CN115428702A - Method for establishing celery wilt resistance identification artificial disease nursery - Google Patents
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Abstract
The invention discloses a method for establishing an artificial disease nursery for identifying celery wilt resistance, which comprises the following steps: culturing pathogenic bacteria of celery blight; (2) artificially inoculating the celery blight disease nursery; (3) investigating the disease condition of celery blight; and (4) evaluating the resistance of celery to blight. The celery blight disease nursery established by the invention has uniform and consistent morbidity, and can ensure the accurate determination of the disease resistance identification result. Compared with the manual inoculation method, the method disclosed by the invention has the advantages that the disease nursery is utilized for resistance identification, the work of pathogen culture inoculation is reduced, the simultaneous detection of a large number of samples is realized, the resistance level of a disease-resistant variety in the whole growth period can be reflected, and the technical support is provided for the work of screening disease-resistant germplasm resources of celery blight, breeding of the disease-resistant variety and the like.
Description
Technical Field
The invention belongs to the technical field of plant disease resistance identification, and particularly relates to a method for establishing an artificial disease nursery for identifying celery wilt resistance.
Background
Celery blight is caused by Fusarium oxysporum (Fusarium oxysporum f.sp.apii), and is a disease which is seriously harmful in celery production all over the world. Celery blight mainly damages roots and stem bases of celery, and finally the whole plant wilts. The field symptoms of celery blight mainly show that plants grow slowly and are remarkably short, leaves are green and yellow, and wilting falls off until the whole plant is died. After the base of the stem of the diseased plant is cut, the vascular bundle becomes yellow brown, the main root is necrotic, and the fibrous root is rotten. Celery blight is a typical soil-borne disease, continuous cropping and continuous cropping are main causes of spread and prevalence of the disease, pathogenic bacteria can survive on soil and disease residues for a long time, once the celery blight occurs, the control is difficult, the yield of celery is reduced, and products are reduced, so that huge economic losses are brought to farmers.
At present, chemical control and soil improvement are mainly used for controlling celery blight, but the celery blight is difficult to control due to strong viability and easy propagation of pathogenic bacteria, celery planting land blocks are continuously replaced, and the planting area is reduced. The popularization and the application of the celery wilt resistant variety are the most direct and effective method for solving the harm of the celery wilt resistant variety. The establishment of an accurate, reliable and rapid disease resistance identification method is a key link of research work such as disease resistance resource screening, variety breeding, resistance evaluation and the like.
The resistance identification work of celery blight varieties is less researched, the root irrigation inoculation and soil bacterium mixing method is mainly used abroad, but the celery blight pathogenic bacteria have long incubation period, so that the disease is slow, the disease grading is unclear and the like. However, the domestic research reports on celery wilt are few, and no research report on identification methods of celery wilt resistance exists so far.
Therefore, how to provide an establishment method for identifying the resistance of celery wilt disease in an artificial disease nursery becomes a problem to be solved urgently by technical personnel in the field.
Disclosure of Invention
The invention aims to establish an artificial disease nursery for identifying the resistance of celery wilt disease aiming at the defects of uneven disease occurrence, long disease occurrence period and unobvious symptoms existing in the artificial inoculation of the celery wilt disease, and provide technical support for the accuracy and repeatability of the evaluation of the resistance of the celery wilt disease.
In order to achieve the purpose, the invention adopts the following technical scheme:
a method for establishing an artificial disease nursery for identifying celery wilt resistance comprises the following steps: culturing pathogenic bacteria of celery blight; (2) artificially inoculating the celery blight disease nursery; (3) investigating the disease condition of celery blight; and (4) evaluating the resistance of celery to blight.
Further, the culture of pathogenic bacteria of celery blight in the step (1) comprises the following specific steps: inoculating celery wilt pathogens to a PDA culture medium for activation culture, carrying out dark culture in an incubator at 26 ℃ for 7d, then punching holes at hyphae at the edges of colonies by using a puncher (with the diameter of 4 mm) to obtain bacterial sheets, inoculating the bacterial sheets on a wheat culture medium, carrying out dark culture in the incubator at 26 ℃ for 7d, naturally airing the wheat grains after the hyphae grow over the wheat grains, and inoculating for later use.
