CN114128680A - High-throughput artificial inoculation identification method for southern rice black-streaked dwarf disease - Google Patents
High-throughput artificial inoculation identification method for southern rice black-streaked dwarf disease Download PDFInfo
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Abstract
The invention relates to the technical field of plant disease resistance identification, and particularly discloses a high-throughput artificial inoculation identification method for southern rice black-streaked dwarf disease, which comprises the following steps: artificially feeding the Pothodes laevigata with the toxicity indoors to serve as an inoculum; under indoor conditions, sowing seeds of different rice varieties to be identified on a seedling tray at the same time, and then culturing to a seedling stage; inoculating in the seedling stage of rice, wherein the average effective inoculation number of each plant with the poisonous insects is 0.5-0.9, transplanting the rice seedlings into an insect-proof net room or a greenhouse for culturing after inoculating for 48-96 h, and performing conventional cultivation management; the incidence rate of the rice is investigated in the full tillering stage of the rice, and the resistance level of the rice material to be detected to the southern rice black-streaked dwarf disease is evaluated according to the incidence rate. The high-throughput artificial inoculation identification method for the southern rice black-streaked dwarf disease can be used for identifying the southern rice of multiple varieties to be detected in a large batch at one time, so that the large-scale identification of the disease resistance of the southern rice black-streaked dwarf disease is realized, and the identification accuracy can be improved.
Description
Technical Field
The invention belongs to the technical field of plant disease resistance identification, and particularly relates to a high-throughput artificial inoculation identification method for southern rice black-streaked dwarf disease.
Background
Rice is one of the main food crops in the world, more than half of the global population takes rice as staple food, and 60% of the national population also takes rice as staple food. Southern rice black streaked dwarf is a disease mainly transmitted by sogatella furcifera in a persistent proliferative manner, and the pathogen thereof is Southern Rice Black Streaked Dwarf Virus (SRBSDV) of Fijivirus (Fijivirus) of Reoviridae (Reoviridae). Since the varieties of rice resisting the disease are rare in production, the occurrence of the disease causes great harm to the rice production in the central south, north vietnamese and south japan in the century. Currently, the control of the disease is mainly to kill the virus-transferring vector sogatella furcifera by using chemical agents so as to block the spread of the disease, but the control effect is poor due to the characteristics of huge population quantity, strong mobility, drug resistance, lasting virus transfer and the like of the sogatella furcifera. Planting disease-resistant varieties is one of the most economic and effective measures for preventing and controlling rice diseases, large-scale resistance source screening is the premise of genetic research on rice resistance and variety improvement, and resistance identification and evaluation on candidate rice seed resources are inevitable measures. However, few research reports on discovery of resistance sources and breeding for disease resistance are reported at present, and identified resistance sources and resistance genes/QTL sites are limited, so that the breeding process of new varieties of southern rice black-streaked dwarf resistant rice is hindered.
At present, a field natural induced disease identification method is mainly used for screening and identifying southern rice black-streaked dwarf virus large-scale germplasm resources, and is easily influenced by factors such as climatic environment conditions, migratory flight number of Sogatella furcifera populations, and virus carrying rate due to the constraint of natural conditions, so that the identification result is large in difference between regions and years, and a reliable result can be obtained only through multi-point repeated identification for many years, so that the workload is increased, the breeding time is prolonged, and the popularization and the application of the method are limited. The problems of low efficiency, uncontrollable property, poor repeatability, low accuracy and the like of the natural induced disease identification method are a major bottleneck of the research in the field at present, and the genetic research and the breeding utilization process of the disease are limited. Through years of researches on inoculation and virus transmission of sogatella furcifera, the laboratory invents a set of high-throughput manual inoculation identification method aiming at the sogatella furcifera, effectively masters the sensitive period of rice seedling inoculation, determines the appropriate effective inoculation insect quantity and the appropriate inoculation time, can identify the disease resistance of a large number of candidate materials at one time, greatly improves the inoculation efficiency and precision, saves the test cost, and provides an effective technical means for resource development screening, variety breeding, resistance genetic rule research and the like of southern rice black-streaked dwarf.
Disclosure of Invention
The invention aims to provide a high-throughput artificial inoculation identification method for southern rice black-streaked dwarf disease, which overcomes the defects and shortcomings of low accuracy, poor repeatability, low efficiency and the like of the conventional natural inoculation identification method for southern rice black-streaked dwarf disease.
