CN112014524A - Method for identifying potato scab resistance - Google Patents
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Abstract
The invention belongs to the technical field of plant diseases, and particularly relates to an identification method of potato scab resistance. The invention provides an identification method of potato scab resistance, which comprises the following steps: sequentially carrying out rooting culture and induction culture of the miniature potatoes on the potato virus-free seedlings, inoculating fermentation liquor of scab pathogenic bacteria in the induction culture, observing the generation condition of the miniature potatoes and the disease condition of plants and the miniature potatoes in the process of continuing the induction culture, and judging the scab resistance of the potatoes according to the area of scab of the miniature potatoes and the necrosis grade of the plants. According to the mechanism that the scab pathogenic bacteria infect potato tubers by using pathogenic toxins to cause disease occurrence, the resistance of the potato scab is identified by combining the techniques of potato virus-free seedling tissue culture and liquid induction of miniature potatoes. The identification period of the identification method provided by the invention is controlled to be about 40d, so that the identification period is greatly shortened.
Description
Technical Field
The invention belongs to the technical field of plant diseases, and particularly relates to an identification method of potato scab resistance.
Background
Potato scab (potatoscab) is an important disease that is caused by streptomyces spp and widely occurs worldwide. The streptomyces scabies, streptomyces acidiscabies causing acidic scab and streptomyces turgidiscabis causing convex scab are the most common 3 strains studied intensively for potato scab. The potato tuber epidermis infected with scab can generate brown spots and gradually expand, tissues around the infection points gradually die, the surface of the tuber becomes rough, the tissues become cork, and the quality of the potato is seriously influenced. According to statistics, the potato scab is more serious in alkaline soil, continuous cropping for many years and extensive greenhouse miniature potato production and cultivation management by taking vermiculite as a substrate. The incidence rate of scab in some areas is more than 90%.
The scab is a soil-borne disease which damages potato tubers, the occurrence process of the disease cannot be directly observed, the potatoes are required to finish the whole growth period, the identification result is observed after the tubers are mature and harvested, the infection condition of pathogenic bacteria to hosts cannot be known in the period that the tubers are not harvested, and the controllability of the whole identification method is low. Common scab resistance identification methods are a field potato inoculation pathogenic bacteria identification method and a pot inoculation pathogenic bacteria suspension identification method, field inoculation identification is close to natural morbidity, but the identification period is the whole growth period of the potatoes, the identification period is as long as 90-120 d according to different maturity times of potato varieties, and meanwhile, the identification process is complex in operation, large in labor consumption, and the defect of field pathogenic bacteria pollution is caused. The inoculation identification of the greenhouse pot is close to natural morbidity, the pathogenic bacteria pollution range is controllable, but the whole growth period of the potato is also identified, and the defects of long period, high cost, complex field management and the like exist. In view of the defects of the identification method, a novel potato scab resistance identification method which is more economical, effective, rapid, visual and strong in controllability is urgently needed so as to better evaluate potato resources resisting scab and perform rapid and accurate screening and identification.
Disclosure of Invention
In order to solve the problems, the invention provides a method for identifying potato scab resistance. The identification method provided by the invention has the advantages of short identification period, strong operability, capability of observing the whole disease generation process, strong identification result consistency and low cost.
In order to achieve the purpose, the invention provides the following technical scheme:
the invention provides an identification method of potato scab resistance, which comprises the following steps:
placing the potato detoxified seedlings in a rooting culture medium for rooting culture to obtain rooted detoxified seedlings;
placing the rootage detoxified seedlings in a miniature potato induction culture medium for induction culture of miniature potatoes to obtain miniature potatoes, inoculating scab pathogenic bacteria fermentation liquor during the induction culture, observing the generation condition of the miniature potatoes and the disease occurrence condition of plants and the miniature potatoes, and judging the scab resistance of the potatoes according to the lesion area grade and the plant necrosis grade of the miniature potatoes;
the miniature potato induction culture medium comprises: 28-32 g/L, MS of sucrose and 9-11 mg/L of choline chloride, and the pH value is 5.6-6.0.