Further, the preparation method of the wheat culture medium comprises the following steps: weighing 1000g of wheat grains, soaking for 24h, pouring off excessive water, putting the completely soaked wheat grains into a culture box (the wheat grains account for 1/2 of the culture box), and sterilizing with steam at 121 deg.C for 30min for later use.
Further, in the step (2), the specific operation of manual inoculation of the celery blight disease nursery is as follows: selecting a land with flat terrain, and culturing a large amount of celery fusarium wilt bacteria cultured in the step (1) according to the ratio of 50g/m 2 Uniformly spreading the mixture in a disease garden, and fully rotary tillage by a rotary cultivator until the rotary tillage depth reaches 10-15 cm underground, so that pathogenic bacteria and soil are fully and uniformly mixed.
Furthermore, in the step (3), the investigation of the incidence of celery blight disease specifically comprises the following steps: planting the variety to be detected and the susceptible variety on the established disease garden, managing the disease garden normally according to the field requirements, investigating the disease condition of the celery of the variety to be detected according to the grading standard of the wilt disease of the celery after the susceptible variety is fully diseased (about 45 days), and calculating the disease index.
Further, the classification standard of the celery blight disease is as follows: grade 0, no symptoms; grade 1, the plant is short and small, and the fibrous root is browned; 2 grade, the plant is short and wilted, and the root browning is obvious; grade 3, plant wilting and yellowing, and obvious browning of stem base; 4 grade, plant withering and yellowing to death, root rot.
Further, the disease index is calculated by the formula: disease index = Σ (number of diseased leaves × number of corresponding diseased stages) × 100/(number of examined total leaves × 4).
Further, in the step (4), the specific operation of celery for evaluating the wilt resistance is as follows: and according to the disease index of the celery blight of each variety, evaluating the resistance of the test variety to the celery blight according to the celery blight resistance evaluation standard.
Further, the grading evaluation standard of the celery wilt resistance is as follows: high resistance, 0< disease index less than or equal to 15; disease resistance, 15< disease index less than or equal to 30; resisting disease, 30< disease index is less than or equal to 45; the disease index is less than or equal to 60 after the disease is infected by 40; high susceptibility, disease index >60.
Further, the established celery wilt resistance identification artificial disease nursery can be applied to identification of wilt resistance of different celery varieties.
The invention has the beneficial effects that:
1. the invention provides a method for establishing an artificial disease nursery for celery blight germs, which adopts soil mixed bacterium inoculation and deep turning and uniform mixing to ensure that pathogens in the artificial disease nursery are uniformly distributed, and the distribution environment of field pathogens is restored to ensure that a resistance identification result is accurate and reliable.
2. Aiming at the defects of large inoculation amount, slow disease, unobvious symptoms and the like of celery wilt potted plant experiments or field artificial inoculation, an industrial disease garden for celery wilt patients is established, the quantity of pathogenic bacteria in soil is artificially enlarged, the field disease state is reduced, the pathogenic incubation period is shortened, the celery wilt disease manifestation time is shortened, and the disease resistance identification efficiency is improved.
4. The invention provides a celery blight disease condition survey grading standard, disease division is carried out according to the damage degree of the pathogen celery after infection, and symptom characteristics are easy to evaluate.
5. The celery wilt disease artificial disease garden established by the invention can be used for evaluating celery wilt disease resistant varieties, the accuracy and reliability of variety resistance evaluation are ensured, the operation method is simple, large samples can be simultaneously evaluated, and the work efficiency of disease resistance identification is improved.
Detailed Description
The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the embodiments of the present invention, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.
In view of the fact that the pathogen of celery wilt has long incubation period, is a systemic infection disease and can cause diseases in the whole growth period, the establishment of the artificial celery wilt disease nursery is an important way for identifying and researching the disease resistance of the celery wilt disease, the technical difficulty of resistance identification is reduced, and an accurate and reliable technical means is provided for screening and identifying the quality resources of large samples.
