CN111436343A - Bacterial wilt identification method - Google Patents
Bacterial wilt identification method Download PDFInfo
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- CN111436343A CN111436343A CN201911314819.4A CN201911314819A CN111436343A CN 111436343 A CN111436343 A CN 111436343A CN 201911314819 A CN201911314819 A CN 201911314819A CN 111436343 A CN111436343 A CN 111436343A
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01G—HORTICULTURE; CULTIVATION OF VEGETABLES, FLOWERS, RICE, FRUIT, VINES, HOPS OR SEAWEED; FORESTRY; WATERING
- A01G22/00—Cultivation of specific crops or plants not otherwise provided for
- A01G22/45—Tobacco
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/02—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving viable microorganisms
- C12Q1/18—Testing for antimicrobial activity of a material
Abstract
The invention belongs to the technical field of crop disease resistance identification, and discloses a bacterial wilt identification method which comprises the steps of manufacturing a dense inoculation disc, installing a heating temperature control device, preparing bacterial liquid, treating tobacco seedlings, inoculating the bacterial liquid, investigating and grading the disease condition and the like. The method uses the seedling stage identification result to replace the traditional field identification result, identifies a large amount of materials in a small space, improves the identification efficiency and the reliability of the result, shortens the identification period, saves the field and the test cost, has the characteristics of rapidness, accuracy, stability, high efficiency and the like, can effectively eliminate the influence of natural environmental conditions, is favorable for the reproduction and comparison of the results between years and different laboratories, and has important significance for the screening of disease-resistant germplasm resources, the research of resistance genetic characteristics and the disease-resistant breeding.
Description
Technical Field
The invention belongs to the technical field of crop disease resistance identification, and particularly relates to a bacterial wilt identification method.
Background
Currently, the closest prior art:
the tobacco bacterial wilt is a soil-borne disease mainly invaded from the root caused by Ralstonia solanacearum, commonly occurs in tobacco areas in south China, and has a tendency of spreading to the north in recent years, so that the tobacco bacterial wilt is a large destructive disease threatening tobacco production in China. The breeding and planting of disease-resistant varieties is an economic and effective prevention and treatment measure for controlling the occurrence and prevalence of bacterial wilt. However, in the research related to breeding for disease resistance, the resistance performance of a large amount of materials needs to be accurately and rapidly evaluated.
At present, the identification of the bacterial wilt resistance of tobacco varieties is mainly carried out by a method of field natural condition identification and artificial seedling stage inoculation identification. The identification of natural conditions in fields is influenced by natural environmental conditions, the disease degree of bacterial wilt in different areas and different years is greatly different, the identification period is long, the requirement on required test conditions is high, and the consumption of manpower and material resources is high.
The artificial seedling stage inoculation identification can better solve the problems existing in the field natural condition identification, and the requirement of quickly, accurately, stably and efficiently identifying the bacterial wilt resistant varieties is met by using the lowest cost. However, the existing artificial seedling stage inoculation identification method for tobacco bacterial wilt usually has certain problems, such as difficult control of disease onset temperature, complicated inoculation steps and low inoculation efficiency, so that the final identification effect is not ideal enough.
Therefore, a simple and efficient rapid identification method is urgently needed in the identification of the resistance to the tobacco bacterial wilt.
In summary, the problems of the prior art are as follows:
(1) in the prior art, the inoculation identification of a natural disease garden in a field is easily influenced by environment and other pathogenic bacteria, and the accuracy of a resistance identification result is directly influenced.
(2) In the prior art, the inoculation condition of a field natural disease garden for inoculation also has the problems of uneven distribution of pathogenic bacteria and the like, so that the phenomenon of uneven bacterial wilt disease occurrence is caused, and the reliability of an identification result is influenced.
(3) In the prior art, common greenhouse artificial seedling inoculation is directly used for inoculating bacteria in soil, the soil temperature is difficult to control, and the reliability of a resistance identification result cannot be ensured.
(4) In the prior art, the greenhouse identification period is long, the number of tobacco seedlings is small in one-time identification, and the identification efficiency is low.
The difficulty of solving the technical problems is as follows: the pathogenic bacteria disease temperature of the technical problems is controlled, and the identification efficiency and the identification accuracy are improved with certain difficulty.
The significance of solving the technical problems is as follows: the method for quickly identifying the tobacco bacterial wilt resistance has important significance for screening of disease-resistant germplasm resources, research of resistance genetic characteristics and disease-resistant breeding.