Preferably, the method for calculating the scab index according to the area grade of the miniature potato scab and the necrosis grade of the plant and judging the resistance of the potato scab according to the scab index comprises the following steps:
determining the area grade of the disease spots of the miniature potatoes according to the area of the disease spots of the miniature potatoes; the judging method for determining the area grade of the diseased potato spots according to the areas of the diseased potato spots comprises the following steps: observing the lesion area on the surface of the miniature potato, wherein when the surface of the miniature potato is asymptomatic and healthy, the lesion area of the miniature potato is graded as 0; when the area of the disease spots of the miniature potatoes is more than 0 and less than or equal to 5 percent, the area grade of the disease spots of the miniature potatoes is 1 grade; when the area of the disease spots of the miniature potatoes is more than 5 and less than or equal to 12.5 percent, the area grade of the disease spots of the miniature potatoes is 2 grade; when the area of the disease spots of the miniature potatoes is more than 12.5 percent and less than or equal to 25 percent, the area grade of the disease spots of the miniature potatoes is 3 grade; when the area of the disease spots of the miniature potatoes is more than 25 percent and less than or equal to 50 percent, the area grade of the disease spots of the miniature potatoes is 4 grade; when the disease spot area of the miniature potato is more than 50 percent, the disease spot area of the miniature potato is graded as 5;
the method for judging the plant necrosis grade comprises the following steps: observing the necrotic part of the plant, wherein when the plant is healthy, the grade of the plant necrosis is 1 grade; when the plant necrosis is more than 0 and less than or equal to 25 percent, the grade of the plant necrosis is grade 2; when the plant necrosis is more than 25% and less than or equal to 50%, the grade of the plant necrosis is 3 grade; when the plant necrosis is more than 50%, the grade of the plant necrosis is 4;
determining a scab index according to a scab index formula, wherein the scab index formula is as follows: the scab index is the scale of the area of the miniature potato lesion multiplied by the scale of plant necrosis/20 multiplied by 100; determining resistance to potato scab based on the scab index;
the method for judging the plant resistance grade comprises the following steps: when the scab index is more than 0 and less than or equal to 5, the resistance grade of the plant is high resistance; when the scab index is more than 5 and less than or equal to 10, the resistance grade of the plant is resistant; when the scab index is more than 10 and less than or equal to 15, the resistance grade of the plant is low; when the scab index is more than 15 and less than or equal to 30, the resistance grade of the plant is in a neutral feeling; when the scab index is more than 30 and less than or equal to 60, the resistance grade of the plant is high; when scab index > 60, the plant resistance rating was severe.
Preferably, the rooting medium comprises: 28-32 g/L, MS of sucrose, 4-5 g/L of culture medium, 0.04-0.06 mg/L of naphthylacetic acid and 2-3 g/L of agar, and the pH value is 5.6-6.0.
Preferably, the preparation method of the scab pathogenic bacteria fermentation liquid comprises the following steps: taking the concentration as 106culturing 100 mul of bacterial suspension of cfu/mL of strong potato scab pathogenic strain streptomyces. scabies in 100mL of trypticase soy peptone liquid culture medium, shaking for 5-7 days at 26-30 ℃ and 180-250 rpm, and filtering hyphae to obtain scab pathogenic bacteria fermentation liquor.
Preferably, the potato-detoxified seedling is a stem tip-detoxified tissue culture seedling from the same potato.
Preferably, when the potato virus-free seedlings are test-tube seedlings, the inoculation amount of the scab pathogenic bacteria fermentation liquid is 2-4% of the volume of the induction culture medium of the miniature potatoes.
Preferably, the rooting culture conditions comprise: culturing for 12-16 d under the conditions of 22-26 ℃ and 14-18 h/d photoperiod.
Preferably, the conditions for induction culture before inoculation of the scab pathogenic bacteria fermentation broth comprise: culturing for 7-10 days at 22-26 ℃ and the photoperiod of 14-18 h/d.
Preferably, the conditions for induction culture after inoculation of the scab pathogenic bacteria fermentation broth comprise: culturing for 20-30 days in a dark environment at 16-20 ℃.
