CN112111551A - Method for identifying resistance of potato to black shank - Google Patents
Method for identifying resistance of potato to black shank Download PDFInfo
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Abstract
The invention provides an identification method of potato black shank resistance, which comprises the following steps: selecting a potato plant with 10 completely unfolded leaves, and selecting the petioles of 4 th to 6 th mature healthy leaves as the petioles to be detected; placing the petiole to be measured at a concentration of 103 Inoculating and culturing in a CFU/mL potato black shank pathogenic bacteria suspension, carrying out potato black shank resistance evaluation after inoculating and culturing for 36h, carrying out grading according to a grading standard and calculating an illness index, and then dividing the potato black shank resistance into four grades according to the calculated illness index for evaluation. The identification method provided by the invention has the advantages of simple operation, low test cost, accurate classification, short identification period and reliable result, and provides technical support for breeding of potato black shank resistance and prevention and control of potato black shank.
Description
Technical Field
The invention relates to an identification method of potato pathogenic bacteria resistance, in particular to an identification method of potato black shank resistance.
Background
The potato black shank is caused by Pectobacterium spp (and Dickeya spp.) and has serious diseases on the base of the stem and the tuber of a potato plant, the diseases mainly infect vascular bundle tissues, and the diseases can be developed from the germination of a seed potato to the later growth stage, and the diseases are most abundant in the seedling stage. The phytophthora parasitica can be transmitted by irrigation and rainwater, infected by wounds caused by insect harm, and infected by mechanical wounds caused by agricultural operation, and typical phytophthora parasitica symptoms are generated on infected plants. The germs on the later-stage diseased plants can quickly infect the potatoes in the soil, so that the potato blocks carry germs. During storage, pathogens are transmitted by contact with healthy pieces of the wound and can survive within the diseased tuber. Meanwhile, pathogenic bacteria are left on the diseased potatoes which are not completely rotted in the field during the tuber planting or harvesting process to live through the winter, and become a source of initial infection in the next year. Therefore, the potato black shank is an important disease in potato planting in the world, and in recent years, the potato black shank is more and more serious in China, and the healthy development of the potato industry is threatened.
Because the potato has no main disease-resistant gene for resisting black shank and only some possible QTL (quantitative trait locus) plays a role, once black shank occurs in a potato field, the control is difficult to obtain, and the prevention and control of the disease are mainly performed in the current production. Thus, there is an urgent need for early diagnosis of potato black shank, and for breeding and planting resistant varieties or materials of potato. And the screening of the potato resistant variety or material cannot be separated from the identification of the potato black shank. Therefore, a method capable of rapidly and stably identifying the black shank resistance of the potatoes is needed at present, so as to identify the degree of the potatoes infected with the black shank, analyze the resistance of the potatoes to the black shank, and be beneficial to the control of the black shank of the potatoes and the breeding of the potatoes for resisting the black shank.
At present, the most common mode for screening the resistance of the potato black shank is to use a living plant as a material and inject pathogenic bacteria suspension into a main stem of the potato. However, this method has the following problems: firstly, the bacteria can cause the death of the whole experimental material to result in high experimental cost, secondly, the land resource waste is caused due to the large occupied area for planting the experimental material, and thirdly, the rotten symptom which is difficult to observe by naked eyes can appear in the process of disease-resistant identification, so that the qualitative grading analysis is not accurate. To solve the above problems, researchers have successively created a method for inoculating isolated tissues or organs of potato with black shank, in which leaves are inoculated, by perforating potato leaves and dropping a suspension of pathogenic bacteria. The method can protect potato plants from being damaged, and can not cause resource waste and cost increase. However, this method has the following disadvantages: the poor water retention capacity of the leaves leads to premature dehydration and death of the leaves, and the invasion of diseases cannot be judged qualitatively; the bacterial suspension easily slips off the leaf to cause inoculation failure; the black shank is most accurately embodied in the vascular bundle tissue of the potatoes, so the method is sometimes inconsistent with the field test result.
In conclusion, the existing commonly used method for identifying the potato black shank is unstable, so that the resistance results of the screened materials are inconsistent, breeding materials with some non-major QTLs are omitted, and some candidate materials for providing resistance by improving the autoimmunity of plants are missed; and may not provide further accurate prognosis for the control of the potato black shank.
Disclosure of Invention
In view of the above, the present invention provides a method for identifying the resistance to black shank of potato, so as to solve the above problems.