Disclosure of Invention
Aiming at the problems in the prior art, the invention provides a bacterial wilt identification method.
The invention is realized in such a way, and the bacterial wilt identification method comprises the following steps:
firstly, manufacturing a dense inoculation tray, fixing plastic centrifuge tubes in a tray, wherein a space of 1-1.5cm is reserved between every two rows of small tubes, and a space for placing a heating temperature control device is reserved.
And step two, cleaning and damaging the roots of the tobacco seedlings, selecting a plurality of plants of the materials to be identified, cleaning soil at the roots of the plants and cutting one third of the roots for standby application when the tobacco seedlings grow to the plant height of 10-12cm and the seedling age of 55-60 d.
Step three, preparing bacterial liquid, namely activating the ralstonia solanacearum strain stored at the temperature of-80 ℃ on a TTC culture medium for 48 hours before use, selecting a typical single bacterial colony to an NB culture solution, performing shake culture at the temperature of 28 ℃ for 36 hours, and performing OD (optical density) culture on the bacterial liquid600Diluting the bacteria liquid to 1.5-1.8 × 10 with clear water8cfu/mL。
And step four, inoculating the plantlets, placing the treated plantlets in a centrifugal tube, filling diluted bacteria liquid, heating water contained in a tray, and keeping the water temperature and the bacteria liquid temperature at 28.0-30.0 ℃.
And step five, disease condition investigation and grading, after inoculation, investigating the disease condition every 2d, recording the disease rates of different varieties, and taking the result when the disease control disease rate is stable as a variety resistance evaluation standard.
Further, in step one, 150 plastic centrifuge tubes of 50ml were mounted in a 62 × 42 × 9.5.5 cm plastic tray using hot melt glue.
A space of 10 × 10 and 10 × 9.5.5 cm is reserved for placing a heating temperature control device.
Further, in the fourth step, the treated plantlets are placed in a centrifuge tube, three plantlets are placed in each centrifuge tube, and 45ml of diluted bacteria liquid is poured into the centrifuge tube. The white tray is filled with clear water for heating the bacterial liquid in water bath, and the water temperature and the bacterial liquid temperature are kept within the range of 28.0-30.0 ℃.
Further, in the fifth step, the disease severity grading standard refers to a CORESTA method:
level 0: the whole plant is disease-free.
Level 1: the stem part has chlorosis stripe no more than one third of the plant height, or only one leaf turns yellow or withers.
And 2, stage: more than 2 leaves turned yellow or withered and more than 3 healthy leaves, or the stem part has a chlorotic stripe at the height of 1/3-1/2.
And 3, level: 1-3 leaves of jianye, or stem chlorosis stripe at the height of 1/2-2/3.
4, level: the plants are basically withered, the stem parts have the chlorosis and the leaf spots are higher than those of 2/3 plants, and healthy leaves are not generated.
Further, in the fifth step, in the resistance evaluation standard, the relative disease indexes of high resistance HR, disease resistance R, medium resistance MR, medium susceptibility MS, medium susceptibility S and high susceptibility HS are respectively 0, 0.1-20, 20.1-40, 40.1-60, 60.1-80 and 80.1-100.
The invention also aims to provide a bacterial wilt identification device for implementing the bacterial wilt identification method.
In summary, the advantages and positive effects of the invention are:
the method replaces the traditional field natural condition identification with the tobacco seedling-stage wounded root soaking constant-temperature water bath inoculation identification, eliminates the influence of the natural environment on the identification result, and obviously improves the efficiency of identifying the bacterial wilt resistance of tobacco varieties.
Compared with the field with difficult control of natural conditions and high cost of a constant temperature greenhouse or an artificial climate chamber, the invention realizes the water bath temperature control of the bacterial liquid by the heating temperature control device and the self-made intensive inoculation disc device, and solves the temperature problem of bacterial wilt attack at lower cost.
Compared with the common artificial inoculation in the seedling stage, the direct inoculation in the soil is difficult to control the soil temperature, the root-injury soaking method is easier to control the temperature condition of the disease, and the invasion of pathogenic bacteria from the root is similar to the natural infection process in the disease field, thereby ensuring the reliability of the resistance identification result.
The invention only needs about 12d for one identification periodOne set of device only occupies less than 0.3m2The 450 tobacco seedlings can be identified at one time on the area, so that the identification efficiency is greatly improved. Meanwhile, each tube has no influence on each other, so that the accuracy of the test is ensured.
