CN108048601B - Seedling inoculation method for pepper cucumber mosaic virus and application thereof - Google Patents
Seedling inoculation method for pepper cucumber mosaic virus and application thereof Download PDFInfo
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Abstract
The invention relates to a seedling inoculation method for pepper cucumber mosaic virus and application thereof. Is used for rapidly identifying and screening germplasm resources with cucumber mosaic virus disease resistance in pepper resources of China. And (3) manually injecting and inoculating the purified cucumber mosaic virus source on the scorched three-generation tobacco, collecting diseased leaves 12-15 days after inoculation, adding phosphate buffer solution into the diseased leaves, homogenizing in an ice bath, filtering or centrifuging by using double-layer gauze, and taking the filtrate or supernatant as inoculation liquid. The inoculation can be carried out from the cotyledon stage of the pepper to the 5-6 leaf stage. Wherein the 1-2 leaf stage is the best inoculation seedling age, sucking 1mL of inoculation liquid with a disposable syringe (1mL), pricking 2 pinholes on the cotyledon with a needle, removing the needle, and slowly injecting the inoculation liquid at the pinhole on the back of the cotyledon to fill two cotyledons with virus inoculation liquid. Disease investigation was performed 14 days after inoculation. The method has the advantages that the virus inoculation amount is easy to control, mesophyll cells are directly injected during inoculation, observation is easy, and missing inoculation is reduced.
Description
Technical Field
The invention belongs to the field of plant protection, and relates to a seedling inoculation method for pepper cucumber mosaic virus and application thereof.
Background
The temperate zone is the main distribution area of Cucumber Mosaic Virus (CMV), and tropical areas show a trend of increasing year by year. Up to now, CMV has a host range of over 100 families of 1200 plants, and is particularly harmful to a variety of vegetable crops and ornamental plants. CMV is transmitted primarily by a variety of aphids in a non-persistent manner, and some plants can be seed-borne. The CMV can survive annually due to wide host range, small aphid transmission frequency, small crop variety replacement frequency and the like, so that the CMV is difficult to control. The detection rate of CMV in pepper main producing areas in China is very high, and the detection rates of Beijing, Shaanxi, lake Guang and Jiangzhe areas are all over 50 percent.
CMV infection of pepper shows a progressive disease process. The basal part of the young and tender leaves in the early stage of the onset of disease is yellowed, and then the yellowing symptoms gradually extend to the whole leaves; the newly born leaf then showed a pronounced mosaic; as the plants developed, the leaves appeared severely shrunken, the plants were stunted, and the leaf color appeared dull dark green. The severity of the symptoms of the pepper infected with CMV depends on the plant age at the time of infection, generally, the infection of CMV at the seedling stage can cause baldness, dwarfing and yield reduction, and the severe infection can cause abstinence; while symptoms after adult CMV infection are relatively mild.
The disease resistance identification is the basis of disease-resistant material screening, resistance genetic rule research and disease-resistant variety breeding, and the key point is that the resistance level of the material can be accurately reflected. The identification of pepper anti-CMV mostly adopts an artificial inoculation identification method. Current CMV artificial inoculation methods include finger rub inoculation, frosted glass spoon inoculation, spray gun inoculation, and viral sap dip inoculation. In the first 3 methods, a thin layer of 600 mesh (about 23 μm in diameter) carborundum is sprayed on the leaf surface before inoculation, and excess inoculum is washed away after 30min of inoculation. Finger rub inoculation and frosted glass spoon inoculation are easy to handle, but the inoculation is not uniform. The spray gun used for spray gun inoculation is a paint spray gun, the distance between the spray gun and the seedling is about 2cm during inoculation, the working pressure of an air compressor is 2.1-2.5 kg-cm, the spray gun inoculation is suitable for resistance identification of a large number of materials, and the problems of uneven inoculation and missing exist. The method has partial application in the screening of pepper CMV-resistant materials in China. The virus sap root-soaking method is to wash the soil at the root of the seedling and soak the soil in the inoculation liquid, and the method has the best inoculation effect, but has large workload, difficult operation and little use. Aiming at the problems in the inoculation method, a new and more effective pepper anti-CMV inoculation method is imperative to be established.
