened to less than 35 days, effectively solving the problems of low disease incidence, long cycle, and poor accuracy of traditional methods. It can efficiently meet the needs of rapid screening of disease-resistant rice varieties and provide strong technical support for the prevention and control of rice black-streaked dwarf disease in southern China. Attached Figure Description

[0030] Figure 1 This is a flowchart of an identification method for improving the incidence of black-streaked dwarf disease in southern rice according to the present invention. Detailed Implementation

[0031] The technical solutions in the embodiments of the present invention will be clearly and completely described below. 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.

[0032] Example 1: (1) Cultivation of infected white-backed planthoppers: A healthy white-backed planthopper rearing system was established. The rearing environment temperature was 27℃, the relative humidity was 70%, and the photoperiod was 16h / 8h (light / dark). Rice seedlings that were not infected with Southern Rice Black-Streaked Dwarf Virus were fed. After the white-backed planthoppers reproduced to the F1 generation, adult insects were selected and transferred to diseased plants infected with Southern Rice Black-Streaked Dwarf Virus. The insects were fed on the sap of the diseased plants for 48 hours to obtain the virus. The insects were then transferred back to healthy rice seedlings. After 10 days, the adult insects were individually tested using qRT-PCR detection technology. Individuals with a high virus carrying rate were selected to obtain the virus-carrying insects. The weight of each virus-carrying insect was weighed using an electronic balance with an accuracy of 0.01mg. Individuals with a weight of 0.4mg / individual were selected. The number of times each nymph fed within 1 hour was recorded using a high-definition video observation system. Individuals that fed 6 times were selected as virus-carrying white-backed planthoppers for later use.

[0033] (2) Pretreatment of rice seedling leaf sheath micro-trauma: Rice seedlings to be tested were cultivated to the early tillering stage. The plant height was measured to be 18cm and the number of leaves on the main stem was 4, which met the requirements of the early tillering stage. Preparation of salicylic acid-melatonin composite inducer: Salicylic acid and melatonin were mixed at a mass ratio of 3:1, and 5% ethanol aqueous solution was added. The solution was dissolved by ultrasonication at 300W until clear, and then diluted with deionized water to a concentration of 20mg / L (prepared and used immediately). The inducer was sprayed onto the rice seedlings using a manual sprayer with a 60μm atomization particle size. The amount sprayed per plant was 10mL. 24h after spraying, no obvious yellowing of the leaves was observed. The chlorophyll content was detected by ethanol extraction: 0.1g of leaves were added to 10mL of 95% ethanol and soaked in the dark for 24h. The absorbance at 665nm and 649nm was measured by spectrophotometer. The chlorophyll content was calculated to be 2.2mg / g (FW), which met the pretreatment requirements.

[0034] Preparation of composite silver-chitosan modified microneedle array: Medical-grade polylactic acid-glycolic acid copolymer with a lactic acid to glycolic acid molar ratio of 7:3 and intrinsic viscosity of 0.7 dL / g was dissolved in a mixed solvent of dichloromethane and N,N-dimethylformamide in a volume ratio of 4:1 to prepare a 16% (w / w) mixed solution. The mixed solution was cast into a silicon mold with a pre-designed conical microneedle structure and vacuum dried in a vacuum drying oven at 36℃ for 9 h. After the solvent was completely evaporated, the microneedle substrate was peeled off. Preparation of chitosan solution: Chitosan with a degree of deacetylation of 92% and a viscosity of 150 mPa·s was dissolved in a 1% acetic acid aqueous solution to prepare a 1.8% (w / w) chitosan solution. Preparation of nano-silver sol: Mix 0.03 mol / L silver nitrate aqueous solution and 1.6% food-grade pigskin gelatin solution at a mass ratio of 1:2, place in a 60℃ constant temperature water bath and add 0.1 mol / L ascorbic acid solution dropwise at a rate of 1 mL / min until the solution turns brownish-red, and continue stirring for 1 h; then transfer the solution to a dialysis bag with a molecular weight cutoff of 3500 Da, and dialyze with deionized water for 48 h (replace the deionized water every 12 h) to obtain 0.45% nano-silver sol. Chitosan solution and nano-silver sol were mixed at a volume ratio of 1:1 and stirred evenly to obtain a composite coating solution. The composite coating solution was coated on the surface of the microneedle substrate by a spin coater at a speed of 800 r / min for 30 s to form a composite coating with a thickness of 10 μm. The coated microneedle substrate was placed in a 40℃ forced-air drying oven and dried for 2 h to obtain a composite nano-silver-chitosan modified microneedle array (each microneedle is conical with a tip diameter of 6 μm and a body height of 220 μm).