In order to realize the aim, the invention provides a high-throughput artificial inoculation identification method for southern rice black-streaked dwarf disease, which comprises the following steps:
(1) and (3) culturing an inoculum: artificially feeding the Pothodes laevigata with the toxicity indoors to serve as an inoculum;
(2) preparing a rice sample: under indoor conditions, sowing seeds of different rice varieties to be identified on a seedling tray at the same time, and then culturing to a seedling stage;
(3) inoculation: inoculating in the seedling stage of rice, wherein the average effective inoculation number of each plant with the poisonous insects is 0.5-0.9, transplanting the rice seedlings into an insect-proof net room or a greenhouse for culturing after inoculating for 48-96 h, and performing conventional cultivation management;
(4) and (3) resistance identification: the incidence rate of the rice is investigated in the full tillering stage of the rice, and the resistance level of the rice material to be detected to the southern rice black-streaked dwarf disease is evaluated according to the incidence rate.
Preferably, in the southern rice black-streaked dwarf high-throughput artificial inoculation identification method, in the step (1), the inoculum is an artificially fed sogatella furcifera population with a toxicity rate of more than 80%.
Preferably, in the southern rice black-streaked dwarf high-throughput artificial inoculation identification method, in the step (2), 30-50 seeds are sown in each rice product.
Preferably, in the above high-throughput artificial inoculation identification method for southern rice black-streaked dwarf, in the step (2), a rice dibbling device is adopted to sow rice seeds on a seedling tray, the dibbling device dibbles a plurality of uniform and consistent healthy rice seedlings at the same time, and a plurality of dibbling devices are utilized to cultivate large-scale healthy rice seedlings as required.
Preferably, in the southern rice black-streaked dwarf high-throughput artificial inoculation identification method, the rice dibbling device is a rice seed rapid dibbling device disclosed in patent number ZL202021748815.5, and one dibbling device can dibble and culture 660 uniform and consistent healthy rice seedlings.
Preferably, in the above high-throughput artificial inoculation identification method for southern rice black-streaked dwarf, in the step (3), the rice seedling stage is 1-2 leaf 1 heart stage.
Preferably, in the southern rice black-streaked dwarf high-throughput artificial inoculation identification method, in the step (4), the incidence rate (%) is the number of infected plants/total number of plants × 100, the rice plants are dwarf, dark green in leaf color, curled in leaf tips, uneven folds formed on the upper leaves near the basal leaf surfaces, and wax-like milky white or black brown nodular protrusions on leaf sheaths are judged as infected plants.
Preferably, in the above method for identifying high-throughput artificial inoculation of southern rice black-streaked dwarf, the resistance level is classified into the following grades:
grade 0 is immunity, and the incidence rate is 0;
grade 1 is high resistance, and the morbidity is 0.1-5.0%;
grade 3 is medium resistance, and the morbidity is 5.1-15.0%;
grade 5 is moderate, and the incidence rate is 15.1% -30.0%;
grade 7 is susceptible, and the incidence rate is 30.1-60.0%;
grade 9 is high-grade, and the incidence rate is more than 60.1%.
Preferably, in the above method for identifying high-throughput artificial inoculation of southern rice black-streaked dwarf, the specific steps of the inoculum culture include:
s1, collecting nymphs of Sogatella furcifera from a rice field, feeding the nymphs to adults in an insect feeding device for cultivating rice TN1, transferring the adults to TN1 rice seedling cups for spawning, and hatching to obtain the healthy nymphs of the Sogatella furcifera;
s2, transferring 1-2-year-old healthy Sogatella furcifera nymphs to a diseased rice plant infected with southern rice black-streaked dwarf virus to feed the virus;
s3, transplanting the seedlings to healthy rice after 48 hours of virus feeding, after 9-12 days of circulation, randomly extracting 50 sogatella furcifera, detecting the virus carrying rate of the groups by using a Dot-ELISA method or an RT-PCR method, wherein the groups with the virus carrying rate of more than 80 percent are determined as inoculants.
Compared with the prior art, the invention has the following beneficial effects:
1. according to the high-throughput artificial inoculation identification method for the southern rice black-streaked dwarf virus, provided by the invention, the white-backed planthopper with virus is artificially bred indoors, so that a large number of virus-transmitting media (inoculants) can be cultivated at one time, rice seedlings are planted indoors, large-batch and uniform-growth rice seedlings can be sown at one time, large-batch identification can be carried out on southern rice of multiple varieties to be detected at one time, and the disease resistance identification of the southern large-scale rice black-streaked dwarf virus is realized.