The technical scheme provided by the invention is as follows: a method of identifying potato black shank resistance comprising:
preparing the suspension to provide a concentration of 103CFU/mL of potato phytophthora parasitica suspension;
screening a potato plant with 10 completely-unfolded leaves from a leaf stalk to be detected, selecting the leaf stalk of the 4 th to 6 th mature healthy leaves of the potato plant as the leaf stalk to be detected, wherein the length of the leaf stalk to be detected is L0；
Inoculating and observing, and pouring the potato black shank pathogenic bacteria suspension into a tissue culture bottle; inserting a petiole to be detected into the dry flower mud, placing the petiole into the tissue culture bottle containing the potato black shank pathogen suspension, covering the tissue culture bottle with a bottle cap, and placing the tissue culture bottle into an illumination incubator with the temperature of 24-26 ℃ and the light of 16 h/8 h for culture and observation;
resistance identification is realized by observing and recording softened water stain symptom of the petiole to be detected by using a light box and an incandescent lamp, measuring the water stain lesion length of the petiole to be detected, and taking the lesion length of the petiole to be detected after being inoculated and cultured for 36 hours as the final lesion length L of the petioleDisease and illnessGrading the diseases according to a grading standard and calculating disease indexes; evaluating and identifying the resistance of the potato black shank according to the disease index: the disease index is less than or equal to 0.2, the disease index is resistant, the disease index is more than 0.4 and less than or equal to 0.6, the disease index is susceptible, and the disease index is more than 0.8 and is susceptible; wherein, the calculation formula of the disease index is as follows:
the disease grading standard is as follows:
lesion length L is more than or equal to 0Disease and illness＜0.08L0The disease grade is set as 0 grade;
0.08L0less than or equal to the length L of the lesionDisease and illness＜0.15L0The disease grade is set as 1 grade;
0.15L0less than or equal to the length L of the lesionDisease and illness＜0.3L0The disease grade is set as 2 grade;
0.3L0less than or equal to the length L of the lesionDisease and illness＜0.6L0The disease grade is set as 3 grade;
0.6L0less than or equal to the length L of the lesionDisease and illness＜L0And the disease grade is set as 4.
Based on the above, the step of screening the petiole to be tested comprises: selecting potato plants which grow for 5-6 weeks after field planting, or potato plants which grow for 3-4 weeks after budding and are planted with tubers, said potato plants having a growth rate of 10 completely unfolded leaf blade, taking the petiole of 4-6 mature healthy leaves, cutting off the redundant leaf blades around the petiole, and cutting the petiole to uniform length L0As the petiole to be measured.
Based on the above, the length L of the petiole to be measured0Is-6.5 cm.
Based on the above, the step of inoculating and culturing comprises: placing the potato black shank pathogen suspension into the tissue culture bottle; firstly, inserting a plurality of leafstalks to be detected of materials to be detected with the same genetic background into dry flower mud, then placing the leafstalks to be detected into the tissue culture bottle containing the potato black shank pathogen suspension, and covering the bottle cap of the tissue culture bottle; and (3) putting the tissue culture bottle covered with the bottle cap into an illumination incubator at 25 ℃ and 16h light/8 h dark for culture and observation.
Based on the above, there are 3-6 petioles to be tested placed in the same tissue culture bottle.
Based on the above, the step of resistance identification comprises: irradiating the tissue culture bottle in the illumination incubator by using a light source at the same time interval, observing the symptoms of softened water staining of the petiole by naked eyes, measuring the length of water-stained lesion of the petiole by using a ruler, photographing and recording, and taking the length of the lesion of the petiole to be measured after being inoculated and cultured for 36 hours as the final lesion length LDisease and illnessGrading according to the disease grading standard, calculating the disease index, and evaluating and identifying the resistance of the potato black shank according to the disease index.
Based on the above, the length L of the petiole to be measured0The disease grading standard is 6.5cm and is as follows:
lesion length L is more than or equal to 0Disease and illnessLess than 0.5cm, and the disease grade is set to 0 grade;
lesion length L is less than or equal to 0.5cmDisease and illnessIf the size is less than 1cm, the disease grade is set to be 1 grade;
lesion length L is less than or equal to 1cmDisease and illnessIf the size is less than 2cm, the disease grade is set to be 2 grade;
lesion length L is less than or equal to 2cmDisease and illnessIf the size is less than 4cm, the disease grade is set to be 3 grade;
lesion length L is less than or equal to 4cmDisease and illnessLess than 6.5cm, and the disease grade is set to 4.