Example 1
The embodiment provides a method for establishing an artificial disease nursery for identifying resistance of celery wilt disease, which comprises the following steps:
step (1) culturing pathogenic bacteria of celery blight. Inoculating celery wilt pathogens to a PDA culture medium for activation culture, carrying out dark culture in an incubator at 26 ℃ for 7d, then punching holes at hyphae at the edges of colonies by using a puncher (with the diameter of 4 mm) to obtain bacterial sheets, inoculating the bacterial sheets on a wheat culture medium, carrying out dark culture in the incubator at 26 ℃ for 7d, naturally airing the wheat grains after the hyphae grow over the wheat grains, and inoculating for later use.
The preparation method of the wheat culture medium in the step (1) comprises the following steps: weighing 1000g wheat grains, soaking for 24 hr, pouring off excessive water, placing the completely soaked wheat grains into a culture box (the wheat grains account for 1/2 of the culture box), and steam sterilizing at 121 deg.C for 30 min.
And (2) artificially inoculating the celery blight disease nursery. Selecting a land with flat terrain, and culturing a large amount of celery blight bacteria in the step (1) according to the weight of 50g/m 2 Uniformly spreading the mixture in a disease nursery, and fully rotary tillage by a rotary cultivator till the rotary tillage depth reaches 10-15 cm underground, so that pathogenic bacteria and soil are fully and uniformly mixed.
And (3) investigating the disease condition of celery blight. Planting susceptible varieties 01-9B (4 leaf stage) on the established disease nursery, dividing the artificial disease nursery into 6 cells, planting 200 celery seedlings in each cell, normally managing according to field requirements, investigating celery disease conditions according to celery blight disease grading standard after full disease occurrence (42 d), and calculating disease index.
In the step (3), the disease condition grading standard after the celery blight inoculation and outbreak is as follows: grade 0, no symptoms; grade 1, the plant is short and small, and the fibrous root is browned; 2 grade, the plant is short and wilting, and the root browning is obvious; grade 3, plant wilting and yellowing, and obvious browning of stem base; grade 4, plant withering and yellowing to death, root rot.
In the step (3), the disease index calculation formula is as follows: disease index = Σ (number of diseased leaves × number of corresponding diseased steps) × 100/(number of survey total leaves × 4).
And (4) evaluating the blight resistance of celery. And according to the disease indexes of the celery blight of various varieties, evaluating the resistance of the test varieties to the celery blight according to a celery blight resistance grading standard.
The celery wilt resistance classification standard in the step (4) is as follows: high resistance, 0< disease index less than or equal to 15; disease resistance, 15< disease index less than or equal to 30; medium resistance, disease index of 30< is less than or equal to 45; the disease index is less than or equal to 60 after the disease is infected by 40; high-grade, disease index >60.
The celery wilt resistance identification artificial disease garden established by the invention can be applied to identification of wilt resistance of different celery varieties.
Test results show that after the celery is transplanted for 45d, the disease occurrence conditions of celery blight of 6 cells divided on an artificial disease nursery are investigated, the investigation results show that the disease indexes of the celery blight of the 6 cells are 65.25, 69.75, 66.50, 69.44, 64.13 and 65.44 respectively, the average disease index is 66.75 +/-0.35, and the disease indexes of the celery blight among the cells have no obvious difference. Therefore, the establishment of the artificial disease nursery is proved to induce celery blight to occur, the disease of each cell is uniform, and the artificial disease nursery can be used for evaluating the disease resistance of varieties and screening the disease nursery for the control effect of bactericides.
Compared with the artificial inoculation method, the method disclosed by the invention has the advantages that the disease nursery is used for resistance identification, the work of pathogen culture inoculation is reduced, the simultaneous detection of a large number of samples is realized, the resistance level of the disease-resistant variety in the whole growth period can be reflected, and the technical support is provided for the work of screening of disease-resistant germplasm resources of celery blight, breeding of the disease-resistant variety and the like.
Example 2
Identification of resistance of different celery varieties to blight
Celery variety and pathogen for test
The celery variety to be tested is 4 parts of celery germplasm materials and is derived from a celery subject group of vegetable institute of agricultural science and institute of Tianjin; the pathogen of celery wilt is QC1906, and is stored in the seedling disease room of plant protection research institute of Tianjin agricultural academy of sciences.