Taking the disease-resistant variety of 'Weidi No. three-C' and the susceptible variety of Honghuadajinyuan as examples, the comparative analysis of the identification of the natural disease nursery of the field and the identification of the invention is as follows:
the comparative analysis of the field identification and the invention shows that under the same resistance identification result, the invention shortens 111d and reduces 79.76% of the cost when used for identifying a larger field, and the identification method of the invention saves the identification cost while obviously improving the inoculation identification efficiency; the phenomenon of uneven disease of plants caused by uneven distribution of pathogenic bacteria in field inoculation identification is also avoided, and the identification accuracy is improved.
The comparison of field identification and identification effects of the invention is shown in fig. 2, which is a graph comparing the effects of field identification and identification effects of the invention provided by the embodiments of the present invention.
In the figure: (a) the field identification effect is shown in the effect graph of 'Weidi No. three-C' (disease-resistant variety); (b) the effect intention of Honghuadajinyuan (high-sense variety) in the field identification effect; (c) the effect of the invention is shown in the effect graph of 'Weidi No. three-C' (disease-resistant variety); (d) the effect of the invention is the effect intention of Honghuadajinyuan (high-sense variety).
Drawings
FIG. 1 is a flow chart of a bacterial wilt identification method provided by the embodiment of the invention.
FIG. 2 is a graph showing the comparison between the field identification and the identification effect of the present invention.
In the figure: (a) the field identification effect is shown in the effect graph of 'Weidi No. three-C' (disease-resistant variety); (b) the effect intention of Honghuadajinyuan (high-sense variety) in the field identification effect; (c) the effect of the invention is shown in the effect graph of 'Weidi No. three-C' (disease-resistant variety); (d) the effect of the invention is the effect intention of Honghuadajinyuan (high-sense variety).
Detailed Description
In order to make the objects, technical solutions and advantages of the present invention more apparent, the present invention is further described in detail with reference to the following embodiments. It should be understood that the specific embodiments described herein are merely illustrative of the invention and are not intended to limit the invention.
In the prior art, the natural conditions of the field are difficult to control and keep the temperature constant, and the cost of a greenhouse or a phytotron is high. The common artificial seedling inoculation directly inoculates bacteria in soil, the soil temperature is difficult to control, and the reliability of a resistance identification result cannot be ensured. The prior art has long identification period and small quantity of tobacco seedlings identified at one time, so that the identification efficiency is low. Meanwhile, each tube is influenced mutually, and the accuracy of the test cannot be guaranteed.
Aiming at the problems in the prior art, the invention provides a method for identifying bacterial wilt, and the invention is described in detail below with reference to the accompanying drawings.
As shown in fig. 1, the method for identifying bacterial wilt provided by the embodiment of the invention comprises the following steps:
s101, manufacturing a dense inoculation tray, namely fixing 150 plastic centrifuge tubes of 50ml in a plastic tray of 62 × 42 × 9.5.5 cm by using hot melt adhesive, keeping attention to the fact that a space with the width of about 1-1.5cm is reserved between each row of small tubes, so that the bacterial liquid of each tube is heated uniformly, reserving a space of 10 × 10 × 9.5.5 cm for placing a heating temperature control device, filling clear water in the white tray for water bath heating of the bacterial liquid, wherein the water depth is 9cm, and arranging the heating temperature control device to keep the water temperature and the temperature of the bacterial liquid within the range of 28.0-30.0 ℃ according to the suitable disease temperature of bacterial wilt.
S102, tobacco seedling treatment and inoculation: after the tobacco seedlings are about 55-60d old and grow to about 10-12cm high, selecting a plurality of plants of the materials to be identified, cleaning root soil, cutting off one third of roots for standby, inserting three plants of the identification materials into a 50ml centrifugal tube in a group, distributing 15ml bacterial liquid to each seedling on average, and because the bacterial liquids in the centrifugal tubes are mutually separated, the resistance of the tobacco seedlings to different strains can be simultaneously identified, and the method can be repeated according to needs.
S103, preparing and inoculating bacterial liquid: the ralstonia solanacearum strain preserved at-80 ℃ is activated on a TTC culture medium for 48 hours before use,picking a typical single colony at the center in an ultraclean workbench to an NB culture solution, placing the NB culture solution in a shaking table for shake culture at 28 ℃ for 36h, and performing OD (optical density) culture according to a bacterial solution600Diluting the bacteria liquid to 1.5-1.8 × 10 with clear water8cfu/m L was ready for use.