The invention provides an identification method of potato scab resistance, which comprises the following steps: sequentially carrying out rooting culture and induction culture of the miniature potatoes on the potato virus-free seedlings, inoculating fermentation liquor of scab pathogenic bacteria in the induction culture, observing the generation condition of the miniature potatoes and the disease condition of plants and the miniature potatoes in the process of continuing the induction culture, and judging the scab resistance of the potatoes according to the area of scab of the miniature potatoes and the necrosis grade of the plants. According to the mechanism that the scab pathogenic bacteria infect potato tubers by using pathogenic toxins to cause disease occurrence, the resistance of the potato scab is identified by combining the techniques of potato virus-free seedling tissue culture and liquid induction of miniature potatoes. The identification period of the identification method provided by the invention is controlled to be about 40d, so that the identification period is greatly shortened; according to the identification method provided by the invention, the seed potatoes do not need to be cultivated and propagated in the field, the workload can be effectively reduced, one plant can be identified by one test tube, the space can be saved, meanwhile, the identification process is finished in a laboratory, the external environment is controllable, and the pollution can be effectively avoided; the identification method provided by the invention is carried out in a culture container in a laboratory in the whole process, and the whole process can be observed; the plants obtained by the identification method provided by the invention are virus-free seedlings of the same potato plant, the physiological ages are consistent, the detection operation process is standard, the interference of other conditions can be reduced, and the consistency and repeatability of the identification result are ensured; the identification method provided by the invention does not need a large amount of labor, and can effectively reduce the identification cost. According to application examples, the cycle of the identification method provided by the invention is only 44 days, and the identification accuracy is up to 100%.
Drawings
FIG. 1 shows the symptoms of tuber infection after greenhouse inoculation of scab germs in a pot culture of potato resources, wherein A: resistance to scab in japan, high resistance; b: aquila, medium resistance; c: 672 to 50, moderate; d: 897003, in the middle of the above-mentioned formula; e: z292, in the middle; f: bl-1c, high; g: ningshu No. 15, high-quality; h: early Ohio, heavy feeling; in B-H, the upper potato tuber is a Control (CK) inoculated with sterile water, and the lower potato tuber is an infected tuber obtained after inoculation of pathogenic bacteria spore suspension;
fig. 2 shows symptoms of Ningshu No. 15 mini-potato after induction and inoculation of S.scabies fermentation broth; wherein, A: inducing miniature potato in Ningshu No. 15 test tube, inoculating different concentrations of S.scabies fermentation liquor for 20 days, and sequentially inoculating 200 μ l TSB sterile fermentation liquor, 200 μ l pathogenic bacteria fermentation liquor, 400 μ l, 600 μ l and 800 μ l for CK, and pathogenic bacteria fermentation liquor from left to right; b: inoculating the fermentation liquor of S.scabies to Ningshu No. 15 for 20 days, then inoculating 200 mul of TSB sterile fermentation liquor to CK, and inoculating 200 mul, 400 mul, 600 mul and 800 mul of pathogenic bacteria fermentation liquor in sequence from left to right; c: inoculating 200 μ l of sterile culture solution with CK to obtain miniature potato; d: inoculating 400 mu l of S.scabies fermentation liquor to obtain miniature potatoes; e: inoculating 600 μ l of S.scabies fermentation liquor to obtain miniature potato; f: inoculating 800 μ l of S.scabies fermentation liquor to obtain miniature potato;
FIG. 3 shows the symptoms of plants and test-tube potatoes after different potato germplasm resource test-tube plantlets induce the micro-potatoes to inoculate fermentation liquids with scab germs at different concentrations; wherein, the upper part of the graphs A to D is different potato germplasm resource test-tube plantlets (the variety of A is Aquila, the variety of B is 897003, the variety of C is bl-1C, the variety of D is Early Ohio) for inducing the plant symptoms of the micro potatoes inoculated with fermentation liquor with different concentrations of scab bacteria, the lower part is the test-tube potato obtained after inoculation, and in the graphs A to D in figure 3, each column is sequentially from left to right: inoculating 200 mul of TSB sterile fermentation liquor with CK, inoculating 200 mul, 400 mul, 600 mul, 800 mul and 1000 mul of pathogenic bacteria fermentation liquor with CK.
