CN107964560B - Rapid identification method for watermelon fusarium wilt and gummy stem blight combined resistance in seedling stage - Google Patents
Rapid identification method for watermelon fusarium wilt and gummy stem blight combined resistance in seedling stage Download PDFInfo
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Abstract
The invention relates to a rapid identification method of watermelon fusarium wilt and gummy stem blight combined resistance in seedling stage, firstly, soaking seeds of watermelon to be identified in warm water, disinfecting and sowing, and growing to 3-5 true leaves after seedling emergence; taking leaves, carrying out spray inoculation by using a fusarium oxysporum spore suspension, washing the roots of seedlings by using water, soaking the roots of the seedlings in the fusarium oxysporum spore suspension, transplanting the seedlings into a container filled with a matrix, placing the container in a greenhouse for culturing, and observing the disease condition 3-7 days after inoculation. Investigating the incidence of the blight and the gummy stem blight, counting the incidence of the blight, the incidence of the gummy stem blight and the disease index, and predicting the resistance of the watermelon and melon to the blight and the gummy stem blight in the seedling stage according to the incidence and the disease index in a grading manner. The identification method can quickly identify the watermelon germplasm resources with the blight and gummy stem blight resistance on the same plant of the same variety, improves the identification efficiency, shortens the period, saves test materials such as seeds and the like, and is suitable for quickly screening a large number of watermelon resistance sources.
Description
Technical Field
The invention relates to a rapid identification method for watermelon fusarium wilt and gummy stem blight combined resistance in a seedling stage, and belongs to the technical field of plant germplasm identification.
Background
Fusarium oxysporum (Fusarium oxysporum) infection caused blight and melon shell-blight caused by Ascochytacitrutrulluna Smith (asexual generation of the Fusarium oxysporum is Didymela bryoniae) are two major fungal diseases of melon crops, and can occur in winter and spring sunlight greenhouses, early spring and autumn greenhouse cultivation from seedling stage to harvest stage, the plant death rate can reach 30% -40% in production, and serious yield reduction is caused, which becomes a main obstacle for restricting melon production. The cultivation of new high-quality compound disease-resistant watermelon and melon varieties is a main method for effectively controlling watermelon and melon fusarium wilt and gummy stem blight, so that the popularization and use of the watermelon and melon resistant varieties is the most economic and effective method. However, the disease resistance identification is the basis of disease resistance breeding, and the disease resistance identification method is the key of disease resistance identification, so that an accurate, reliable, concise and rapid identification method is made, the breeding efficiency can be improved, and the breeding process can be accelerated.
The conventional domestic and foreign resistance identification of watermelon fusarium wilt and gummy stem blight is mainly identification by a single inoculation method, such as root soaking inoculation, root breaking inoculation and leaf puncture inoculation of fusarium wilt in a seedling stage, and spray inoculation, in-vitro leaf inoculation and the like of a spore suspension of gummy stem blight in the seedling stage. The single inoculation method has low efficiency, consumes more test materials such as seeds and the like, and is unstable in different batches of tests. A small amount of blast disease and gummy stem blight compound inoculation is also reported, namely, a living body is used for sequentially inoculating blast disease and gummy stem blight, the two pathogenic bacteria are simultaneously inoculated by the existing compound inoculation method, the disease incidence interaction is often caused, the disease index is high, error results are easy to generate, moreover, as the blight bacteria are lethal to plants, the plants are basically dead after inoculation, the multi-resistance seedling stage identification test cannot be carried out, and if other disease resistances such as gummy stem blight and the like need to be identified, a separate inoculation test is carried out in the later period.
Disclosure of Invention
The invention aims to provide a rapid identification method for watermelon fusarium wilt and gummy stem blight combined resistance in the seedling stage aiming at the defects of the prior art, the identification method is suitable for screening a large number of rapid resistance sources, can avoid the condition observation and statistical result influenced by the interaction of the onset of different diseases, and is rapid in identification and more accurate in identification result.