S104, disease investigation and grading: the disease onset condition is investigated at 8 days after inoculation, and the disease onset condition is investigated every 2 days later, and the disease onset condition of 12d tobacco seedlings is stable generally. Recording the disease grade and the disease incidence of different varieties, and taking the result when the disease incidence of the susceptible contrast is stable as the evaluation standard of the resistance of the varieties. Disease severity grading standard reference CORESTA method: level 0: the whole plant is disease-free. Level 1: the stem part has chlorosis stripe no more than one third of the plant height, or only one leaf turns yellow or withers. And 2, stage: more than 2 leaves turned yellow or withered and more than 3 healthy leaves, or the stem part has a chlorotic stripe at the height of 1/3-1/2. And 3, level: 1-3 leaves of jianye, or stem chlorosis stripe at the height of 1/2-2/3. 4, level: the plants are basically withered, the stem parts have the chlorosis and the leaf spots are higher than those of 2/3 plants, and healthy leaves are not generated. Evaluation criteria for resistance: the relative disease indexes of High Resistance (HR), disease resistance (R), Medium Resistance (MR), Medium Susceptibility (MS), susceptible disease (S) and High Susceptibility (HS) are respectively 0, 0.1-20, 20.1-40, 40.1-60, 60.1-80 and 80.1-100.
Wherein the content of the first and second substances,
the invention is further described with reference to specific examples.
Example 1
The susceptible control varieties of Honghuadajinyuan and Cuibi I, the disease-resistant control varieties of Dendi III-C and Yanyan 97 are selected as test materials for inoculation identification. And putting three strains of each variety into a centrifuge tube as a bundle, setting two centrifuge tubes as one repetition, and simultaneously setting two same repetitions in other devices, so that 18 tobacco seedlings are identified for each variety. And (3) filling clear water with the height of 9cm into the tray, filling 45ml of diluted bacterium liquid A into the centrifugal tube, and placing a heating temperature control device in the reserved space. After 8 days, the disease grade and the disease incidence of different varieties can be investigated.
Example 2
254 portions of F inoculated with Honghuadajinyuan and 'Dadi' No. III-C2：3The rest of the procedure was the same as in example 1.
TABLE 1 inoculation of four anti-influenza control varieties to identify morbidity on different days (%)
As can be seen from Table 1, the disease rates of the four varieties are stable at 12d, so the investigation disease grade at 12d is more appropriate, and the resistance grades of the four varieties are basically consistent with the expected results, namely the resistance grades of Honghuadajinyuan, Cuibi I, Shuidi III-C and Yanyan 97 are HS, S, R and R respectively.
TABLE 2254 parts of F2：3Distribution number of variety resistance classification
From table 2, it can be seen that the infection resistance grade number of the filial line population of Honghuadajinyuan and Candidi No. III-C tends to be distributed in the infection direction, because the inoculation concentration may be too high or the inoculation amount may be too large, and the subsequent experiment can make the infection resistance grade number distribution of the filial line population substantially conform to the normal distribution by adjusting the bacterial liquid concentration and bacterial liquid amount.
The present invention will be further described with reference to effects.
The method uses the seedling stage identification result to replace the traditional field identification result, identifies a large amount of materials in a small space, improves the identification efficiency and the reliability of the result, shortens the identification period, saves the field and the test cost, has the characteristics of rapidness, accuracy, stability, high efficiency and the like, can effectively eliminate the influence of natural environmental conditions, is favorable for the reproduction and comparison of the results between years and different laboratories, and has important significance for the screening of disease-resistant germplasm resources, the research of resistance genetic characteristics and the disease-resistant breeding.
The invention is further described below in connection with specific experiments.
The method utilizes the combination of rock smoke 97(P1) × safflower Hongjinyuan as a parent (P2) to construct F1 and F2 populations, analyzes the genetic effect of bacterial wilt resistance of the rock smoke 97, provides a basis for breeding new varieties of high-quality bacterial wilt resistant flue-cured tobacco, investigates and analyzes the bacterial wilt disease index of each single plant in 4 generation populations (P1, P2, F1 and F2) of the rock smoke 97 × and safflower Hongjinyuan 4, analyzes by DPS 9.5, the difference of the disease indexes of the P1 and P2 generations reaches a very significant level (P < 0.01), the disease index is higher than the average value of the parent, the F2 phenotypic distribution shows unimodal distribution, and considers the bacterial wilt resistance of the rock smoke 97 to be quantitative trait heredity.
Note: letters are marked as the significance result of the difference at the 0.01 level.