Detailed Description
The invention provides an identification method of potato scab resistance, which comprises the following steps: placing the potato detoxified seedlings in a rooting culture medium for rooting culture to obtain detoxified rooted seedlings;
placing the rootage detoxified seedlings in a miniature potato induction culture medium for induction culture of miniature potatoes to obtain miniature potatoes, inoculating scab pathogenic bacteria fermentation liquor during the induction culture, observing the generation condition of the miniature potatoes and the disease occurrence condition of plants and the miniature potatoes, and judging the scab resistance of the potatoes according to the area of scab of the miniature potatoes and the necrosis grade of the plants;
the miniature potato induction culture medium comprises: 28-32 g/L, MS of sucrose culture medium 4-5 g/L and 10mg/L of choline chloride, and the pH value is 5.6-6.0.
The identification method provided by the invention can effectively reduce the identification period; the identification process is carried out in the test tube, so that not only can the space be saved and the environment be controlled, but also the whole process of disease occurrence can be observed, and the identification result is more visual; and the identification method provided by the invention does not need a large amount of manual operation, and can greatly reduce the identification cost.
In the present invention, the culture medium for rooting culture preferably includes: 28-32 g/L, MS of sucrose, 4-5 g/L of culture medium, 0.04-0.06 mg/L of naphthylacetic acid and 2-3 g/L of agar, and the pH value is 5.6-6.0. The rooting culture medium used by the invention has high rooting speed and is beneficial to shortening the identification period.
In the present invention, the preparation method of the fermentation liquid of scab pathogenic bacteria preferably comprises: taking the concentration as 106cfu/mL of 100. mu.l bacterial suspension of a strong pathogenic strain streptomyces. scabies of potato scab,culturing in 100ml trypticase soy peptone liquid medium, shaking at 26-30 ℃ and 180-250 rpm for 5-7 days, and filtering mycelia to obtain scab pathogenic bacteria fermentation liquor. The preparation method of the fermentation liquor can improve the efficiency of potato infection of scab after inoculation of the scab pathogenic bacteria fermentation liquor.
In the present invention, the potato-detoxified seedling is preferably a stem-tip-detoxified tissue culture seedling from the same potato. The plants taken by the method are virus-free seedlings with consistent physiological age, and the interference of other conditions in the identification process can be effectively reduced by matching with a standard operation process, so that the consistency and repeatability of the identification result are ensured.
In the invention, when the potato virus-free seedling is a test-tube seedling, the volume ratio of the inoculation amount of the scab pathogenic bacteria fermentation liquid to the minitype potato induction culture medium is preferably 2-4%. In the embodiment of the invention, the inoculation amount is preferably 400-800 μ l/strain, and more preferably 500-600 μ l/strain; the caliber and the length of the test tube used for the test tube seedling are preferably (18-20) mmX (180-200) mm. The setting of the inoculation amount can not only ensure the morbidity of plants, but also can not reduce the potato bearing rate of miniature potatoes to cause deviation of identification results.
In the present invention, the rooting culture conditions preferably include: the temperature is preferably 22-26 ℃, more preferably 24 ℃, the photoperiod is preferably 16-18 h/d, more preferably 16h/d, the culture is preferably 12-16 d, and more preferably 14 d.
In the present invention, the method for inducing culture before inoculation of scab pathogenic bacteria preferably comprises: the temperature is preferably 22-26 ℃, more preferably 24 ℃, the photoperiod is preferably 16-18 h/d, more preferably 16h/d, the culture is preferably carried out for 7-10 d, more preferably 10 d. The culture conditions and the induction culture method provided by the invention can ensure that the test-tube plantlet of the potato grows well, so that the test-tube plantlet can root strongly, and the development of the work of identifying the potato scab is facilitated.
In the present invention, the method for inducing culture after inoculation of scab pathogenic bacteria preferably comprises: the culture is preferably carried out at 16-20 ℃, more preferably at 18 ℃ for 20-30 days, and more preferably for 20 days in a dark environment.
According to the method, the generation process of the plant miniature potatoes and the disease occurrence conditions of the plants and the miniature potatoes are observed every 10 days during dark culture, the disease occurrence period and the severity are recorded one by one, and the scab index is calculated according to the scab area of the miniature potatoes and the necrosis grade of the plants, so that the scab resistance grade is determined.