In order to achieve the purpose, the invention adopts the technical scheme that:
a method for rapidly identifying watermelon fusarium wilt and gummy stem blight combined resistance in a seedling stage comprises the following steps:
(1) preparation of test plant seedlings: soaking watermelon seeds in disinfectant, washing with flowing water to remove residual disinfectant on the surface, soaking the seeds in warm water at 50-60 ℃, stirring to room temperature, standing for 3-6h, taking out, disinfecting, sowing, and growing to 3-5 true leaves after seedling emergence for later use;
(2) preparation of pathogenic bacteria:
preparing fusarium wilt pathogens: inoculating watermelon wilt bacteria to PDA solid culture medium, culturing at 26-28 deg.C for one week, and culturing to obtain the final productInoculating the bacteria in PDB liquid culture medium, culturing at 26-28 deg.C and 150r/min for one week, filtering, adding sterile distilled water, and making into 5 × 105Spore/ml spore suspension for later use;
preparing gummy stem blight bacteria: inoculating gummy stem blight bacteria to PDA solid culture medium, culturing at 26-28 deg.C for one week, selecting fresh cucumber without wound, washing with flowing water, wiping surface with 75% alcohol, air drying, slightly removing epidermis on cucumber surface, beating cultured strain with sterilization puncher with diameter of 0.7cm to obtain mycelium block, placing the mycelium block on cucumber, culturing at 20 deg.C for 5-7 days, scraping with blade, grinding, filtering, adding sterile distilled water, and making into 5 × 105Spore/ml spore suspension for later use;
(3) preparing a blade: taking a culture dish, paving filter paper soaked by benzimidazole plant fresh-keeping aqueous solution at the bottom of the culture dish, disinfecting, and putting the 3 rd to 5 th true leaves of the plant seedlings in the step (1) on the filter paper for later use;
(4) inoculation of pathogenic bacteria:
inoculation of blight disease bacteria: digging out the plant seedlings in the step (1), cleaning roots, absorbing water by using absorbent paper, soaking the roots in the spore suspension in the step (2), transplanting the seedlings into a container filled with a matrix after 5min, culturing the seedlings in a greenhouse at 26 +/-1 ℃, and observing the state of illness 7d after inoculation;
inoculation of gummy stem blight bacteria: spraying the suspension of the cranberry spores obtained in the step (2) onto the leaves obtained in the step (3), wherein each leaf is sprayed with 500 mu 0, and then placing the culture dish in an illumination incubator for storage, wherein the culture conditions are as follows: the temperature is 26 +/-1 ℃, the illumination period is 16h illumination/8 h darkness, the relative humidity is about 90%, and the state of the patient is observed after 3 d;
(5) survey and evaluation:
and (3) investigation and grading of blight: recording according to the disease observation result of the blight in the step (4), and investigating and recording the resistance grading according to a grade 5 standard, namely:
level 0: no disease symptoms;
level 1: leaf wilting or slight wilting of part of the cotyledons and true leaves:
and 2, stage: 1 true leaf wilting or heavier leaf wilting;
and 3, level: leaf and more than 60% of true leaves will wither;
and 4, stage 4: the whole plant is slightly wilted, more than 60 percent of the plant withers, but heart leaves still survive;
and 5, stage: the whole plant withers and withers severely;
wherein, the disease-resistant plants are below grade 2, and the disease-susceptible plants are above grade 3; resistance evaluation was performed according to the incidence of disease (number of diseased plants/total investigated) x 100%, and the calculation results were divided into four grades, and the disease (S): the withering rate is 80-100%; light antibody (LR): the withering rate is 51-80%; anti-Medium (MR): the withering rate is 21-50%; high Resistance (HR): the withering rate is less than 20 percent;
investigating and grading gummy stem blight: grading according to the sizes of the disease spots on the leaves in the step (4), wherein the grading standard of each disease grade is as follows:
level 0: the leaves have no scab;
level 1: the area of the leaf scab is less than or equal to 25 percent;
and 2, stage: 25 percent of leaf lesion area is less than or equal to 50 percent;
and 3, stage: 50% of leaf lesion area is less than or equal to 75%;
4, level: the area of the leaf lesion spots is less than or equal to 100 percent in percentage by weight.
Calculating the disease index according to the examined disease grade, wherein the formula is as follows:
disease index ∑ (number of onset at each stage × representative value at each stage)/(total number of investigated plants × representative number at highest stage) × 100%
The calculation results are divided into 6 grades: high feeling (HS): 80.1-100; infection (S): 60.1-80; (iii) feeling (MS): 40.1-60; anti-Medium (MR): 20.1-40.0; disease resistance (R): 10.1-20.0; high Resistance (HR): 1-10.