Based on the above, the step of preparing the suspension comprises: carrying out whole dish streak culture on potato black shank pathogenic bacteria on a TSA (TSA-derived food transfer) plate culture medium, and diluting to obtain OD (origin-destination) bacteria600The concentration corresponding to the value is 103CFU/mL of the potato phytophthora parasitica suspension.
Under the same condition, compared with the existing method for injecting the main stems in the field, the method for identifying the black shank resistance of the potatoes provided by the invention has the advantages that the morbidity after the disease is inoculated by the method is basically consistent with the morbidity of the main stems in the field injected by the same material; the identification method provided by the invention only needs about 2 days, and the identification period is short; the identification method provided by the invention adopts in-vitro inoculation culture, and the disease condition is classified into 5 grades, so that the problem that the quality cannot be determined due to the fact that the rot symptom which is difficult to observe by naked eyes or the condition of unstable disease attack can be avoided in the disease-resistant identification process of the field main stem injection material, the analysis result is inaccurate, and the identification material result is unclear; in addition, the method adopts in-vitro culture and is not used in field tests, so that the pollution of field soil by phytophthora parasitica to environment pollution is avoided.
Further, it was found through many experiments that when 10 is used3When the potato petiole is infected by CFU/mL potato phytophthora parasitica suspension for 36h in inoculation mode, the result of the length of the petiole lesion is stable and repeatable, the time of the petiole lesion is shortened due to too low concentration, the error value is large, and the time of the petiole lesion is too fast due to too high concentration, so that some materials with low resistance are missed.
Therefore, the method for identifying the black shank resistance of the potato has the advantages of simple operation, low test cost, accurate grading, short identification period and reliable result, and can provide technical support for breeding the black shank resistance of the potato and controlling the black shank of the potato.
Drawings
FIG. 1 is a flow chart of the operation of the method for identifying the resistance to phytophthora parasitica of potato according to the embodiment of the present invention.
FIG. 2 shows the plating of bacterial suspensions at different dilution ratios used in the examples of the present invention.
FIG. 3 is a graph of lesion petiole length at different times of infecting a potato petiole with suspensions of potato phytophthora parasitica at different dilution factors according to an embodiment of the present invention.
FIG. 4 is a bar graph of lesion petiole length between infestations with different potato petioles and potato phytophthora parasitica suspensions of the present invention.
FIG. 5 is a graph showing the black shank resistance rating of potato petiole inoculated for 36h according to an embodiment of the present invention.
Detailed Description
The technical solution of the present invention is further described in detail by the following embodiments.
Referring to fig. 1, an embodiment of the present invention provides a method for identifying resistance to black shank of potato, including:
step one, preparing suspension
The method mainly comprises the steps of determining the concentration of potato phytophthora parasitica and preparing a suspension of potato phytophthora parasitica.
(1) Determining the concentration of potato phytophthora parasitica (provided by the plant protection institute of the university of Henan agriculture)
a. Inoculating potato black shank pathogenic bacteria (pathogenic bacteria from pectobacterium carotovorum subspecies solani) taken out from an ultralow temperature refrigerator at minus 80 ℃ into 1mL of sterilized 30g/L TSB liquid culture medium according to a ratio of 1:500, and performing shake culture at 28 ℃ for 3.5 h; firstly, sucking 50 mu L of bacterial suspension onto a sterilized TSA culture medium plate by using a pipette together with a sterilized gun head, wherein the TSA culture medium consists of 30g/L of TSB culture medium and 17g/L of agar powder, uniformly coating the TSA culture medium plate with a disposable sterile coating rod, carrying out dark culture in a 28 ℃ incubator for 12-16 hours, then selecting a single colony to streak the culture dish on the sterilized 90mm TSA culture medium plate, and carrying out dark culture in the 28 ℃ incubator for 36 hours; b. preparing 100mL of MES buffer solution for diluting pathogenic bacteria, adjusting the pH value to 5.8 by using KOH, and sterilizing for 20min at 121 ℃, wherein the MES buffer solution consists of MS 4.3g/L and MES 2.13 g/L;
c. referring to FIG. 2, the MES buffer solution was used to dilute the plates of 36 h-cultured pathogens in a gradient manner until the cultured pathogens became flat after 36hPlate dilution to 10-9The bacterial suspension of (4); and for the higher concentration 10-1、10-2、10-3Determination of OD in the bacterial suspension600The values (concentration gradient for accurate determination of OD) give the different OD values shown in Table 1600Carrying out plate coating on the last bacterial suspension with lower concentration and gradient according to the bacterial liquid concentration corresponding to the value, and carrying out dark culture for 16h to count the number of bacterial colonies;
d. the pipette tips sterilized at high temperature and high pressure are respectively matched with a pipette tip from 10-7、10-8、10-950 microliter of bacterial suspension is absorbed from the bacterial solution (the adding amount is too small, so that the plate coating result with lower dilution multiple is inaccurate) and is respectively added into 1/10 TSA culture medium plates, the bacterial suspension is uniformly coated in the culture dish by using a disposable sterile triangular coating rod until the bacterial suspension is dried, corresponding marks are made on a dish cover, the bacterial colony number is counted for 12 hours by dark culture, and each concentration is repeated for three times;
e. the estimated concentration of the bacterial suspension from the number of colonies grown in overnight culture on the plate is shown in Table 1.