Step (1) culturing pathogenic bacteria of celery blight. Inoculating celery wilt pathogen QC1906 to a PDA culture medium for activation culture, carrying out dark culture in an incubator at 26 ℃ for 7d, then punching holes at hypha positions at the edges of bacterial colonies by using a puncher (with the diameter of 4 mm), inoculating obtained bacterial sheets to a wheat culture medium, carrying out dark culture in the incubator at 26 ℃ for 7d, naturally airing the wheat grains after the hypha grows over the wheat grains, and inoculating for later use.
The preparation method of the wheat culture medium related in the step (1) comprises the following steps: weighing 1000g of wheat grains, soaking for 24h, pouring off excessive water, putting the completely soaked wheat grains into a culture box (the wheat grains account for 1/2 of the culture box), and sterilizing with steam at 121 deg.C for 30min for later use.
And (2) artificially inoculating the celery blight disease nursery. Selecting a land with flat terrain, and culturing a large amount of celery blight bacteria in the step (1) according to the weight of 50g/m 2 Uniformly spreading the mixture in a disease garden, and fully rotary tillage by a rotary cultivator until the rotary tillage depth reaches 10-15 cm underground, so that pathogenic bacteria and soil are fully and uniformly mixed.
And (3) investigating the disease condition of celery blight. Transplanting and planting 4 celery varieties (4 leaf stages) on the established disease nursery, planting 50 celery seedlings on each variety, managing normally according to field requirements, investigating celery disease conditions according to the classification standard of celery blight disease after the disease varieties are fully ill (48 d), and calculating disease index.
In the step (3), the disease condition grading standard after the celery blight inoculation and outbreak is as follows: grade 0, no symptoms; grade 1, the plant is short and small, and the fibrous root is browned; 2 grade, the plant is short and wilting, and the root browning is obvious; grade 3, the plant wilts and turns yellow, and the base of the stem is obviously browned; grade 4, plant withering and yellowing to death, root rot.
In the step (3), the disease index calculation formula is as follows: disease index = Σ (number of diseased leaves × number of corresponding diseased steps) × 100/(number of survey total leaves × 4)
And (4) evaluating the blight resistance of celery. And according to the disease indexes of the celery blight of various varieties, evaluating the resistance of the test varieties to the celery blight according to a celery blight resistance grading standard.
The celery wilt resistance classification standard in the step (4) is as follows: high resistance, 0< disease index less than or equal to 15; disease resistance, 15< disease index less than or equal to 30; resisting disease, 30< disease index is less than or equal to 45; the disease index is less than or equal to 60 after the disease is infected by 40; high-grade, disease index >60.
The identification results of the wilt resistance of the germplasm resources of 4 celery are shown in a table 1:
| variety of (IV) C | Index of disease condition | Evaluation of resistance |
| 01-9B | 68.75 | Feeling of height |
| 08-11B | 53.75 | Infection of the disease |
| S428 | 20.83 | Disease-resistant |
| S405 | 12.08 | Gao Kang |
TABLE 1
Table 1 shows that 4 celery varieties show different levels of resistance to blight pathogen QC1906, wherein the celery germplasm 01-9B belongs to a high-sensitive material, the disease index is 68.75, and the celery germplasm S405 shows a higher resistance level to celery blight, and the disease index is 12.08. Therefore, the celery wilt resistance evaluation method established by the invention can be applied to screening and identifying celery germplasm resources.
In the present specification, the embodiments are described in a progressive manner, each embodiment focuses on differences from other embodiments, and the same and similar parts among the embodiments are referred to each other. The device disclosed by the embodiment corresponds to the method disclosed by the embodiment, so that the description is simple, and the relevant points can be referred to the method part for description.
The previous description of the disclosed embodiments is provided to enable any person skilled in the art to make or use the present invention. Various modifications to these embodiments will be readily apparent to those skilled in the art, and the generic principles defined herein may be applied to other embodiments without departing from the spirit or scope of the invention. Thus, the present invention is not intended to be limited to the embodiments shown herein but is to be accorded the widest scope consistent with the principles and novel features disclosed herein.