2. The high-throughput artificial inoculation identification method for southern rice black-streaked dwarf virus can avoid the inconsistent virus-transmission effects caused by different environmental conditions of the virus-transmission of sogatella furcifera, and improves the identification accuracy by carrying out inoculation identification on materials to be detected at the same time under the same condition.
3. The southern rice black-streaked dwarf virus high-throughput artificial inoculation identification method determines the most appropriate inoculation period, effective inoculation insect quantity and inoculation time through repeated tests, shortens the cultivation and inoculation time of rice seedlings to be tested, reduces the using amount of virus-transmitting media and greatly improves the inoculation efficiency. The high-throughput manual requirement of southern rice black-streaked dwarf virus inoculation identification is realized, the bottlenecks that the conventional identification is difficult to scale, has low efficiency and low accuracy are broken through, and the method can be applied to not only rice resistance evaluation, development of disease-resistant resources and breeding of new disease-resistant varieties, but also phenotypic identification of disease-resistant gene positioning, research on resistance genetic law and other fields, thereby providing powerful technical support for research on disease-resistant rice.
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FIG. 1 is a photograph of a rice sample prepared in example 1 of the present invention.
FIG. 2 is a photograph of an inoculation in example 1 of the present invention.
Detailed Description
The following detailed description of specific embodiments of the invention is provided, but it should be understood that the scope of the invention is not limited to the specific embodiments.
Example 1
A high-throughput artificial inoculation identification method for southern rice black-streaked dwarf disease comprises the following steps:
(1) and (3) culturing an inoculum: artificially feeding the sogatella furcifera with toxicity indoors, wherein the sogatella furcifera population with the toxicity rate of more than 80 percent is used as an inoculum;
(2) preparing a rice sample: under indoor conditions, a rice dibbling device (a rice seed rapid dibbling device with the patent number of ZL 202021748815.5) is adopted to sow a plurality of seeds of rice varieties to be identified on seedling trays, the planting quantity is 30 seeds, and then the seeds are cultivated to the seedling stage, as shown in figure 1;
(3) inoculation: inoculating rice seedlings of a plurality of rice varieties in the same inoculation chamber at the 2-leaf 1-heart stage of the rice seedling age as shown in figure 2, wherein the effective average inoculation amount of each rice seedling is 0.5, and after inoculating insects for 72 hours, transplanting the rice seedlings into an insect-proof net chamber for culture and carrying out conventional cultivation management;
(4) and (3) resistance identification: investigating the disease incidence of rice in the full tillering period of the rice, wherein rice plants are dwarf, have dark green leaf color, curled leaf tips, uneven folds formed on the upper leaves close to the basal leaf surface, wax-like milky white or black brown nodular protrusions are arranged on leaf sheaths and are judged as infected plants, the disease incidence (%) < infected plant number/total plant number multiplied by 100, and the resistance level of the rice material to be detected to southern rice black-streaked dwarf is evaluated according to the disease incidence;
the resistance levels were classified into the following classes:
grade 0 is immunity, and the incidence rate is 0;
grade 1 is high resistance, and the morbidity is 0.1-5.0%;
grade 3 is medium resistance, and the morbidity is 5.1-15.0%;
grade 5 is moderate, and the incidence rate is 15.1% -30.0%;
grade 7 is susceptible, and the incidence rate is 30.1-60.0%;
grade 9 is high-grade, and the incidence rate is more than 60.1%.
The specific steps of inoculum culture are as follows:
s1, preparing a toxic source:
suspected diseased plants expressing symptoms of southern rice black-streaked dwarf are collected from the field, and RT-PCR detection is utilized to determine whether the suspected diseased plants carry southern rice black-streaked dwarf viruses or not, and the method comprises the following specific steps: total RNA from rice was extracted according to the method described in the instruction of the RNA extraction kit (Trizol Reagent). Primers were synthesized according to published sequences (synthesized by Biotechnology (Shanghai) GmbH):
SRB-S10-F 5’-CCACATCGCGTCATCTCAAACTAC-3’
SRB-S10-R 5’-CGGTCTTACGCAACGATGAACC-3’
reverse transcription was performed with reference to the reverse transcriptase PrimeScriptTM RT-PCR Kit (TAKARA) instructions.