TABLE 1 plate colony counts or OD after plating at different dilution concentrations600Numerical value
| Concentration of dilution | Bacterial colony count/dish | Bacterial concentration/ | OD | 600 |
| 10-9 | ≈1 | 102CFU/mL | - | |
| 10-8 | ≈6 | 103CFU/mL | - | |
| 10-7 | ≈200 | 104CFU/mL | - | |
| 10-6 | ＞1800 | 105CFU/mL | - | |
| 10-5 | - | 106CFU/mL | - | |
| 10-4 | - | 107CFU/mL | ≈0.04 | |
| 10-3 | - | 108CFU/mL | ≈0.23 | |
| 10-2 | - | 109CFU/mL | ≈0.89 | |
| 10-1 | - | 1010CFU/mL | ≈2.56 |
Wherein, the overnight cultured plate is diluted to 10-7Then 50 μ L of the bacterial suspension was plated overnight, and the number of colonies counted on the petri dish was about 200, and the original bacterial concentration after 12 hours of overnight culture was calculated to be 1010CFU/mL; taking the dilution concentration of 10-8Then 50. mu.L of the bacterial suspension was plated overnight, and the number of colonies counted on the petri dish was about 6, and the original concentration of the overnight-cultured bacteria was also estimated to be 1010CFU/mL; taking the dilution concentration of 10 by the same principle-9Then 50 μ L of the bacterial suspension was plated overnight and the number of colonies was 1 by counting the colonies on the petri dish, and the concentration of the original bacteria in the overnight culture was calculated to be 1010CFU/mL; as described above, after the pathogen is cultured overnight at 28 ℃ for 12 hours in a plate, the original bacterial suspension is diluted to 10-2The OD measured later was about 0.89, and the diluted concentration was 10-3The initial bacterial suspension concentration was 10 at a later measured OD of about 0.8910CFU/mL。
(2) Preparation of a suspension of potato phytophthora parasitica
The potato phytophthora parasitica is subjected to whole dish streaking on a TSA solid culture medium, and dark culture is carried out at 28 ℃ for 36 h. The liquid transfer gun is matched with a gun head sterilized at 121 ℃ for 15min to absorb 9mL of MES buffer solution, the MES buffer solution is dripped into a whole dish of TSA solid culture medium after potato black shank pathogenic bacteria are cultured for 36h, a disposable sterile triangular coating rod is used for rolling back and forth until all thalli are scraped, and a spectrophotometer is used for measuring OD of the bacterial liquid at the moment600The corresponding concentration is obtained, and MES buffer solution is used for gradient dilution until the concentration required by the test is reached.
In this embodiment, the OD is obtained by the above method600The values are 10CFU/mL and 10 respectively2CFU/mL、103CFU/mL、104CFU/mL and 105A suspension of CFU/mL of phytophthora parasitica.
Step two, screening the petioles to be detected
The potato seedlings with 10 fully-unfolded leaves and grown for 4 weeks after stem emergence and tubers are selected, the petioles of 4-6 mature healthy leaves are taken as test materials for each plant, redundant leaves around the petioles are cut off, and the petioles are cut to be uniform in length of about 6.5 cm.
The test materials in this example were derived from 14 different potato materials #1- #14 shown in table 2, and were planted in the scientific and educational park of southern river agriculture university by tuber planting from 2 months in 2019 to 5 months in 2019, and were sown and field-managed in the manner of in-place main planting.