Disclosure of Invention
The invention aims to: aiming at the problems existing in the current pepper anti-CMV seedling stage identification method in China, a new, more effective and more complete seedling stage inoculation method and the application thereof are provided.
The purpose of the invention can be realized by the following technical scheme:
the pepper cucumber mosaic virus seedling inoculation method comprises the following steps:
(1) taking the purified cucumber mosaic virus strain, manually injecting and inoculating the cucumber mosaic virus strain on a three-generation tobacco with a dead spot (Nicotiana tabacum CVS Samun NN), and harvesting diseased leaves 12-15 days after inoculation; adding the diseased leaves into a phosphate buffer solution, carrying out ice bath homogenization, and filtering or centrifuging to obtain a filtrate or a supernatant, namely inoculation liquid;
(2) after the pepper seeds are disinfected, artificially culturing until 2 true leaves of the seedlings are completely unfolded, and then inoculating;
(3) sucking 1mL of bacterial liquid by using a 1mL sterile syringe, pricking 2 pinholes on cotyledons by using a needle head, removing the needle head, slowly injecting the inoculation liquid into the pinholes on the backs of the cotyledons to ensure that two cotyledons are filled with the virus inoculation liquid, keeping the temperature at 25 ℃ day and night after inoculation, and managing the normal growth of seedlings.
Wherein the phosphate buffer is preferably 0.02 mol.L with a pH value of 7-1The phosphate buffer of (1); 5mL of the phosphate buffer was added per gram of diseased leaves for ice bath homogenization.
The filtering method is preferably double-layer gauze filtering; the centrifugation condition is 3500 rpm-1Centrifuge for 15 minutes.
The pepper seeds are sterilized in 5% sodium hypochlorite for 20 minutes and then washed with sterile water for 3-4 times.
The method for artificially culturing the pepper seeds is preferably as follows: the pepper seeds germinate in a culture dish under the dark condition of 25 ℃ after being sterilized, the seeds are sowed in a nutrition pot of 8 multiplied by 8 to grow after germinating, the temperature of the growing period is 25 ℃ in the daytime/20 ℃ at night, and the inoculation can be carried out when 2 true leaves of the seedlings are completely unfolded.
The method disclosed by the invention is applied to rapidly identifying and screening germplasm resources with cucumber mosaic virus resistance in pepper resources.
The pepper seedlings are preferably inoculated according to the pepper cucumber mosaic virus seedling inoculation method in the seedling stage, and disease condition investigation is carried out on the pepper seedlings 14 days after inoculation.
The application is further preferable, the disease condition is preferably graded according to a grading standard established by a disease-resistant breeding and attacking group of 'eight five' hot peppers in China, and the grade is 0-no symptom; grade 1-heart vein clearing or inoculated leaf acute small withered spot; grade 3-necrotic spots on systemic mosaic or stem; grade 5-necrotic streaks on systemic floral leaves, malformations, or stems; grade 7-most leaf malformation, fern leaves, plant dwarfing or necrosis of the stem, branch and vein system; grade 9-plants were severely stunted, stopped growing or severely systemically necrotic, until the whole plant died; disease index ∑ (disease grade × number of strains at that grade)/(total number of investigated strains × 9) ] × 100. The material or population disease resistance division criteria were: immunization (I): DI is 0; high Resistance (HR): 0 < DI < 5; disease resistance (R): 5 < DI < 20; anti-Medium (MR): 20 < DI < 40; infection (S): DI > 40.