The method for judging the resistance of the potato scab according to the area of the miniature potato scab and the necrosis grade of the plant preferably comprises the following steps:
determining the area grade of the disease spots of the miniature potatoes according to the area of the disease spots of the miniature potatoes;
determining a scab index according to a scab index formula, wherein the scab index formula is as follows: the scab index is the scale of the area of the miniature potato lesion multiplied by the scale of plant necrosis/20 multiplied by 100;
and determining the resistance of the potato scab according to the scab index.
In the invention, the judgment standard of the lesion area and the plant necrosis grade of the miniature potato, the calculation of the scab index and the method for determining the scab resistance grade preferably refer to the classification standard of Mishra KK, 2001, Genchenyingying, 2015 and the like, and as shown in tables 1-2, the method is divided into 5 grades according to the lesion area grade Mean (MA) and the plant necrosis grade Mean (MN):
TABLE 1 micro potato lesion area rating
TABLE 2 plant necrosis rating
Scab index si (scab index) for each repetition of each resource: SI ═ MA × MN)/20 × 100, the potato scab resistance rating was divided into 6 ratings as shown in table 3:
TABLE 3 plant resistance rating
The present invention preferably performs multiple sets of parallel experiments in the actual identification process, such as 20 biological replicates per inoculum size in the examples. Mean values of scab indices (MSI) were calculated for each variety using Microsoft Office Excel 2007. The potato scab index can be more effectively judged by taking the average value of a plurality of scab indexes, and a more accurate identification method is provided.
For further illustration of the present invention, the method for identifying potato scab resistance provided by the present invention is described in detail below with reference to the drawings and examples, but they should not be construed as limiting the scope of the present invention.
Example 1
Culturing S.scabies strain in Gao's No. 1 culture medium for 7 days, washing spore with sterile water to obtain 10% spore6cfu/mL bacterial suspension, 100 μ l bacterial suspension is added into 100mL sterilized Tryptone Soy Broth (TSB) culture medium (30 g of TSB finished product, 1000mL of distilled water), the mixture is shaken for 7d at 28 ℃ and 180rpm, and after hyphae are filtered from the fermentation filtrate, the obtained scab bacterial fermentation filtrate is stored in a refrigerator at 4 ℃.
Taking potato virus-free seedlings (original seeds of Japanese scab resistant, Aquila, 672-50, 16-8, 897003, Z292, bl-1c and Early Ohio, provided by the national potato improvement center of Keshan division of the Black Longjiang academy of agriculture, and Ningshu No. 15 provided by the solid original division of the Ningxia agriculture and forestry academy of sciences), cutting and taking normal and regular stem sections, inoculating the stem sections into rooting medium of 30g/L of sucrose, 4.42g/L of MS, 0.05mg/L of NAA0, 2.2g/L of agar and 5.8 of pHs, and culturing for 14d in 16h/d photoperiod at 24 ℃ to induce the stem sections of the potatoes to root. Taking the rooted detoxicated seedling out of rooting culture medium, washing the root culture medium with sterile water, absorbing water with sterile filter paper, inoculating to 1.5cm glass test tube containing 15mL of miniature potato inducing culture medium (30 g/L sucrose, 4.42g/L MSC, 10mg/L CCC, pH5.8), and culturing at 24 deg.C for 10d in 16h/d photoperiod.
After 10 days of culture in the minipotato induction medium, 400. mu.l of Elsinoe scaber fermentation filtrate per tube was inoculated under sterile conditions, 20 replicates per inoculum size. 400 μ l of sterile TSB medium was used as a Control (CK). After inoculation, the cells were cultured in a dark environment at 18 ℃ for 20 days. And (3) regularly observing the generation process of the miniature potatoes of the plants and the disease conditions of the plants and the miniature potatoes during dark culture, recording the disease development period and the severity one by one, referring to tables 1-3, calculating the scab index according to the scab area of the miniature potatoes and the necrosis grade of the plants, and determining the scab resistance grade.
Example 2
The same test as in example 1 was carried out, except that 600. mu.l of the Eleoboderma scaber fermentation filtrate was inoculated per test tube, and 600. mu.l of TSB sterile culture medium was used as a Control (CK).