Step three, inoculation culture
Pouring 40mL of the potato black shank pathogen suspension into a square tissue culture bottle with the thickness of 60mm 90mm, quickly inserting 5 leaf stalks of the pretreated potato material with the same material (the same genetic background) into square dry flower mud with the length of 5.8cm and the thickness of 0.5cm, covering a tissue culture cover, putting the bottle into a light incubator with the temperature of 25 ℃ and the dark of 16h light/8 h for culture and observation, wherein 40mL of MES solution is used in the tissue culture bottle of a blank control group.
In the potato black shank resistance identification method provided by the invention, the concentration of the potato black shank pathogen suspension and the infection time of the petiole to be detected (namely the inoculation culture time of the potato black shank pathogen suspension) have important influences on the reliability of the identification method provided by the invention. The effect of concentration and inoculation time on the identification method is further illustrated below.
(1) Concentration test analysis of potato phytophthora parasitica suspension
The effect of concentration is mainly shown by the 5-group alignment test. Specifically, the petioles to be tested obtained from the susceptible material # 1 were used as test materials at concentrations of 10CFU/mL and 10CFU/mL, respectively2CFU/mL、103CFU/mL、104CFU/mL and 105Repeating the step three and the inoculation culture of the suspension of the CFU/mL potato phytophthora parasitica, wherein each group containsThe inoculation culture time of the test is 72h, and the lesion length of the petiole to be tested is recorded every 2h of measurement. And (3) carrying out statistical analysis according to the measured lesion length of the petiole to be detected, and drawing a curve chart of the lesion petiole length of the potato petiole infected by the potato phytophthora parasitica suspension with different dilution times as shown in figure 3.
As can be seen in fig. 3: when the concentration of the suspension of the potato phytophthora parasitica is 103And in CFU/mL, the lesion length error of the leaf stalks of 5 potatoes in the same tissue culture bottle is small, and the potato leaf stalks have stability and renewability. When the concentration of the suspension of the potato phytophthora parasitica is higher, for example, 104CFU/mL and 105CFU/mL, the potato petioles develop more rapidly and some less resistant material is missed. When the concentration of the potato phytophthora parasitica suspension is higher, such as 10CFU/mL and 10CFU/mL2In CFU/mL, the incidence time of the petiole is reduced, the error value is large, and the stability and the repeatability of the length of the petiole are poor. Therefore, a concentration of 10 is used3The consistency and stability of the test data of the CFU/mL potato phytophthora parasitica suspension infecting the potato petioles are the best.
(2) Experimental analysis of infection time of petiole to be tested
The effect of concentration is mainly shown by the 5-group alignment test. Specifically, the petioles to be tested obtained from potato materials #1- #5 shown in table 2 were used as test materials, and the petioles to be tested from the 5 sources were placed at a concentration of 103And (3) inoculating and culturing the suspension of the potato phytophthora parasitica in CFU/mL for 48 h. Wherein, the lesion length of the petiole to be measured is measured and recorded every 12 h. Statistical analysis was performed based on the measured lesion length of the petioles to be tested, and a histogram of lesion petiole length between potato petioles of different materials and time of infestation was plotted as shown in fig. 4.
As can be seen in fig. 4: when the leaves to be detected, which are derived from potato materials #1- #5, are infected for 24 hours, the leaves are basically free from pathological changes; after being infected for 36h, the petiole to be tested is diseased. Compared with 48h of infection, the difference of the lesion length data of the petioles to be detected, which are derived from the potato materials #1- #5, is smaller when the petioles to be detected are infected for 36h, the data are relatively stable, and the repeatability is good. Therefore, the optimum infection time of the potato petioles is 36 h.
Therefore, the embodiment of the present invention is shown as "103CFU/mL potato phytophthora parasitica pathogen suspension and dip dyeing time 36 h' are used as test conditions of the third step, and petioles to be tested, which are derived from 14 different potato materials #1- #14 shown in the table 2, are used as test objects to perform inoculation culture so as to prepare for the subsequent resistance identification step test.
Step four, resistance identification
Visually observing the softened water stain symptom of the petioles by using a lamp box and an incandescent lamp every 12h, measuring the water stain lesion length of the petioles by using a ruler, taking a picture and recording, classifying according to a classification standard and calculating an illness index by using the inoculated 36h lesion length as a final lesion length, and finally determining the resistance according to the calculated illness index. Wherein, the susceptibility is determined by using susceptible material # 3 as susceptible control and disease-resistant material # 12 as disease-resistant control.