In the step (1), the disinfectant is a 10% sodium hypochlorite solution.
In the step (2), in the processes of preparing blight bacteria and gummy stem blight bacteria, the filtration refers to filtration by using 4 layers of gauze.
In the step (3), the concentration of the benzimidazole plant fresh-keeping aqueous solution is 11.8 mg/L.
In the step (4), inoculation is carried out on the same plant of the same variety at the same time.
Has the beneficial effects that: the method for identifying the combined resistance inoculation of the living body and the in vitro leaf in the seedling stage has the advantages of overcoming the defects that the fusarium wilt pathogens are lethal to plants after the compound inoculation in the prior art, the plants are basically dead after the inoculation, the subsequent multi-resistance seedling stage identification test cannot be carried out, and the single inoculation test is required to be carried out on fusarium wilt-sensitive materials if other disease resistances such as gummy stem blight and the like need to be continuously identified. The combined resistance identification technology for the inoculation of the living bodies and the in vitro leaves in the seedling stage can realize the simultaneous identification of two kinds of disease resistance of the same plant, improve the identification efficiency, shorten the period, save the test materials such as seeds and the like, is suitable for the screening of a large number of rapid resistance sources, and does not influence the subsequent screening of the variety on the resistance identification of other diseases when the variety is lethal to any pathogenic bacteria.
Detailed Description
The invention is further illustrated by the following examples.
Examples
A method for rapidly identifying watermelon fusarium wilt and gummy stem blight combined resistance in a seedling stage comprises the following steps:
(1) preparing test plant seedlings: soaking kardise (resisting blight and gummy stem blight), Sumi No. 1 (resisting blight and gummy stem blight) watermelon seeds and W7 (resisting blight and gummy stem blight) in 10% sodium hypochlorite solution for 5min, washing with flowing water to remove residual disinfectant on the surface, soaking seeds in warm water at 50-60 ℃, stirring to room temperature, standing for 4h, taking out for disinfection and sowing, and growing to 3-5 true leaves after seedling emergence for later use;
(2) preparation of pathogenic bacteria:
preparing fusarium wilt germs: inoculating watermelon Fusarium oxysporum to PDA solid culture medium, culturing at 26-28 deg.C for one week, inoculating the cultured Fusarium oxysporum to PDB liquid culture medium, culturing at 26-28 deg.C for one week at 150r/min, filtering with 4 layers of gauze, adding sterile distilled water, and making into 5 × 105Spore/ml spore suspension for later use;
preparing gummy stem blight bacteria: inoculating gummy stem blight bacteria to PDA solid culture medium 26Culturing at-28 deg.C for one week, selecting fresh cucumber without wound, washing with flowing water, wiping surface with 75% alcohol, air drying, slightly removing epidermis on cucumber surface, beating mycelium block with diameter of 0.7cm with sterilized punch, placing the mycelium block on cucumber, placing into incubator, culturing at 20 deg.C for 5-7 days to generate conidium, scraping with blade, grinding, filtering with 4 layers of gauze, adding sterile distilled water, and making into 5 × 105Spore/ml spore suspension for later use;
(3) preparing a blade: taking a culture dish with the diameter of 9cm, paving filter paper soaked by 11.8mg/L benzimidazole plant fresh-keeping aqueous solution at the bottom of the culture dish, disinfecting, and putting the 3 rd to 5 th true leaves of the plant seedlings in the step (1) on the filter paper for later use;
(4) inoculation of pathogenic bacteria:
inoculation of blight disease bacteria: digging out the plant seedlings in the step (1), cleaning roots, absorbing water by using absorbent paper, soaking the roots in the spore suspension in the step (2), transplanting the seedlings into a container filled with a matrix after 5min, culturing the seedlings in a greenhouse at 26 +/-1 ℃, and observing the state of illness 7d after inoculation;