The pepper anti-CMV seedling stage inoculation identification method comprises the following steps:
(1) taking the purified cucumber mosaic virus strain, manually injecting and inoculating the cucumber mosaic virus strain on a three-generation withered spot tobacco (Nicotiana tabacum CVS Samun NN), harvesting diseased leaves 12-15 days after inoculation, and adding 5ml of 0.02 mol.L with the pH value of 7 into each 1g of diseased leaves-1The phosphate buffer solution is homogenized in an ice bath, and filtered by double-layer gauze or 3500 rpm-1Centrifuging for 15 minutes, wherein the filtrate or supernatant is inoculation liquid;
(2) sterilizing pepper seeds in 5% sodium hypochlorite for 20 minutes, and washing with sterile water for 3-4 times;
(3) pepper seeds germinate in a culture dish at 25 ℃ in the dark after being sterilized, the seeds are sowed in a nutrition pot of 8 multiplied by 8 to grow after being germinated, the temperature of the growth period is 25 ℃/20 ℃ in the day, and the inoculation can be carried out when 2 true leaves of the seedlings are completely unfolded;
(4) sucking 1mL of bacterial liquid by using a disposable syringe, pricking 2 pinholes on the cotyledon by using a needle head, removing the needle head, and slowly injecting the inoculation liquid into the pinholes on the back of the cotyledon to fill two cotyledons with the virus inoculation liquid. After inoculation, keeping the temperature at 25 ℃ day and night, and managing the normal growth of seedlings;
(5) disease condition investigation is carried out on the pepper seedlings after inoculation for 14 days, grading is carried out according to the grading standard established by the 'eighty-five' pepper disease-resistant breeding and attacking group in China, and disease condition indexes are calculated; the classification standard is as follows: grade 0-no symptoms; grade 1-heart vein clearing or inoculated leaf acute small withered spot; grade 3-necrotic spots on systemic mosaic or stem; grade 5-necrotic streaks on systemic floral leaves, malformations, or stems; grade 7-most leaf malformation, fern leaves, plant dwarfing or necrosis of the stem, branch and vein system; grade 9-plants were severely stunted, stopped growing or severely systemically necrotic, until the whole plant died.
The disease index calculation method is preferably as follows: disease index ∑ (disease grade × number of strains at that grade)/(total number of investigated strains × 9) ] × 100. The material or population disease resistance division criteria were: immunization (I): DI is 0; high Resistance (HR): 0 < DI < 5; disease resistance (R): 5 < DI < 20; anti-Medium (MR): 20 < DI < 40; infection (S): DI > 40.
The invention has the advantages that: compared with the conventional common inoculation method, the method does not need to spray corundum, reduces the operation steps, is directly injected by a needle, is easy to control the virus inoculation amount, has small damage to the leaf, can directly inject the inoculation liquid into mesophyll cells, is easy to observe, and reduces the missing inoculation. The method has satisfied results for identifying part of the existing pepper germplasm resources in a research laboratory. The method can be used for rapidly screening and identifying the pepper germplasm resources with the CMV resistance in pepper resources in China, and the CMV resistance resource utilization of the peppers is widened.
Detailed Description
Example 1
(1) And (3) breeding the purified cucumber mosaic virus strain on a three-leaf blight cigarette (Nicotiana tabacum CVS Samunn NN), manually injecting and inoculating, and harvesting diseased leaves 12-15 days after inoculation. Adding 5ml of 0.02 mol.L with pH value of 7 into 1g of diseased leaves-1The phosphate buffer solution is homogenized in an ice bath, and filtered by double-layer gauze or 3500 rpm-1Centrifuging for 15 minutes, whichThe filtrate or supernatant is the inoculation liquid;
(2) sterilizing pepper seeds in 5% sodium hypochlorite for 20 minutes, and washing with sterile water for 3-4 times;
(3) the pepper seeds germinate in a culture dish under the dark condition of 25 ℃ after being sterilized, the seeds are sowed in a nutrition pot of 8 multiplied by 8 to grow after germinating, the temperature of the growing period is 25 ℃ in the daytime/20 ℃ at night, and the inoculation can be carried out when 2 true leaves of the seedlings are completely unfolded.
(4) Sucking 1mL of bacterial liquid by using a disposable syringe (1mL), removing a needle head, and slowly injecting the bacterial liquid into the back of the cotyledon of the pepper to fill two cotyledons with the virus inoculation liquid. After inoculation, the temperature is kept at 25 ℃ day and night, and the normal growth management of seedlings is realized.