It is determined mainly according to the following criteria:
(1) the length of the diseased petiole is more than or equal to 0 and less than 0.5cm, and the disease grade is set to be 0 grade; the length of the diseased petiole is less than 1cm and is less than or equal to 0.5cm, and the disease grade is set to be 1 grade; the length of the lesion petiole is more than or equal to 1cm and less than 2cm, and the disease grade is set to be 2 grade; the length of the lesion petiole is less than or equal to 2cm and less than 4cm, and the disease grade is set to be 3 grade; the length of the lesion petiole is more than or equal to 4cm and less than 6.5cm, and the disease grade is set to be 4 grade.
(2) The disease index was calculated according to the following formula:
(3) the level of potato resistance was determined according to the following disease index: the high disease resistance index is less than or equal to 0.2; disease resistance index is more than 0.2 and less than or equal to 0.4; the disease resistance index is more than 0.4 and less than or equal to 0.6; the disease index is more than 0.6 and less than or equal to 0.8; susceptibility is 0.8 < disease index, and evaluation grades of black shank resistance of 4 potato materials with disease resistance, susceptibility and susceptibility are shown in figure 5.
The results of the evaluation of the black shank resistance of 14 potato materials according to the above criteria are shown in table 2: susceptible materials include #3 (susceptible control), #11, # 13; the susceptible materials are #1, #6, # 10; the medium-resistant materials are #2, #5, #7 and # 8; the disease-resistant materials are #4, #12 (disease-resistant control) and # 14.
Table 214 potato materials black shank resistance ratings provided using embodiments of the present invention
| Test materials | Infected plant | Blank control | Index of disease condition | Evaluation of |
| # | ||||
| 1 | 13 |
5 plants | 0.788 | Infection of the |
| # | ||||
| 2 | 10 strains of Bacillus subtilis | 5 plants | 0.4 | Resistance to |
| #3 | 15 |
5 plants | 0.983 | Susceptibility to |
| #4 | 15 |
5 plants | 0.233 | |
| # | ||||
| 5 | 10 strains of Bacillus subtilis | 5 plants | 0.425 | Resistance to |
| #6 | 13 |
5 plants | 0.634 | Infection of the |
| # | ||||
| 7 | 13 |
5 plants | 0.404 | Resistance to |
| #8 | 7 |
5 plants | 0.429 | Resistance to |
| #9 | 13 |
5 plants | 0.942 | Susceptibility to |
| #10 | 12 |
5 plants | 0.75 | Infection of the disease |
| #11 | 13 |
5 plants | 0.846 | Susceptibility to |
| #12 | 13 |
5 plants | 0.481 | Disease resistance |
| #13 | 13 |
5 plants | 0.923 | Susceptibility to |
| #14 | 20 |
5 plants | 0.363 | Disease resistance |
Note: in order to ensure the consistency of the comparative tests, the potato plants using the petiole grafting disease technique shown in the test results of this time were the same batch of test material as the potato plants using the main stem injection technique shown in table 2. Multiple tests show that the leaf stalk grafting disease technology in the identification method provided by the invention is simple and convenient to operate, the result is stable, and the planting number of the grafted plants and the blank control can be respectively reduced to 5 plants and 3 plants. When sampling, mature and healthy petioles of 4 th leaf to 6 th leaf can be selected for each test material for inoculation identification.
Evaluation result verification and comparison test
Under the same other conditions, the traditional main stem injection technology and the identification method provided by the invention are adopted to carry out a comparative verification test. Specifically, compared with the identification method provided by the embodiment of the present invention, the main differences of the traditional main stem injection technology are as follows:
a. main stem injection inoculation culture: respectively puncturing the main stems of 4 weeks after sprouting with a disposable sterile syringe needle, sucking 10 μ L of potato with concentration of 1.5 × 108The method comprises the following steps of (1) inoculating CFU/mL pathogenic bacteria suspension to a puncture part, and inoculating each main stem of the potato at 5 different sites with the distance of about 3 cm; after inoculation, the inoculated part is covered with a preservative film for 12h to keep the humidity so as to stabilize the morbidity, and 10 mu L of MES solution is used for inoculation after the plants of the blank control group are punctured.