inoculation of gummy stem blight bacteria: spraying the suspension of the cranberry spores obtained in the step (2) onto the leaves obtained in the step (3), wherein each leaf is sprayed with 500 mu 0, and then placing the culture dish in an illumination incubator for storage, wherein the culture conditions are as follows: the temperature is 26 +/-1 ℃, the illumination period is 16h illumination/8 h darkness, the relative humidity is about 90%, and the state of the patient is observed after 3 d;
(5) investigation and evaluation:
and (3) investigation and grading of blight: recording according to the disease observation result of the blight in the step (4), and investigating and recording the resistance grading according to a grade 5 standard, namely:
level 0: no disease symptoms;
level 1: leaf wilting or slight wilting of partial cotyledon and true leaf:
stage 2: 1 true leaf wilting or heavier leaf wilting;
and 3, level: leaf and more than 60% of true leaves will wither;
and 4, stage 4: the whole plant is slightly wilted, more than 60 percent of the plant withers, but heart leaves still survive;
and 5, stage: the whole plant withers and withers severely;
wherein, the plants below grade 2 are disease-resistant plants, and the plants above grade 3 are susceptible plants; resistance evaluation was performed according to the incidence of disease (number of diseased plants/total investigated) x 100%, and the calculation results were divided into four grades, and the disease (S): the withering rate is 80-100%; light antibody (LR): the withering rate is 51-80%; anti-Medium (MR): the withering rate is 21-50%; high Resistance (HR): the withering rate is less than 20 percent;
investigating and grading gummy stem blight: grading according to the sizes of the disease spots on the leaves in the step (4), wherein the grading standard of each disease grade is as follows:
level 0: the leaves have no scab;
level 1: the area of the leaf scab is less than or equal to 25 percent;
stage 2: 25 percent of leaf lesion area is less than or equal to 50 percent;
and 3, level: 50% of leaf lesion area is less than or equal to 75%;
4, level: the area of the leaf lesion spots is less than or equal to 100 percent in percentage by weight.
Calculating the disease index according to the examined disease grade, wherein the formula is as follows:
disease index ═ Σ (number of onset at each stage × representative value at each stage)/(total number of investigated plants × representative number at the highest stage) × 100%
The calculation results are divided into 6 grades: high feeling (HS): 80.1-100; infection (S): 60.1-80; (iii) feeling (MS): 40.1-60; anti-Medium (MR): 20.1-40.0; disease resistance (R): 10.1-20.0; high Resistance (HR): 1-10.
The results are shown in Table 1:
TABLE 1 watermelon variety wilt and gummy stem blight combined resistance identification in seedling stage
As can be seen from table 1: the morbidity of Sumi No. 1 is 81.25 percent, the Sumi No. 1 is identified as a fusarium wilt susceptible variety, the morbidity of the carmine is 18.75 percent, and the Sumi No. 1 is identified as a fusarium wilt high-resistance variety; the watermelon variety resistance to the gummy stem blight is identified by using the watermelon seedling in vitro leaves, the disease index of Sumi No. 1 is 81.48, the gummy stem blight highly susceptible variety, the disease index of W7 is 39.81, and the gummy stem blight resistant variety is obtained. The identification result shows that the Sumi No. 1 is a fusarium wilt disease susceptible variety, the carmine is a fusarium wilt disease high resistant variety, the Sumi No. 1 is a gummy stem blight high susceptible variety, and the W7 is a gummy stem blight resistant variety, which is in line with the expectation.
The method for identifying the combined resistance of the wilt disease inoculation of the living body in the seedling stage and the gummy stem blight inoculation of the leaves in vitro can quickly identify the watermelon germplasm resources with the wilt disease resistance and the gummy stem blight resistance at the same time on the same plant of the same variety, and obtain accurate identification results of the wilt disease gummy stem blight resistance of different watermelon variety resource materials in the seedling stage.
The above description is only for the purpose of illustrating the technical solutions of the present invention and not for the purpose of limiting the same, and other modifications or equivalent substitutions made by those skilled in the art to the technical solutions of the present invention should be covered within the scope of the claims of the present invention without departing from the spirit and scope of the technical solutions of the present invention.