The PCR amplification system is as follows: template RNA 2. mu.L, PrimeScript 1Step Enzyme Mix 2.0. mu.L, 2X 1Step Buffer (Dye Plus) 25. mu.L, 5 '-end primer 1.0. mu.L, 3' -end primer 1.0. mu.L, RNase Free dH2O 19. mu.L, totaling 50. mu.L.
The reaction procedure is as follows: reverse transcription is carried out for 30min at 50 ℃; pre-denaturation at 94 ℃ for 2 min; denaturation at 94 ℃ for 30s, annealing at 58 ℃ for 30s, extension at 72 ℃ for 1min, and 35 cycles; finally, the extension is carried out for 10min at 72 ℃ and the product is stored at 4 ℃.
Detecting a target strip (920bp) of a PCR amplification product by 1% agarose gel electrophoresis, selecting rice plants infected with southern rice black-streaked dwarf virus (containing the 920bp amplification strip) for amplification and propagation, and using the rice plants for virus feeding of a virus-transmitting mediator, namely sogatella furcifera;
s2, collecting nymphs of the sogatella furcifera from a rice field, feeding the nymphs to adults in an insect feeding room cultivated with rice TN1, capturing the adults by using test tubes, transferring the adults to TN1 rice seedling cups for spawning, and hatching to obtain the healthy nymphs of the sogatella furcifera; temperature adjusting, humidity adjusting and light adjusting equipment is arranged in the insect breeding room, so that the temperature is kept at 26 +/-1 ℃, the humidity is kept between 70% and 80%, and the illumination time is 12 hours per day; the cup for planting TN1 rice seedlings is a 1000mL glass beaker;
s3, transferring the newly hatched healthy Sogatella furcifera nymphs to the rice diseased plants infected with the southern rice black-streaked dwarf virus obtained in S1 for virus feeding;
s4, transplanting the seedlings to healthy rice after 48 hours of virus feeding, after 9-12 days of circulation, randomly extracting 50 sogatella furcifera, detecting the virus carrying rate of the groups by using a Dot-ELISA method or an RT-PCR method, and determining the groups with the virus carrying rate of more than 80 percent as inoculants.
Cultivating inoculants, wherein 300 white-backed planthoppers can be bred in each cup, about 30000 white-backed planthoppers can be bred in 1 batch (100 cups), the predicted toxicity rate is more than 80%, 48000 rice seedlings (30000 heads multiplied by 80%/0.5 head/plant is 48000) can be inoculated in each batch according to 0.5 effective inoculation insect quantity, 660 seedlings can be quickly dibbled on a 75cm multiplied by 30cm seedling tray by using a dibbling device for processing, 48000 seedlings can be sown only by 73 seedling trays, and 1600 materials can be identified once by counting 30 seedlings of each material to be detected, so that large-scale material identification is realized, and the inoculation efficiency of the southern rice black-streaked dwarf virus resistance identification is greatly improved.
Test example 1 identification Effect of different effective amounts of inoculated insects
The effective inoculation worm amount is determined according to the toxic rate, and is set to be 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9 and 1.0 head/plant 10 treatment groups, and repeated for 3 times.
The test process comprises the following steps: after accelerating germination and exposing white seeds of a disease-sensitive rice variety TN1, selecting seeds with uniform growth vigor, sowing the seeds into 1000ml beakers paved with nutrient soil with the thickness of 2cm, placing 30 seeds in each material, and culturing in a light incubator (26 +/-1 ℃, 12h of light/12 h of dark). Inoculating adult white-backed planthopper with toxicity passing through the cycle at the age of 2.5 leaves, sealing by using gauze, removing the white-backed planthopper after 48h, transplanting the rice seedlings to an insect-proof net room or a greenhouse, and performing normal water and fertilizer management.
The incidence of disease was investigated after 20 days, every 7 days, 3 times continuously, and the incidence of disease was counted for each treatment. Randomly extracting leaves of 30 suspected diseased plants for RT-PCR virus-carrying detection, judging that the rice plants are dwarf, dark green in leaf color, curled in leaf tips, uneven folds formed on the upper leaves close to the basal part of the leaves and wax-like milky white or black brown nodular bulges on leaf sheaths are all judged to be infected plants according to the detection results.