Example 3
The same test as in example 1 was carried out, except that the Eleosphaera scaber fermentation filtrate was inoculated with 800. mu.l per test tube, and 800. mu.l of TSB sterile culture medium was used as a Control (CK).
Comparative example 1
The same test as in example 1 was carried out, except that the Eleosphaera scaber fermentation filtrate was inoculated with 200. mu.l per test tube, and 200. mu.l of TSB sterile culture medium was used as a Control (CK).
Comparative example 2
The same test as in example 1 was carried out, except that the Eleosphaera scabiosa fermentation filtrate was inoculated with 1000. mu.l per test tube, and 1000. mu.l of TSB sterile culture medium was used as a Control (CK).
Comparative example 3
Taking potato virus-free test-tube seedlings (Japanese scab-resistant, Aquila, 672-50, 16-8, 897003, Z292, bl-1c and Early Ohio original seeds provided by the national potato improvement center of Keshan division of Black Longjiang agricultural institute, Ningshu No. 15 provided by the solid original division of Ningxia agricultural and forestry academy of sciences), carrying out field conventional planting, sowing the harvested potato planting resource original seeds into flowerpots with the diameter of about 15cm and filled with a thoroughly decomposed matrix and vermiculite in a ratio of 1:1, selecting plants with consistent growth and root irrigation for 10 inoculation when the seedlings grow to about 10cm6200mL of CFU/mL S.scabies bacterial suspension. Sowing each variety in 4 pots, repeating for 3 times, setting clear water as Control (CK), culturing in greenhouse,and (3) keeping short-term drought after 4-6 weeks of inoculation, harvesting after 11 weeks, detecting the incidence condition of the new potato blocks (the result is shown in figure 1), evaluating the scab resistance grades of different potato resources, and obtaining the evaluation result shown in table 4.
TABLE 4 identification of resistance to Potato resources by greenhouse Pot culture inoculation
As can be seen from Table 4, the identification period of the potato scab resistance identification method provided by the prior art is as long as 11 weeks, the identification process is time-consuming and labor-consuming, and the identification process has the defect of opacity.
Application example 1
And observing the generation condition of the miniature potatoes obtained after the induction culture of the application examples 1-3 and the comparative examples 1-2 and the disease occurrence condition of the plants and the miniature potatoes. The observation results are shown in tables 5 to 13 and fig. 2 and 3. Wherein, fig. 2 shows the symptoms of Ningshu No. 15 mini-potato after induction and inoculation of S.scabies fermentation broth; wherein, A: inducing miniature potato in Ningshu No. 15 test tube, inoculating different concentrations of S.scabies fermentation liquor for 20 days, and sequentially inoculating 200 μ l TSB sterile fermentation liquor, 200 μ l pathogenic bacteria fermentation liquor, 400 μ l, 600 μ l and 800 μ l for CK, and pathogenic bacteria fermentation liquor from left to right; b: inoculating the fermentation liquor of S.scabies to Ningshu No. 15 for 20 days, then inoculating 200 mul of TSB sterile fermentation liquor to CK, and inoculating 200 mul, 400 mul, 600 mul and 800 mul of pathogenic bacteria fermentation liquor in sequence from left to right; c: inoculating 200 μ l of sterile culture solution with CK to obtain miniature potato; d: inoculating 400 mu l of S.scabies fermentation liquor to obtain miniature potatoes; e: inoculating 600 μ l of S.scabies fermentation liquor to obtain miniature potato; f: inoculating 800 μ l of S.scabies fermentation liquor to obtain miniature potato;
FIG. 3 shows the plant and test-tube potato symptoms after different potato germplasm resources test-tube plantlets induce micro potato to inoculate fermentation liquor with scab germs in different concentrations; wherein, the upper part of the graphs A to D is different potato germplasm resource test-tube plantlets (the variety of A is Aquila, the variety of B is 897003, the variety of C is bl-1C, the variety of D is Early Ohio) for inducing the plant symptoms of the micro potatoes inoculated with fermentation liquor with different concentrations of scab bacteria, the lower part is the test-tube potato obtained after inoculation, and in the graphs A to D in figure 3, each column is sequentially from left to right: inoculating 200 mul of TSB sterile fermentation liquor with CK, inoculating 200 mul, 400 mul, 600 mul, 800 mul and 1000 mul of pathogenic bacteria fermentation liquor with CK.