(5) The pepper seedlings were investigated 14 days after inoculation. The method is mainly based on the classification standard established by the 'eighty-five' hot pepper disease-resistant breeding and attacking group in China, and has 0 grade to no symptom; grade 1-heart vein clearing or inoculated leaf acute small withered spot; grade 3-necrotic spots on systemic mosaic or stem; grade 5-necrotic streaks on systemic floral leaves, malformations, or stems; grade 7-most leaf malformation, fern leaves, plant dwarfing or necrosis of the stem, branch and vein system; grade 9-plants were severely stunted, stopped growing or severely systemically necrotic, until the whole plant died. Disease index ∑ (disease grade × number of strains at that grade)/(total number of investigated strains × 9) ] × 100. The disease resistance of the variety group is divided into the following standard: immunization (I): DI is 0; high Resistance (HR): 0 < DI < 5; disease resistance (R): 5 < DI < 20; anti-Medium (MR): 20 < DI < 40; infection (S): DI > 40. The identification test was performed in triplicate, 10 strains per treatment. The test materials used were susceptible C.anumn G29 and resistant C.fractures PBC688(G.J.Guo, S.B.Wang, J.B.Liu et al, Rapid identification and characterization of QTLs exploiting Resistance to Current Mobile Virus in Pepper (Capsule fractures.) the or applied Gene, 2016, 130,1:41-52.) as control varieties.
And (4) evaluating the pepper resources according to the disease degree investigation result, the disease classification and the disease index.
In this test, some of the material identification results are shown in Table 1, and among 140 identified pepper materials, 15 of the materials have disease indexes between 5 and 20 and belong to resistant materials; the disease index of 47 parts of material is between 20 and 40, and the material belongs to an anti-material; the disease index of 78 parts of materials is more than 40, and the materials belong to susceptible materials; no pepper resource for CMV immunity and high resistance is found, which indicates that the germplasm resource screening range needs to be further expanded in the pepper CMV resistance research, and provides a material basis for the pepper CMV resistance research.
TABLE 1 identification result of pepper germplasm resources for resisting CMV
Claims (9)
1. The seedling inoculation method for pepper cucumber mosaic virus is characterized by comprising the following steps:
(1) taking the purified cucumber mosaic virus strain, manually injecting and inoculating the cucumber mosaic virus strain on a three-generation tobacco with a dead spot (Nicotiana tabacum CVS Samun NN), and harvesting diseased leaves 12-15 days after inoculation; adding the diseased leaves into a phosphate buffer solution, carrying out ice bath homogenization, and filtering or centrifuging to obtain a filtrate or a supernatant, namely inoculation liquid;
(2) pepper seeds germinate in a culture dish at 25 ℃ in the dark after being sterilized, the seeds are sowed in a nutrition pot of 8 multiplied by 8 to grow after being germinated, the temperature of the growing period is 25 ℃ in the daytime/20 ℃ at night, and the inoculation can be carried out when 2 true leaves of the seedlings are completely unfolded;
(3) sucking 1mL of bacterial liquid by using a 1mL sterile syringe, pricking 2 pinholes on cotyledons by using a needle head, removing the needle head, slowly injecting the inoculation liquid into the pinholes on the backs of the cotyledons to ensure that two cotyledons are filled with the virus inoculation liquid, keeping the temperature at 25 ℃ day and night after inoculation, and managing the normal growth of seedlings.
2. The method according to claim 1, wherein the phosphate buffer is 0.02 Mol-L at pH 7-1The phosphate buffer of (1); 5mL of the phosphate buffer was added per gram of diseased leaves for ice bath homogenization.
3. The method of claim 1 wherein the filtration method is double gauze filtration; the centrifugation condition is 3500 rpm-1Centrifuge for 15 minutes.
4. The method as claimed in claim 1, wherein the pepper seeds are sterilized in 5% sodium hypochlorite for 20 minutes and rinsed with sterile water 3-4 times.
5. The use of the method of claim 1 for rapidly identifying and screening germplasm resources with cucumber mosaic virus disease resistance from pepper resources.
6. Use according to claim 5, characterized in that pepper seedlings are inoculated according to the pepper cucumber mosaic virus seedling inoculation method of any one of claims 1 to 4, and the pepper seedlings are investigated for disease 14 days after inoculation.
7. The use of claim 6, wherein the disease condition is graded according to the grading standard established by the disease-resistant breeding and attacking group of 'eight five' hot pepper in China, grade 0-no symptom; grade 1-heart vein clearing or inoculated leaf acute small withered spot; grade 3-necrotic spots on systemic mosaic or stem; grade 5-necrotic streaks on systemic floral leaves, malformations, or stems; grade 7-most leaf malformation, fern leaves, plant dwarfing or necrosis of the stem, branch and vein system; grade 9-plants were severely stunted, stopped growing or severely systemically necrotic, until the whole plant died; disease index ∑ (disease grade × number of strains at that grade)/(total number of investigated strains × 9) ] × 100; the disease resistance of the variety group is divided into the following standard: immunization (I): DI is 0; high Resistance (HR): 0 < DI < 5; disease resistance (R): 5 < DI < 20; anti-Medium (MR): 20 < DI < 40; infection (S): DI > 40.