[0035] The microneedle array was used to puncture the base of the leaf sheath of the main stem of rice at a distance of 2.5 cm from the ground. The puncture depth was controlled at 60 μm, and the puncture density was controlled at 2.5 punctures / mm. 2 This creates micron-sized wound channels on the leaf sheath.

[0036] (3) Inoculation and transmission of the virus-carrying white-backed planthopper: The virus-carrying white-backed planthoppers obtained in step (1) were inoculated onto rice seedlings treated in step (2) at a density of 1 planthopper per seedling. The inoculated rice seedlings were then placed in an artificial climate chamber for virus transmission. The temperature of the artificial climate chamber was set to 26℃, the relative humidity was set to 75%, the light cycle was set to 14h / 10h (light / dark), the carbon dioxide concentration was controlled at 550ppm, and the virus transmission time lasted for 48h.

[0037] After the transmission of the virus is completed, the effectiveness will be verified:

[0038] ① One hundred white-backed planthoppers were randomly selected, and the SRBSDV load in vivo was detected using a qRT-PCR kit. The result was 1.0 μg / mL.

[0039] ② Take 0.1g of tissue from the wound site of rice leaf sheath, cut it into small pieces according to GB4789.2-2022, add 0.9mL of sterile physiological saline and shake well. Dilute serially to 10⁻³, spread on nutrient agar medium, and incubate at 37℃ for 24h. Count the bacterial colonies at the wound site and calculate that it is 22 CFU / mm². 2 ;

[0040] ③ The leaf sheath puncture area was observed using a stereomicroscope, and the proportion of necrotic area to puncture area was measured to be 3%; all three indicators met the requirements.

[0041] (4) Rice transplanting and disease incidence phenotypic statistics: Virus-carrying white-backed planthoppers were gently removed from rice seedlings with a soft brush. The seedlings were then transplanted to an identification nursery and cultured under standard field management conditions (watering twice a week, manual weeding, and no application of antiviral agents). Two replicate nurseries were set up at each experimental site, with each replicate nursery covering an area of ​​10 m². 2 Fifty rice seedlings were planted in each replicate nursery. Disease incidence was assessed 32 days after transplanting.

[0042] ① Use a ruler with an accuracy of 0.1cm to measure the height of each rice plant and determine whether it meets the criteria of "severely stunted rice plants with a height ≤ 1 / 3 of the normal plant height";

[0043] ② Use a ruler to measure the length of the heart leaf extending beyond the lower leaf sheath, and determine whether it meets the requirement of "the heart leaf breaking through the lower leaf sheath and the breakthrough length ≥ 5mm";

[0044] ③ Observe whether there are phenomena such as "the plant extending spirally from the lower leaf occipital" and "the leaf tip curling spirally". Statistics show that 41 plants in the first replicate nursery were infected, with an infection rate of 82%; 40 plants in the second replicate nursery were infected, with an infection rate of 80%; the average of 81% was taken as the infection rate result of Example 1.

[0045] Example 2: (1) Cultivation of infected white-backed planthoppers: The rearing environment for healthy white-backed planthoppers was 28℃, relative humidity 75%, and photoperiod 16h / 8h (light / dark). They were fed rice seedlings that were not infected with Southern Rice Black-Streaked Dwarf Virus. After the F1 generation adults were transferred to infected plants and fed for 48 hours, they were transferred back to healthy rice seedlings. After 10 days, the adults were tested using qRT-PCR to screen individuals with high virus carrying rates. Individuals with high virus carrying rates were selected. Individuals weighing 0.5mg / head were selected using an electronic balance with an accuracy of 0.01mg. Individuals with a high-definition video observation system were selected to feed 7 times per hour as infected white-backed planthoppers for later use.