The method is used for screening out bacterial wilt resistant tobacco germplasm OX2028 (flue-cured tobacco), Dada grind (sun-cured tobacco), Shudisan-C (flue-cured tobacco) and N.debneyi (wild tobacco), and the germplasm can provide donor parents or gene resources for tobacco bacterial wilt resistant breeding for utilization; thereby widening the breeding parent source and laying a foundation for accelerating the bacterial wilt breeding process and improving the current condition of single bacterial wilt resistant variety.
As shown in FIG. 2, the effect of field identification provided by the embodiment of the present invention is compared with the effect of identification provided by the present invention. In the figure: (a) the field identification effect is shown in the effect graph of 'Weidi No. three-C' (disease-resistant variety); (b) the effect intention of Honghuadajinyuan (high-sense variety) in the field identification effect; (c) the effect of the invention is shown in the effect graph of 'Weidi No. three-C' (disease-resistant variety); (d) the effect of the invention is the effect intention of Honghuadajinyuan (high-sense variety).
The above description is only for the purpose of illustrating the preferred embodiments of the present invention and is not to be construed as limiting the invention, and any modifications, equivalents and improvements made within the spirit and principle of the present invention are intended to be included within the scope of the present invention.
Claims (6)
1. A bacterial wilt identification method is characterized by comprising the following steps:
firstly, manufacturing a dense inoculation plate, fixing plastic centrifuge tubes in a tray, wherein a space of 1-1.5cm is reserved between each row of small tubes, and a space for placing a heating temperature control device is reserved;
cleaning tobacco seedlings and damaging roots, selecting a plurality of plants of materials to be identified to clean root soil and cutting off one third of roots for standby when the tobacco seedlings grow to be 10-12cm high and 55-60d seedling age;
step three, preparing bacterial liquid, namely activating the ralstonia solanacearum strain stored at the temperature of-80 ℃ on a TTC culture medium for 48 hours before use, selecting a typical single bacterial colony to an NB culture solution, performing shake culture at the temperature of 28 ℃ for 36 hours, and performing OD (optical density) culture on the bacterial liquid600Diluting the bacteria liquid to 1.5-1.8 × 10 with clear water8cfu/mL；
Step four, inoculating the plantlets, placing the treated plantlets in a centrifugal tube, filling diluted bacteria liquid, heating water contained in a tray, and keeping the temperature of the water temperature and the bacteria liquid to be 28.0-30.0 ℃;
and step five, disease condition investigation and grading, after inoculation, investigating the disease condition every 2d, recording the disease rates of different varieties, and taking the result when the disease control disease rate is stable as a variety resistance evaluation standard.
2. The method for identifying bacterial wilt according to claim 1, wherein in step one, 150 plastic centrifuge tubes of 50ml are fixed in a plastic tray of 62 × 42 × 9.5.5 cm by using hot melt adhesive;
a space of 10 × 10 and 10 × 9.5.5 cm is reserved for placing a heating temperature control device.
3. The method for identifying bacterial wilt according to claim 1, wherein in step four, treated seedlings are placed in a centrifuge tube, three seedlings are placed in each tube, and 45ml of diluted bacterial liquid is poured in. The white tray is filled with clear water for heating the bacterial liquid in water bath, and the water temperature and the bacterial liquid temperature are kept within the range of 28.0-30.0 ℃.
4. The method for identifying bacterial wilt according to claim 1, wherein in step five, the disease severity grading standard refers to the CORESTA method:
level 0: the whole plant is disease-free;
level 1: the stem part has chlorosis and streak spots not more than one third of the plant height, or only one leaf turns yellow or withers;
and 2, stage: more than 2 leaves turn yellow or wither and more than 3 healthy leaves, or the stem part has a chlorotic stripe at the height of 1/3-1/2;
and 3, level: 1-3 healthy leaves or stem chlorotic stripe spots are arranged at the height of 1/2-2/3;
4, level: the plants are basically withered, the stem parts have the chlorosis and the leaf spots are higher than those of 2/3 plants, and healthy leaves are not generated.
5. The method for identifying bacterial wilt according to claim 1, wherein in the fifth step, the relative disease indexes of high resistance HR, resistance R, resistance MR, medium resistance MS, susceptible S and high sensitivity HS in the resistance evaluation criteria are 0, 0.1-20, 20.1-40, 40.1-60, 60.1-80 and 80.1-100 respectively;
6. a bacterial wilt identification apparatus for implementing the method for identifying bacterial wilt according to claim 1.
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