Claims (10)
1. A method for establishing an artificial disease nursery for identifying celery wilt resistance is characterized by comprising the following steps: culturing pathogenic bacteria of celery blight; (2) artificially inoculating the celery blight disease nursery; (3) investigating the disease condition of celery blight; and (4) evaluating the resistance of celery to blight.
2. The method for establishing the celery blight resistance identification artificial disease nursery as claimed in claim 1, wherein the step (1) of culturing pathogenic bacteria of celery blight comprises the following specific steps: inoculating celery wilt pathogens to a PDA culture medium for activation culture, carrying out dark culture in an incubator at 26 ℃ for 7d, punching holes at hypha positions at the edges of bacterial colonies by using a puncher, inoculating obtained bacterial sheets to a wheat culture medium, carrying out dark culture in the incubator at 26 ℃ for 7d, naturally airing the wheat grains after the hypha grows over the wheat grains, and inoculating for later use.
3. The method for establishing the celery wilt resistance identification artificial disease nursery as claimed in claim 2, wherein the preparation method of the wheat culture medium comprises the following steps: weighing 1000g of wheat grains, soaking for 24h, pouring off excessive water, putting the completely soaked wheat grains into a culture box, and performing steam sterilization at 121 ℃ for 30min for later use.
4. The method for establishing the celery blight resistance identification artificial disease nursery as claimed in claim 3, wherein in the step (2), the specific operation of artificial inoculation of the celery blight disease nursery is as follows: selecting a land with flat terrain, and culturing a large amount of celery fusarium wilt bacteria cultured in the step (1) according to the ratio of 50g/m 2 Uniformly spreading the mixture in a disease nursery, and fully rotary tillage by a rotary cultivator till the rotary tillage depth reaches 10-15 cm underground, so that pathogenic bacteria and soil are fully and uniformly mixed.
5. The method for establishing the celery wilt resistance identification artificial disease nursery as claimed in claim 1, wherein in the step (3), the investigation of the morbidity of celery wilt disease specifically comprises the following operations: transplanting the variety to be tested and the susceptible variety on the established disease garden, normally managing according to field requirements, investigating the disease condition of the celery of the variety to be tested according to the classification standard of the blight condition of the celery after the susceptible variety is sufficiently diseased, and calculating the disease index.
6. The method for establishing the celery blight resistance identification artificial disease nursery as claimed in claim 5, wherein the celery blight disease grading standard is as follows: grade 0, no symptoms; grade 1, the plant is short and small, and the fibrous root is browned; 2 grade, the plant is short and wilted, and the root browning is obvious; grade 3, the plant wilts and turns yellow, and the base of the stem is obviously browned; 4 grade, plant withering and yellowing to death, root rot.
7. The method for establishing the celery wilt resistance identification artificial disease nursery as claimed in claim 6, wherein a disease index calculation formula is as follows: disease index = Σ (number of diseased leaves × number of corresponding diseased steps) × 100/(number of survey total leaves × 4).
8. The method for establishing the celery wilt resistance identification artificial disease nursery as claimed in claim 7, wherein in the step (4), the celery resistance evaluation specific operation is that: and according to the disease index of the celery blight of each variety, evaluating the resistance of the test variety to the celery blight according to the celery blight resistance evaluation standard.
9. The method for establishing the celery wilt resistance identification artificial disease nursery as claimed in claim 7, wherein the grading evaluation standard of celery wilt resistance is as follows: high resistance, 0< disease index less than or equal to 15; disease resistance, disease index is less than or equal to 30 after 15; resisting disease, 30< disease index is less than or equal to 45; the disease index is less than or equal to 60 after the disease is infected by 40; high susceptibility, disease index >60.
10. The method for establishing the celery wilt resistance identification artificial disease nursery as claimed in claim 1, wherein the established celery wilt resistance identification artificial disease nursery can be applied to identification of wilt resistance of different celery varieties.
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| CN107980499A (en) * | 2017-12-25 | 2018-05-04 | 河北省农林科学院粮油作物研究所 | A kind of method of sesame droop disease resistance field test |
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| CN105432381A (en) * | 2015-12-07 | 2016-03-30 | 江苏省农业科学院 | Prevention and control method of pepper phytophthora blight |
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