8. The pepper anti-CMV seedling stage inoculation identification method is characterized by comprising the following steps of:
(1) taking the purified cucumber mosaic virus strain, manually injecting and inoculating the cucumber mosaic virus strain on a three-generation withered spot tobacco (Nicotiana tabacum CVS Samun NN), harvesting diseased leaves 12-15 days after inoculation, and adding 5ml of 0.02 mol.L with the pH value of 7 into each 1g of diseased leaves-1The phosphate buffer solution is homogenized in an ice bath, and filtered by double-layer gauze or 3500 rpm-1Centrifuging for 15 minutes, wherein the filtrate or supernatant is inoculation liquid;
(2) sterilizing pepper seeds in 5% sodium hypochlorite for 20 minutes, and washing with sterile water for 3-4 times;
(3) pepper seeds germinate in a culture dish at 25 ℃ in the dark after being sterilized, the seeds are sowed in a nutrition pot of 8 multiplied by 8 to grow after being germinated, the temperature of the growth period is 25 ℃/20 ℃ in the day, and the inoculation can be carried out when 2 true leaves of the seedlings are completely unfolded;
(4) sucking 1mL of bacterial liquid by using a disposable syringe, pricking 2 pinholes on cotyledons by using a needle head, removing the needle head, slowly injecting the inoculation liquid into the pinholes on the back surfaces of the cotyledons to ensure that two cotyledons are filled with the virus inoculation liquid, keeping the temperature of 25 ℃ day and night after inoculation, and managing the normal growth of seedlings;
(5) disease condition investigation is carried out on the pepper seedlings after inoculation for 14 days, grading is carried out according to the grading standard established by the 'eighty-five' pepper disease-resistant breeding and attacking group in China, and disease condition indexes are calculated; the classification standard is as follows: grade 0-no symptoms; grade 1-heart vein clearing or inoculated leaf acute small withered spot; grade 3-necrotic spots on systemic mosaic or stem; grade 5-necrotic streaks on systemic floral leaves, malformations, or stems; grade 7-most leaf malformation, fern leaves, plant dwarfing or necrosis of the stem, branch and vein system; grade 9-plants were severely stunted, stopped growing or severely systemically necrotic, until the whole plant died.
9. The method for identifying pepper anti-CMV seedling stage inoculation as claimed in claim 8, wherein the method for calculating disease index is: disease index ∑ (disease grade × number of strains at that grade)/(total number of investigated strains × 9) ] × 100; the disease resistance of the variety group is divided into the following standard: immunization (I): DI is 0; high Resistance (HR): 0 < DI < 5; disease resistance (R): 5 < DI < 20; anti-Medium (MR): 20 < DI < 40; infection (S): DI > 40.
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| CN108990586A (en) * | 2018-06-15 | 2018-12-14 | 云南农业大学 | The method that injection inoculation obtains infection 4 kinds of pathogen diseased plants of Yunnan tobacco clump top sickness |
| CN109937736A (en) * | 2019-04-11 | 2019-06-28 | 江苏省农业科学院 | A kind of sponge gourd cucumber mosaic virus Seedling Inoculation, method of resistance identification and application |
| CN112538516A (en) * | 2020-12-18 | 2021-03-23 | 山东省华盛农业股份有限公司 | Seedling-stage injection inoculation method for cucurbita pepo mosaic virus |
| CN113125643A (en) * | 2021-04-02 | 2021-07-16 | 宁波市农业科学研究院 | Method for rapidly identifying tomato bacterial wilt seedling stage resistance by injection inoculation method |
| CN114561424B (en) * | 2022-03-24 | 2024-01-26 | 浙江中医药大学 | Method for injection inoculation of red sage root cucumber mosaic virus at seedling stage |
| CN116076252A (en) * | 2022-11-28 | 2023-05-09 | 华中农业大学 | Precise application method of exogenous substances for strong seedling cultivation of grafted seedlings |
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