[0046] (2) Pretreatment of rice seedling leaf sheaths with micro-trauma: Rice seedlings to be tested were cultivated to the early tillering stage, with a plant height of 20 cm and 5 leaves on the main stem. A salicylic acid-melatonin composite inducer was prepared: salicylic acid and melatonin were mixed at a mass ratio of 3:1, dissolved in 5% ethanol aqueous solution using ultrasonication at 300W, and then diluted with deionized water to a concentration of 25 mg / L (prepared immediately before use). The solution was sprayed using a manual sprayer with an atomizing particle size of 80 μm, with 10 mL sprayed per plant. 24 h after spraying, no obvious yellowing of the leaves was observed; the chlorophyll content, as determined by ethanol extraction, was 2.4 mg / g (FW).

[0047] Preparation of composite silver nanoparticle-chitosan modified microneedle array: A polylactic acid-glycolic acid copolymer with a lactic acid to glycolic acid molar ratio of 7:3 and an intrinsic viscosity of 0.8 dL / g was selected and dissolved in a mixed solvent of dichloromethane and N,N-dimethylformamide in a volume ratio of 4:1 to prepare a mixed solution with a mass fraction of 18%; after casting into a silicon mold, it was vacuum dried at 38℃ for 10 h to obtain the microneedle substrate. Chitosan with a degree of deacetylation of 95% and a viscosity of 200 mPa·s was selected and dissolved in a 1% acetic acid aqueous solution to prepare a 2.0% (w / w) chitosan solution. For the preparation of the nano-silver sol, the silver nitrate concentration was 0.04 mol / L and the gelatin solution concentration was 1.8%, ultimately yielding a 0.6% nano-silver sol. The chitosan solution and nano-silver sol were mixed at a 1:1 volume ratio to obtain a composite coating solution. Spin coating (800 r / min, 30 s) was then applied to form a 12 μm thick coating, which was dried at 40℃ for 2 h to obtain a microneedle array (needle tip diameter 8 μm, needle height 250 μm). This microneedle array was used to puncture the base of rice leaf sheaths at a distance of 3 cm from the ground, with a puncture depth of 80 μm and a puncture density of 3 needles / mm. 2 .

[0048] (3) Inoculation and transmission of virus-carrying white-backed planthoppers: One virus-carrying white-backed planthopper was inoculated per plant. The artificial climate chamber was maintained at 28℃, 80% relative humidity, a 14h / 10h light / dark cycle, and a carbon dioxide concentration of 600ppm. Transmission was carried out for 48 hours. Efficacy verification: The SRBSDV load in the white-backed planthopper was 1.2 μg / mL, and the bacterial colony count at the wound site was 18 CFU / mm². 2 The necrotic area ratio is 2%, which meets the requirements.

[0049] (4) Statistical analysis of rice transplanting and disease incidence: Transplanting conditions were the same as in Example 1 (2 replicates, 10m² per plot). 2 (50 plants), and routine field management. Disease incidence was counted 35 days after transplanting: in replication 1, 42 plants were infected with the disease, with a disease incidence rate of 85%; in replication 2, 41 plants were infected with the disease, with a disease incidence rate of 83%; the average of 84% was taken as the disease incidence rate result for Example 2.

[0050] Example 3: (1) Cultivation of infected white-backed planthoppers: The rearing environment for healthy white-backed planthoppers was 26℃, relative humidity 65%, and photoperiod 16h / 8h (light / dark). They were fed rice seedlings that were not infected with Southern Rice Black-Streaked Dwarf Virus. After the F1 generation adults were transferred to diseased plants and fed for 48 hours, they were transferred back to healthy rice seedlings. After 10 days, the adults were tested using qRT-PCR technology. Individuals with high virus carrying rates were selected to obtain virus-carrying individuals. Individuals with a weight of 0.3mg / head and feeding 5 times per hour were selected as virus-carrying white-backed planthoppers for later use.

[0051] (2) Pretreatment of leaf sheath micro-trauma in rice seedlings: Rice seedlings to be tested were cultivated to the early tillering stage, with a plant height of 15cm and 4 leaves on the main stem. The salicylic acid-melatonin composite inducer was applied at a concentration of 22mg / L using a manual sprayer with a particle size of 50μm, 10mL per plant; 24h after spraying, the chlorophyll content was 2.1mg / g (FW), and there was no obvious yellowing.