Claims (1)
1. A method for rapidly identifying watermelon fusarium wilt and gummy stem blight combined resistance in a seedling stage is characterized by comprising the following steps:
(1) preparing test plant seedlings: soaking watermelon and muskmelon seeds in disinfectant for 5min, washing with flowing water to remove residual disinfectant on the surface, soaking seeds in warm water at 50-60 ℃, stirring to room temperature, standing for 3-6h, taking out, sterilizing, sowing, and growing to 3-5 true leaves after seedling emergence for later use; the disinfectant is a sodium hypochlorite solution with the concentration of 10%;
(2) preparation of pathogenic bacteria:
preparing fusarium wilt germs: inoculating watermelon Fusarium oxysporum to PDA solid culture medium, culturing at 26-28 deg.C for one week, inoculating the cultured Fusarium oxysporum to PDB liquid culture medium, culturing at 26-28 deg.C for one week at 150r/min, filtering, adding sterile distilled water, and making into the final product with concentration of 5 × 105Spore/ml spore suspension for later use;
preparing gummy stem blight bacteria: gummosis of tendrilleaf blightInoculating bacteria to PDA solid culture medium, culturing at 26-28 deg.C for one week, selecting fresh cucumber without wound, washing with flowing water, wiping surface with 75% alcohol, air drying, slightly removing epidermis on cucumber surface, beating cultured strain with sterilized puncher with diameter of 0.7cm to obtain mycelium block, placing the mycelium block on cucumber, culturing at 20 deg.C for 5-7 days, generating conidium, scraping with blade, grinding, filtering, adding sterile distilled water, and making into 5 × 105Spore/ml spore suspension for later use; the filtration refers to the filtration by 4 layers of gauze
(3) Preparing a blade: taking a culture dish, paving filter paper soaked by benzimidazole plant fresh-keeping aqueous solution with the concentration of 11.8mg/L at the bottom of the culture dish, disinfecting, and putting the 3 rd to 5 th true leaves of the plant seedlings in the step (1) on the filter paper for later use;
(4) inoculation of pathogenic bacteria:
inoculation of blight disease bacteria: digging out the plant seedlings in the step (1), cleaning roots, absorbing water by using absorbent paper, soaking the plant seedlings in the spore suspension in the step (2), transplanting the seedlings into a container filled with a matrix after 5min, culturing the seedlings in a greenhouse at 26 +/-1 ℃, and observing the state of illness 7d after inoculation;
inoculation of gummy stem blight bacteria: spraying the spore suspension of the gummy stem blight bacteria in the step (2) onto the leaves in the step (3), wherein each leaf is sprayed with 500 mu l, and then placing the culture dish in an illumination incubator for storage, wherein the culture conditions are as follows: the temperature is 26 +/-1 ℃, the illumination period is 16h illumination/8 h darkness, the relative humidity is 90%, and the state of the patient is observed after 3 d;
(5) investigation and evaluation:
and (3) investigation and grading of blight: recording according to the disease observation result of the blight in the step (4), and investigating and recording the resistance grading according to a grade 5 standard, namely:
level 0: no disease symptoms;
level 1: leaf wilting or slight wilting of partial cotyledon and true leaf;
and 2, stage: 1 true leaf wilting or heavier leaf wilting;
and 3, level: leaf and more than 60% of true leaves will wither;
4, level: the whole plant is slightly wilted, more than 60 percent of the plant is withered, and heart leaves still survive;
and 5, stage: the whole plant withers and withers severely;
wherein, the disease-resistant plants are below grade 2, and the disease-susceptible plants are above grade 3; resistance evaluation was performed according to the incidence rate (number of diseased plants/total investigated plants) × 100%, the calculation results were divided into four grades, and the disease was: the withering rate is 80-100%; light resistance: the withering rate is 51-80%; resisting: the withering rate is 21-50%; high resistance: the withering rate is less than 20 percent;
investigating and grading gummy stem blight: grading according to the sizes of the disease spots on the leaves in the step (4), wherein the grading standard of each disease grade is as follows:
level 0: the leaves have no scab;
level 1: the area of the leaf scab is less than or equal to 25 percent;
and 2, stage: 25 percent, the area of the leaf scab is less than or equal to 50 percent;
and 3, level: 50 percent of the area of the leaf scab is less than or equal to 75 percent;
4, level: 75 percent of the area of the leaf scab is less than or equal to 100 percent;
calculating the disease index according to the examined disease grade, wherein the formula is as follows:
disease index ═ Σ (number of onset at each stage × representative value at each stage)/(total number of investigated plants × representative number at the highest stage) × 100%
The calculation results are divided into 6 grades: high feeling: 80.1-100; the infection: 60.1-80; sensing: 40.1-60; resisting: 20.1-40.0; disease resistance: 10.1-20.0; high resistance: 1-10.
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