b. Evaluating and identifying by using the traditional main stem injection technology: taking the susceptible material # 3 as susceptible control, taking the disease-resistant material # 12 as disease-resistant control, pulling out potato plants with roots after 10-15 days, longitudinally cutting the main stem part close to the ground, observing the color of vascular bundle, wherein the main stem section of the plants exceeding the injection inoculation number of 2/3 is black as susceptible material, the main stem section of the plants lower than the injection inoculation number of 1/3 is black as disease-resistant material, and the main stem section exceeding the injection inoculation number of 1/2 but not exceeding 2/3 is black as susceptible material? A material; according to the above criteria, as shown in table 3, the evaluation results of the blackleg resistance of 14 potato materials revealed that? Materials, #1, #2, #3, #6, #9, #10, #11, #13 are susceptible materials.
TABLE 314 Black shank resistance rating of potato materials using conventional king stalk injection
| Test materials | Infected plant | Blank control | Main stem section plane | Evaluation of |
| # | ||||
| 1 | 13 |
5 |
10/13 black color | Infection of the |
| # | ||||
| 2 | 10 strains of Bacillus subtilis | 5 |
6/10 black color | Infection of the |
| # | ||||
| 3 | 15 |
5 plants | 13/15 black color | Infection of the |
| # | ||||
| 4 | 15 |
5 |
3/15 black color | |
| # | ||||
| 5 | 10 strains of Bacillus subtilis | 5 |
5/10 black color | Is the disease affected? |
| #6 | 13 |
5 |
10/13 black color | Infection of the |
| # | ||||
| 7 | 13 |
5 |
4/10 black color | Disease resistance |
| #8 | 7 |
5 |
3/7 black color | Is the disease affected? |
| #9 | 13 |
5 plants | 13/13 black color | Infection of the |
| # | ||||
| 10 | 12 |
5 plants | 11/12 black color | Infection of the disease |
| #11 | 13 |
5 |
12/13 black color | Infection of the |
| # | ||||
| 12 | 13 |
5 |
2/13 black color | Disease resistance |
| #13 | 13 |
5 plants | 11/13 black color | Infection of the disease |
| #14 | 20 |
5 |
4/20 black color | Disease resistance |
It can be seen from tables 2 and 3 that the morbidity of petiole inoculation in the identification method provided by the invention is basically consistent with the morbidity of field stem injection of the same material, but the field stem injection material has the problem of long identification period, and besides, the rot symptom which is difficult to observe by naked eyes or the morbidity of the field stem injection material is unstable in the disease-resistant identification process, so that the qualitative grading can not be realized, the analysis result is not accurate, and the identification material result is unclear. In addition, the identification method provided by the invention adopts a petiole in-vitro culture method, and can avoid the problems of soil pollution and the like caused by a field main stem injection method.
Finally, it should be noted that: the above examples are only intended to illustrate the technical solution of the present invention and not to limit it; although the present invention has been described in detail with reference to preferred embodiments, those skilled in the art will understand that: modifications to the specific embodiments of the invention or equivalent substitutions for parts of the technical features may be made; without departing from the spirit of the present invention, it is intended to cover all aspects of the invention as defined by the appended claims.
Claims (8)
1. A method of identifying potato black shank resistance comprising:
preparing the suspension to provide a concentration of 103CFU/mL of potato phytophthora parasitica suspension;
screening a potato plant with 10 completely-unfolded leaves from a leaf stalk to be detected, selecting the leaf stalk of the 4 th to 6 th mature healthy leaves of the potato plant as the leaf stalk to be detected, wherein the length of the leaf stalk to be detected is L0；
Inoculating and observing, and pouring the potato black shank pathogenic bacteria suspension into a tissue culture bottle; inserting a petiole to be detected into the dry flower mud, placing the petiole into the tissue culture bottle containing the potato black shank pathogen suspension, covering the tissue culture bottle with a bottle cap, and placing the tissue culture bottle into an illumination incubator with the temperature of 24-26 ℃ and the light of 16 h/8 h for culture and observation;
resistance identification is realized by observing and recording softened water stain symptom of the petiole to be detected by using a light box and an incandescent lamp, measuring the water stain lesion length of the petiole to be detected, and taking the lesion length of the petiole to be detected after being inoculated and cultured for 36 hours as the final lesion length L of the petioleDisease and illnessGrading the diseases according to a grading standard and calculating disease indexes; evaluating and identifying the resistance of the potato black shank according to the disease index: the disease index is less than or equal to 0.2, the disease index is resistant, the disease index is more than 0.4 and less than or equal to 0.6, the disease index is susceptible, and the disease index is more than 0.8 and is susceptible; wherein, the calculation formula of the disease index is as follows:
the disease grading standard is as follows:
lesion length L is more than or equal to 0Disease and illness＜0.08L0The disease grade is set as 0 grade;
0.08L0less than or equal to the length L of the lesionDisease and illness＜0.15L0The disease grade is set as 1 grade;
0.15L0less than or equal to the length L of the lesionDisease and illness＜0.3L0The disease grade is set as 2 grade;
0.3L0less than or equal to the length L of the lesionDisease and illness＜0.6L0The disease grade is set as 3 grade;
0.6L0less than or equal to the length L of the lesionDisease and illness＜L0And the disease grade is set as 4.