[0052] Preparation of composite silver-chitosan modified microneedle array: A polylactic acid-glycolic acid copolymer with an intrinsic viscosity of 0.6 dL / g was mixed with a solution containing 15% by mass and dried under vacuum at 35℃ for 8 h to obtain the microneedle substrate. A chitosan solution with a degree of deacetylation of 90% and a viscosity of 100 mPa·s was prepared, containing 1.5% by mass. For the preparation of the silver nanoparticle sol, the silver nitrate concentration was 0.02 mol / L and the gelatin solution concentration was 1.5%, resulting in a 0.3% silver nanoparticle sol. After coating with the composite coating solution, an 8 μm thick coating was formed, and dried at 40℃ for 2 h to obtain the microneedle array (needle tip diameter 5 μm, needle height 200 μm). The microneedles were punctured at the base of the leaf sheath 2 cm above the ground, with a puncture depth of 50 μm and a puncture density of 2 per mm. 2 .

[0053] (3) Inoculation and transmission of the virus-carrying white-backed planthopper: Inoculation was carried out at a density of 1 planthopper per plant. The artificial climate chamber temperature was 25℃, relative humidity was 70%, light cycle was 14h / 10h (light / dark), carbon dioxide concentration was 500ppm, and the transmission time was 48h. Efficacy verification: The SRBSDV load in the white-backed planthopper was 0.9μg / mL, and the bacterial colony count at the wound site was 24CFU / mm². 2 The necrotic area ratio is 4%, which meets the requirements.

[0054] (4) Rice transplanting and disease incidence phenotype statistics: The transplanting conditions were the same as in Example 1. The disease incidence was counted 30 days after transplanting: 38 plants in Repeat 1 were infected with the disease, with a disease incidence of 76%; 37 plants in Repeat 2 were infected with the disease, with a disease incidence of 74%; the average value of 75% was taken as the disease incidence result of Example 3.

[0055] Comparative Example 1: The same treatment method as in Example 1 was used, except that the microneedle array used for the pretreatment of micro-trauma in the leaf sheath of rice seedlings in step (2) was a pure polylactic acid-hydroxyacetic acid copolymer microneedle array that had not undergone composite nano-silver-chitosan modification.

[0056] (4) Statistical analysis of rice transplanting and disease incidence: Transplanting conditions (2 replicate nurseries, 10m² per nursery) 2 The number of plants (50 plants) and the statistical methods were the same as in Example 1. Statistics were collected 32 days after transplanting: in Duplicate 1, 21 plants were infected with the disease, with a disease rate of 42%; in Duplicate 2, 22 plants were infected with the disease, with a disease rate of 43%; the average rate of 42.5% was used as the disease rate result for Comparative Example 1.

[0057] Comparative Example 2: The same treatment method as in Example 1 was used, except that in step (1), the poisonous white-backed planthoppers were not screened by weight and feeding frequency, and the third instar poisonous nymphs were directly selected.

[0058] (4) Rice transplanting and disease incidence phenotypic statistics: The transplanting and statistical conditions were the same as in Example 1. Statistics were collected 32 days after transplanting: 16 plants were infected in replicate 1, with a disease incidence of 32%; 17 plants were infected in replicate 2, with a disease incidence of 33%; the average of 32.5% was used as the disease incidence result for comparative example 2.

[0059] Comparative Example 3: The same treatment method as in Example 1 was used, except that the pretreatment of micro-trauma to the leaf sheath of rice seedlings was not performed in step (2).

[0060] (4) Rice transplanting and disease incidence phenotypic statistics: The transplanting and statistical conditions were the same as in Example 1. Statistics were collected 32 days after transplanting: 11 diseased plants in replicate 1, with a disease incidence of 22%; 12 diseased plants in replicate 2, with a disease incidence of 23%; the average value of 22.5% was used as the disease incidence result for comparative example 3.

[0061] test:

[0062] Experiment on the incidence and viral load of Southern Black-streaked Dwarf Disease in Rice;

[0063] Rice seedlings treated in Examples 1-3 and Comparative Examples 1-3 were divided into two replicates, with 50 seedlings in each replicate. Untreated rice seedlings that grew normally (blank control) were divided into one replicate (50 seedlings).