TABLE 5 resistance identification of the miniature potato scab of Japanese scab-resistant test-tube plantlet
TABLE 6 evaluation of resistance to Potato Eleocharitis in miniature Aquila test-tube plantlets
TABLE 7672-50 identification of resistance of test-tube plantlet to potato scab
TABLE 816-8 TEST TUBULAR plantlet MINI-SOYA CHUAN scab RESISTANCE IDENTIFICATION
TABLE 9897003 test-tube plantlet mini potato scab resistance identification
TABLE 10Z 292 test-tube plantlet minitype potato scab resistance identification
TABLE 11 bl-1c test-tube plantlet mini potato scab resistance identification
TABLE 12 Ningshu No. 15 test-tube plantlet minitype potato scab resistance identification
TABLE 13 Early Ohio test-tube plantlet resistance identification of miniature potato scab
As can be seen from tables 5 to 13, the inoculum size of the fermentation broth provided in examples 1 to 3 is the optimum inoculum size for identifying the resistance of the plant. As can be seen from the comparative example 3, tables 5-13, figures 2 and 3, the accuracy of the potato scab identification method provided by the invention is as high as 100%, the identification time is short, the result can be obtained only in 44 days, the conventional detection method provided by the comparative example 3 can obtain the identification result after 11 weeks, and the identification method provided by the embodiment of the invention effectively shortens the time of the potato scab identification method.
Although the present invention has been described with reference to the preferred embodiments, it should be understood that various changes and modifications can be made therein by those skilled in the art without departing from the spirit and scope of the invention as defined in the appended claims.
Claims (9)
1. A method for identifying potato scab resistance comprises the following steps:
placing the potato detoxified seedlings in a rooting culture medium for rooting culture to obtain rooted detoxified seedlings;
placing the rootage detoxified seedlings in a miniature potato induction culture medium for induction culture of miniature potatoes to obtain miniature potatoes, inoculating scab pathogenic bacteria fermentation liquor during the induction culture, observing the generation condition of the miniature potatoes and the disease occurrence condition of plants and the miniature potatoes, and judging the scab resistance of the potatoes according to the lesion area grade and the plant necrosis grade of the miniature potatoes;
the miniature potato induction culture medium comprises: 28-32 g/L, MS of sucrose and 9-11 mg/L of choline chloride, and the pH value is 5.6-6.0.
2. The method as claimed in claim 1, wherein the method for calculating the scab index according to the micro potato scab area grade and the plant necrosis grade comprises the following steps:
determining the area grade of the disease spots of the miniature potatoes according to the area of the disease spots of the miniature potatoes; the judging method for determining the area grade of the diseased potato spots according to the areas of the diseased potato spots comprises the following steps: observing the lesion area on the surface of the miniature potato, wherein when the surface of the miniature potato is asymptomatic and healthy, the lesion area of the miniature potato is graded as 0; when the area of the disease spots of the miniature potatoes is more than 0 and less than or equal to 5 percent, the area grade of the disease spots of the miniature potatoes is 1 grade; when the area of the disease spots of the miniature potatoes is more than 5 and less than or equal to 12.5 percent, the area grade of the disease spots of the miniature potatoes is 2 grade; when the area of the disease spots of the miniature potatoes is more than 12.5 percent and less than or equal to 25 percent, the area grade of the disease spots of the miniature potatoes is 3 grade; when the area of the disease spots of the miniature potatoes is more than 25 percent and less than or equal to 50 percent, the area grade of the disease spots of the miniature potatoes is 4 grade; when the disease spot area of the miniature potato is more than 50 percent, the disease spot area of the miniature potato is graded as 5;
the method for judging the plant necrosis grade comprises the following steps: observing the necrotic part of the plant, wherein when the plant is healthy, the grade of the plant necrosis is 1 grade; when the plant necrosis is more than 0 and less than or equal to 25 percent, the grade of the plant necrosis is grade 2; when the plant necrosis is more than 25% and less than or equal to 50%, the grade of the plant necrosis is 3 grade; when the plant necrosis is more than 50%, the grade of the plant necrosis is 4;
determining a scab index according to a scab index formula, wherein the scab index formula is as follows: the scab index is the scale of the area of the miniature potato lesion multiplied by the scale of plant necrosis/20 multiplied by 100; determining resistance to potato scab based on the scab index;
the method for judging the plant resistance grade comprises the following steps: when the scab index is more than 0 and less than or equal to 5, the resistance grade of the plant is high resistance; when the scab index is more than 5 and less than or equal to 10, the resistance grade of the plant is resistant; when the scab index is more than 10 and less than or equal to 15, the resistance grade of the plant is low; when the scab index is more than 15 and less than or equal to 30, the resistance grade of the plant is in a neutral feeling; when the scab index is more than 30 and less than or equal to 60, the resistance grade of the plant is high; when scab index > 60, the plant resistance rating was severe.