And (3) test results: the disease condition of the rice variety TN1 under different effective inoculation insect quantity is shown in the table 1. The incidence of TN1 is improved with the increase of the effective inoculation worm quantity, when the effective inoculation worm quantity is 0.9 head/plant and 1 head/plant, the incidence of TN1 is 100%, the effective inoculation worm quantity is 0.5 head/plant to 1 head/plant, no significant difference exists between treatments, the effective inoculation worm quantity of the invention is 0.5 head/plant to 0.9 head/plant, the inoculation efficiency can be improved, and the identification accuracy is improved.
TABLE 1 incidence of TN1 rice variety with different effective inoculation insect amounts
Test example 2 evaluation Effect of different seedling ages to be inoculated
Selecting a disease-susceptible rice variety TN1, setting the effective inoculation insect quantity to be 1 head per plant, setting 5 treatment groups of 1-leaf 1-heart stage, 2-leaf 1-heart stage, 3-leaf 1-heart stage, 4-leaf 1-heart stage and 5-leaf 1-heart stage inoculation in the inoculation seedling age, and setting the specific test process and the investigation method to be the same as those in test example 1.
Table 2 shows the incidence of rice inoculation at different seedling ages, wherein the incidence of TN1 is not significantly different at the 1-leaf 1 heart stage, the 2-leaf 1 heart stage and the 3-leaf 1 heart stage, but the incidence of rice gradually decreases from the 3-leaf 1 heart stage, and the incidence of rice at the 4-leaf 1 heart stage is significantly different, which indicates that the incidence of rice is decreased with the increase of the seedling age of the inoculation. The inoculated seedling age is 1 leaf 1 heart stage-2 leaf 1 heart stage, and the identification effect can be improved.
TABLE 2 incidence of rice inoculation at different seedling ages
| Age of inoculated seedling | Incidence (%) | Significance of difference (1%) |
| 1 leaf 1 heart stage | 100 | A |
| 2 leaf 1 heart stage | 100 | A |
| 3 leaf 1 Heart stage | 95.56 | A |
| 4 leaf 1 heart stage | 84.44 | B |
| 5 leaf 1 Heart stage | 56.67 | C |
Test example 3 Effect of evaluation of different inoculation time
Selecting a disease-susceptible rice variety TN1, setting inoculation time to be 5 treatment groups of 24h, 48h, 72h, 96h and 120h, treating 30 seedlings each, wherein the seedling age is 2 leaves and 1 heart stage, effectively inoculating 1 head/plant of insects, and repeating for 3 times. The specific test procedure and the investigation method were the same as those in test example 1. The disease condition of TN1 under different inoculation times is shown in Table 3, and as the inoculation time increases, the disease rate of TN1 tends to gradually increase, and the disease rates of 4 periods of inoculation of 48h, 72h, 96h and 120h have no significant difference, so that the suitable inoculation time is determined to be 48-96 h.
TABLE 3 morbidity of TN1 at different inoculation times
Test example 4 comparison of the effects of the identification of the Rice variety by Artificial inoculation and the identification of the Rice variety in the heavily diseased region
Field identification in severe disease areas: selecting Guangxi Fengchong heavy disease area as an identification garden, using susceptible varieties TN1 and Zhenshan 97 as research materials, setting 4 rows of areas for each material, planting 13 holes in each row, transplanting each seed for 3 times, investigating the disease incidence of the rice in the tillering full period of the rice, investigating 2 times after 7 days and 30 days, calculating the disease incidence, and the investigation and the disease incidence calculation methods are the same as the above. According to the investigation of 8 months and 15 days, the quantity of the sogatella furcifera is 1032.1 heads/100 strains, the toxicity rate is 4.36 percent, and the effective inoculation quantity is 0.45 heads/strain.
Artificial inoculation: by adopting the high-throughput artificial inoculation identification method for southern rice black-streaked dwarf disease provided in example 1, the incidence rate of rice is investigated at the full tillering stage of rice, and is further investigated 2 times after 7 days and 30 days, and the incidence rate is calculated.
The results of the artificial inoculation identification and the field identification are shown in table 4, and compared with the results of the field identification in the seriously ill region, the results of the identification by the method have no significant difference in the morbidity of the rice variety TN1, and are all high in sensitivity, so that the indoor artificial inoculation identification method established by the research can simulate the effect of the field natural induction identification in the seriously ill region, and the resistance identification result is accurate and reliable.