[0064] Test method:

[0065] (1) Disease incidence statistics: The disease incidence was statistically analyzed according to the transplanting time of each treatment (32 days for Example 1 and Comparative Examples 1-3, 35 days for Example 2 and 30 days for Example 3) and the average value of the two replicate nurseries was taken. The disease incidence was statistically analyzed 32 days after transplanting for the blank control.

[0066] (2) Detection of viral load in rice: On the day of incidence rate statistics, 10 diseased rice plants were randomly selected from each treatment (10 healthy rice plants were selected as blank control). 0.5g of leaf sheath tissue was taken from each plant, and viral protein was extracted using a qRT-PCR kit. The absorbance value at 450nm was detected according to the kit instructions. The viral load was calculated based on the standard curve, and the average value of the 10 plants was taken. The results are as follows:

[0067] Table 1

[0068] Example 1 81 0.75 Example 2 84 0.82 Example 3 75 0.68 Comparative Example 1 42.5 0.32 Comparative Example 2 32.5 0.25 Comparative Example 3 22.5 0.18

[0069] As shown in Table 1, the incidence rate and viral load in rice in Examples 1-3 were significantly higher than those in all comparative examples and blank controls, indicating that the technical solution of the present invention can synergistically improve the incidence rate of rice black-streaked dwarf disease in southern China and meet the requirements for efficient identification.

[0070] The preferred embodiments of the present invention disclosed above are merely illustrative of the invention. These preferred embodiments do not describe all details exhaustively, nor do they limit the invention to the specific implementations described. Clearly, many modifications and variations can be made based on the content of this specification.

Claims

1. A method for identifying diseases that increase the incidence of rice black-streaked dwarf virus in southern China, characterized in that, Includes the following steps: (1) Cultivation of white-backed planthoppers carrying the Southern Rice Black-streaked Dwarf Virus: Cultivate white-backed planthopper populations carrying the Southern Rice Black-streaked Dwarf Virus, and screen for individuals carrying the virus. The weight of the screened individuals carrying the virus is 0.3-0.5 mg / person, and the number of feeding times per hour is ≥5 times. (2) Pretreatment of rice seedling leaf sheaths with micro-trauma: The rice seedlings to be tested in the early tillering stage were subjected to pretreatment of leaf sheaths with micro-trauma. The seedlings in the early tillering stage were 15-20 cm tall and had 4-5 leaves on the main stem. During the pretreatment, a microneedle array modified with composite nano-silver-chitosan was used to puncture the base of the leaf sheath of the main stem of the rice at a distance of 2-3 cm from the ground. The microneedles of the array were conical with a tip diameter of 5-8 μm, a needle height of 200-250 μm, a puncture depth of 50-80 μm, and a puncture density of 2-3 per mm. 2 A micrometer-scale wound channel is created through puncture; (3) Inoculation and transmission of the virus-carrying white-backed planthopper: The virus-carrying white-backed planthoppers obtained in step (1) were inoculated on rice seedlings treated in step (2) at a density of 1-2 planthoppers per plant. The virus was transmitted in a transmission environment for 48 hours. The temperature of the transmission environment was 25-28℃, the relative humidity was 70-80%, the photoperiod was 14h / 10h, and the carbon dioxide concentration was 500-600ppm. (4) Statistics on rice transplanting and disease incidence phenotype: After removing white-backed planthoppers, rice seedlings were transplanted to the identification nursery and cultured under conventional field management conditions. Disease incidence was counted 30-35 days after transplanting. In step (2), 24 hours before the micro-trauma treatment, the rice seedlings need to be sprayed with a salicylic acid-melatonin composite inducer. The specific spraying method is as follows: spray with a manual sprayer with an atomization particle size of 50-80μm. After spraying, the rice leaves are tested, and the test results must meet the requirements of no obvious yellowing and chlorophyll content FW≥2.0mg / g.