2. The method for identifying potato phytophthora parasitica resistance according to claim 1, wherein the step of screening the petioles to be tested comprises: selecting potato plants which grow for 5-6 weeks after field planting or potato plants which grow for 3-4 weeks after budding and are planted with tubers, wherein the potato plants have 10 completely-unfolded leaves, taking the petioles of 4-6 mature and healthy leaves, cutting off the redundant leaves around the petioles, and cutting the petioles to uniform length L0To serve as the petiole to be measured.
3. The method for identifying potato phytophthora parasitica resistance as claimed in claim 2, wherein the length L of the petiole to be tested is0Is 4-6.5 cm.
4. The method of identifying potato phytophthora parasitica resistance according to claim 1, 2 or 3, wherein the step of inoculating the culture comprises: placing the potato black shank pathogen suspension into the tissue culture bottle; firstly, inserting a plurality of leafstalks to be detected of materials to be detected with the same genetic background into dry flower mud, then placing the leafstalks to be detected into the tissue culture bottle containing the potato black shank pathogen suspension, and covering the bottle cap of the tissue culture bottle; and (3) putting the tissue culture bottle covered with the bottle cap into an illumination incubator at 25 ℃ and 16h light/8 h dark for culture and observation.
5. The method for identifying the resistance of potato to phytophthora parasitica according to claim 4, wherein 3 to 6 petioles to be tested are placed in the same tissue culture bottle.
6. The method of identifying potato phytophthora parasitica resistance according to claim 5, wherein the step of identifying the resistance comprises: irradiating the tissue culture bottle in the illumination incubator by using a light source at the same time interval, observing the symptoms of softened water staining of the petiole by naked eyes, measuring the length of water-stained lesion of the petiole by using a ruler, photographing and recording, and taking the length of the lesion of the petiole to be measured after being inoculated and cultured for 36 hours as the final lesion length LDisease and illnessGrading according to the disease grading standard, calculating the disease index, and evaluating and identifying the resistance of the potato black shank according to the disease index.
7. The method for identifying potato phytophthora parasitica resistance according to claim 6, wherein the length L of the petiole to be tested0The disease grading standard is 6.5cm and is as follows:
lesion length L is more than or equal to 0Disease and illnessLess than 0.5cm, and the disease grade is set to 0 grade;
lesion length L is less than or equal to 0.5cmDisease and illnessIf the size is less than 1cm, the disease grade is set to be 1 grade;
lesion length L is less than or equal to 1cmDisease and illnessIf the size is less than 2cm, the disease grade is set to be 2 grade;
lesion length L is less than or equal to 2cmDisease and illnessIf the size is less than 4cm, the disease grade is set to be 3 grade;
lesion length L is less than or equal to 4cmDisease and illnessLess than 6.5cm, and the disease grade is set to 4.
8. The method of identifying potato phytophthora parasitica according to claim 4, wherein the step of preparing the suspension comprises: carrying out whole dish streak culture on potato black shank pathogenic bacteria on a TSA (TSA-derived food transfer) plate culture medium, and diluting to obtain OD (origin-destination) bacteria600The concentration corresponding to the value is 103CFU/mL of the potato phytophthora parasitica suspension.
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| CN114451163A (en) * | 2022-01-18 | 2022-05-10 | 河北农业大学 | Walnut black spot pathogen infection test method |
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| 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 |
| CN114451163A (en) * | 2022-01-18 | 2022-05-10 | 河北农业大学 | Walnut black spot pathogen infection test method |
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