TABLE 4 results of the identification of the artificial inoculation and the identification of the field
The foregoing descriptions of specific exemplary embodiments of the present invention have been presented for purposes of illustration and description. It is not intended to limit the invention to the precise form disclosed, and obviously many modifications and variations are possible in light of the above teaching. The exemplary embodiments were chosen and described in order to explain certain principles of the invention and its practical application to enable one skilled in the art to make and use various exemplary embodiments of the invention and various alternatives and modifications as are suited to the particular use contemplated. It is intended that the scope of the invention be defined by the claims and their equivalents.
Claims (8)
1. A high-throughput artificial inoculation identification method for southern rice black-streaked dwarf is characterized by comprising the following steps:
(1) and (3) culturing an inoculum: artificially feeding the Pothodes laevigata with the toxicity indoors to serve as an inoculum;
(2) preparing a rice sample: under indoor conditions, sowing seeds of different rice varieties to be identified on a seedling tray at the same time, and then culturing to a seedling stage;
(3) inoculation: inoculating in the seedling stage of rice, wherein the average effective inoculation number of each plant with the poisonous insects is 0.5-0.9, transplanting the rice seedlings into an insect-proof net room or a greenhouse for culturing after inoculating for 48-96 h, and performing conventional cultivation management;
(4) and (3) resistance identification: the incidence rate of the rice is investigated in the full tillering stage of the rice, and the resistance level of the rice material to be detected to the southern rice black-streaked dwarf disease is evaluated according to the incidence rate.
2. The high-throughput artificial inoculation identification method for southern rice black-streaked dwarf disease according to claim 1, wherein in the step (1), the inoculum is an artificially fed sogatella furcifera population with a virus carrying rate of more than 80%.
3. The high-throughput artificial inoculation identification method for southern rice black-streaked dwarf disease according to claim 1, wherein in the step (2), 30-50 seeds are sown in each rice product.
4. The southern rice black-streaked dwarf high-throughput artificial inoculation identification method as claimed in claim 1, wherein in the step (2), a rice dibbling device is adopted to sow rice seeds on a seedling tray, the dibbling device dibbles a plurality of uniform healthy rice seedlings simultaneously, and a plurality of dibbling devices are utilized to cultivate large-scale healthy rice seedlings as required.
5. The high-throughput artificial inoculation identification method for southern rice black-streaked dwarf disease according to claim 1, wherein in the step (3), the rice seedling stage is that the rice seedling is in 1-2 leaf 1 heart stage.
6. The southern rice black-streaked dwarf high-throughput artificial inoculation identification method as claimed in claim 1, wherein in the step (4), rice plants are dwarf, dark green in leaf color, curled in leaf tips, uneven folds formed on the upper leaves near the basal leaves, wax-like milky white or black brown nodular protrusions on leaf sheaths are judged as infected plants, and the incidence (%) is the number of infected plants/total number of plants x 100.
7. The southern rice black-streaked dwarf high-throughput artificial inoculation identification method of claim 1, wherein the resistance level is classified into the following grades:
grade 0 is immunity, and the incidence rate is 0;
grade 1 is high resistance, and the morbidity is 0.1-5.0%;
grade 3 is medium resistance, and the morbidity is 5.1-15.0%;
grade 5 is moderate, and the incidence rate is 15.1% -30.0%;
grade 7 is susceptible, and the incidence rate is 30.1-60.0%;
grade 9 is high-grade, and the incidence rate is more than 60.1%.
8. The high-throughput artificial inoculation identification method for southern rice black-streaked dwarf according to claim 1, wherein the specific steps of the inoculum culture comprise:
s1, collecting nymphs of Sogatella furcifera from a rice field, feeding the nymphs to adults in an insect feeding device for cultivating rice TN1, transferring the adults to TN1 rice seedling cups for spawning, and hatching to obtain the healthy nymphs of the Sogatella furcifera;
s2, transferring 1-2-year-old healthy Sogatella furcifera nymphs to a diseased rice plant infected with southern rice black-streaked dwarf virus to feed the virus;
s3, transplanting the seedlings to healthy rice after 48 hours of virus feeding, after 9-12 days of circulation, randomly extracting 50 sogatella furcifera, detecting the virus carrying rate of the groups by using a Dot-ELISA method or an RT-PCR method, and determining the groups with the virus carrying rate of more than 80 percent as inoculants.
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