2. The method according to claim 1, characterized in that, The preparation method of the composite nano-silver-chitosan modified microneedle array in step (2) includes: Preparation of microneedle substrate: Medical-grade polylactic acid-glycolic acid copolymer (PLA-glycolic acid copolymer) with a lactic acid to glycolic acid molar ratio of 7:3 and an intrinsic viscosity of 0.6-0.8 dL / g was prepared. The PLA-glycolic acid copolymer was then formulated into a 15-18 wt% dichloromethane-N,N-dimethylformamide mixed solution, with a dichloromethane to N,N-dimethylformamide volume ratio of 4:

1. The mixed solution was cast into a silicon mold and vacuum dried at 35-38℃ for 8-10 h to obtain the microneedle substrate. Preparation of composite coating solution: Take chitosan with a degree of deacetylation ≥90% and a viscosity of 100-200 mPa·s; dissolve chitosan in 1% acetic acid aqueous solution to prepare a 1.5-2.0 wt% chitosan solution; take 0.3-0.6 wt% silver nanoparticle sol, and mix the chitosan solution and silver nanoparticle sol at a volume ratio of 1:1 to obtain the composite coating solution; Coating and drying: A composite coating liquid was coated on the surface of the microneedle substrate using a spin coating method. The spin coating speed was 800 r / min and the coating time was 30 s, forming a composite coating with a thickness of 8-12 μm. The microneedle array was obtained by drying in a forced air at 40℃ for 2 h.

3. The method according to claim 2, characterized in that, The nano-silver sol was prepared by ascorbic acid reduction-gelatin stabilization method. The specific steps are as follows: 0.02-0.04 mol / L silver nitrate aqueous solution and 1.5-1.8 wt% food-grade pigskin gelatin solution were mixed at a mass ratio of 1:2; under the condition of keeping warm at 60℃, 0.1 mol / L ascorbic acid solution was added dropwise at a rate of 1 mL / min until the solution turned brownish-red, and then the reaction was continued to be stirred for 1 h; the solution was purified by dialysis with a dialysis bag with a molecular weight cutoff of 3500 Da for 48 h, and the deionized water was replaced every 12 h during the dialysis process, and finally the nano-silver sol was obtained.

4. The method according to claim 1, characterized in that, The preparation method of the salicylic acid-melatonin complex inducer is as follows: take salicylic acid and melatonin, mix them at a mass ratio of 3:1; dissolve them in a 5% (v / v) ethanol aqueous solution, and then dilute with deionized water to a concentration of 20 mg / L. Prepare and use immediately.

5. The method according to claim 1, characterized in that, The concentration of the compound inducer used for spraying is 20-25 mg / L, and the spraying amount is 10 mL per rice plant.

6. The method according to claim 1, characterized in that, After step (3) of the virus transmission is completed, the effectiveness of the virus transmission needs to be verified. The verification method includes the following steps: One hundred white-backed planthoppers were randomly selected, and their SRBSDV load was detected by qRT-PCR. The detection result must meet the requirement that the load is ≥0.8μg / mL, which can ensure that the insect carries sufficient virus.

7. The method according to claim 1, characterized in that, The statistical method for the incidence rate described in step (4) is as follows: observe the phenotype of rice plants. Diseased plants must meet at least one of the following characteristics: the rice plants are severely stunted and the plant height is ≤1 / 3 of the normal plant height; the heart leaf breaks through the leaf sheath of the lower leaf and the breakthrough length is ≥5mm; some plants extend spirally from the lower leaf occipital opening; and some leaf tips show spiral curling. Healthy rice plants are normal in height and have vigorous tillering. When making statistics, the average incidence rate of two replicates at each test site is used as the result of the incidence rate.

8. The method according to claim 7, characterized in that, The incidence rate should be counted 30-35 days after rice transplanting to the test nursery, and the incidence rate statistics must also meet the following requirements: To determine whether a diseased plant is "severely stunted and its height is ≤1 / 3 of the normal height," the plant height must be measured with a ruler with an accuracy of 0.1 cm. The determination of "the heart leaf breaks through the lower leaf sheath and the breakthrough length is ≥5mm" requires measuring the length of the heart leaf that extends beyond the lower leaf sheath, ensuring that the length is ≥5mm.

9. The method according to claim 7, characterized in that, Two replicates were set up at each test site, with each replicate having an area of ​​10m². 2 The number of rice seedlings to be tested planted in each replicate nursery is ≥50.

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