3. The method of claim 1, wherein the rooting medium comprises: 28-32 g/L, MS of sucrose, 4-5 g/L of culture medium, 0.04-0.06 mg/L of naphthylacetic acid and 2-3 g/L of agar, and the pH value is 5.6-6.0.
4. The method for identifying scab-causing pathogenic bacteria according to claim 1, wherein the preparation method comprises: taking the concentration as 106culturing 100 mul of bacterial suspension of cfu/mL of strong potato scab pathogenic strain streptomyces. scabies in 100mL of trypticase soy peptone liquid culture medium, shaking for 5-7 days at 26-30 ℃ and 180-250 rpm, and filtering hyphae to obtain scab pathogenic bacteria fermentation liquor.
5. The method of claim 1, wherein the potato-free seedling is a stem tip-free tissue culture seedling from the same potato.
6. The method according to claim 1, wherein when the potato-free seedling is a test-tube seedling, the inoculation amount of the eschar pathogenic bacteria fermentation broth is 2-4% of the volume of the induction culture medium of the minisize potatoes.
7. The method of claim 1, wherein the rooting culture conditions comprise: culturing for 12-16 d under the conditions of 22-26 ℃ and 14-18 h/d photoperiod.
8. The method as claimed in claim 1, wherein the conditions of the induction culture before inoculation of the fermentation broth of the pathogens of scab comprise: culturing for 7-10 days at 22-26 ℃ and the photoperiod of 14-18 h/d.
9. The method as claimed in claim 1, wherein the conditions for induction culture after inoculation of the fermentation broth of scab-causing pathogenic bacteria include: culturing for 20-30 days in a dark environment at 16-20 ℃.
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Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JP2011229443A (en) * | 2010-04-27 | 2011-11-17 | Nagasaki Prefecture | Method for controlling potato-cyst nematode and potato scab with fusarium oxysporum |
| CN105145356A (en) * | 2015-09-10 | 2015-12-16 | 宁波市农业科学研究院 | Rapid propagation method of purple potato minitubers |
| CN106359094A (en) * | 2016-08-31 | 2017-02-01 | 天津大学 | Method for quickly inducing potato minituber |
| CN109507368A (en) * | 2018-11-16 | 2019-03-22 | 江苏徐淮地区徐州农业科学研究所(江苏徐州甘薯研究中心) | A kind of accurate Resistance Identification method of seedling stage sweet potato black rot |
| CN109744113A (en) * | 2019-01-30 | 2019-05-14 | 云南洪邦薯业发展有限公司 | A kind of implantation methods of micro potato |
-
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Patent Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JP2011229443A (en) * | 2010-04-27 | 2011-11-17 | Nagasaki Prefecture | Method for controlling potato-cyst nematode and potato scab with fusarium oxysporum |
| CN105145356A (en) * | 2015-09-10 | 2015-12-16 | 宁波市农业科学研究院 | Rapid propagation method of purple potato minitubers |
| CN106359094A (en) * | 2016-08-31 | 2017-02-01 | 天津大学 | Method for quickly inducing potato minituber |
| CN109507368A (en) * | 2018-11-16 | 2019-03-22 | 江苏徐淮地区徐州农业科学研究所(江苏徐州甘薯研究中心) | A kind of accurate Resistance Identification method of seedling stage sweet potato black rot |
| CN109744113A (en) * | 2019-01-30 | 2019-05-14 | 云南洪邦薯业发展有限公司 | A kind of implantation methods of micro potato |
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