CN108739147B

CN108739147B - Method for creating clubroot-resistant Chinese cabbage germplasm

Info

Publication number: CN108739147B
Application number: CN201810607729.3A
Authority: CN
Inventors: 孙继峰; 韩太利; 徐立功; 谭金霞; 杨晓东
Original assignee: Shandong Weifang Academy of Agricultural Sciences
Current assignee: Shandong Weifang Academy of Agricultural Sciences
Priority date: 2018-06-13
Filing date: 2018-06-13
Publication date: 2020-09-01
Anticipated expiration: 2038-06-13
Also published as: CN108739147A

Abstract

The invention discloses a method for creating clubroot-resistant Chinese cabbage germplasm. The creation method of the invention comprises the following steps: 1) crossing with the special selfed progeny of Kanggen 51-1 as female parent and the special selfed progeny of Baogen Wang No. 1 as male parent to obtain hybrid progeny; carrying out clubroot resistance identification on the hybrid filial generation, and selecting the clubroot-resistant hybrid filial generation; 2) continuously selfing the filial generation of the clubroot-resistant hybrid for multiple generations to obtain a homozygous clubroot-resistant selfing line; 3) and (3) carrying out the club disease resistance identification of the full growth cycle on the homozygous club disease-resistant inbred line under the alkaline soil environment, and selecting the club disease-resistant inbred line as the club disease-resistant Chinese cabbage germplasm. The invention creates the resistant germplasm with broad-spectrum resistance by character polymerization of different resistant materials; preparing a safe and efficient soil improvement fertilizer; a special nursery garden for identifying the clubroot disease resistance is established; the monitoring of the whole growth cycle is realized, and the identification result of the clubroot resistance is more accurate.

Description

Method for creating clubroot-resistant Chinese cabbage germplasm

Technical Field

The invention belongs to the technical field of agriculture, relates to Chinese cabbage breeding, and particularly relates to a method for creating clubroot-resistant Chinese cabbage germplasm and a method for identifying clubroot resistance of Chinese cabbage.

Background

Clubroot is a worldwide disease of cruciferous plants caused by plasmodiophora brassicae. It was first discovered in 1737 on the west coast of the mediterranean region of the united kingdom and in the south europe, and has now spread worldwide. The susceptible plants include Chinese cabbage, caulis et folium Brassicae Capitatae, radix Brassicae Rapae, cauliflower, caulis et folium Brassicae Junceae, radix Raphani, and kohlrabi etc. In recent years, the incidence of the clubroot disease is continuously increased, and the direct result of the clubroot disease is that the yield of cruciferous plants is reduced in a large area, and the yield of vegetables is seriously influenced. Because the clubroot is dangerous and has large loss and difficult prevention and treatment, the research of the clubroot resistance is very important. Clubroot disease resistance and variety creation and screening are important contents in Chinese cabbage breeding.

Clubroot is a soil-borne disease, and after a plant suffers from the disease, the survival time of pathogenic bacteria in the soil can reach more than 8 years, so that the disease is extremely harmful. The plasmodiophora falcata is divided into a plurality of physiological races, and the domestic main pathogenic races comprise No. 2 physiological race, No. 4 physiological race and No. 7 physiological race. The plasmodiophora brassicae mainly infects roots of plants, and lumps are formed at the infected parts, so that the transportation of water and nutrient substances is hindered, the plants are wilted, the growth and development are hindered, and the yield is reduced or even the plants are completely harvested. At present, clubroot occurs in many places in China, the clubroot of Chinese cabbages in Yunnan, Sichuan and Shandong fields exceeds 65 percent of the planting area, the loss of partial field blocks exceeds 50 percent, the production of the Chinese cabbages is severely restricted, the disease occurrence range is continuously enlarged, and the harm is continuously aggravated. For the area with diseases, the method is no longer suitable for planting cruciferous crops, the economic loss is serious, the land pollution is also serious, and even the vegetable safety is endangered.

The method for preventing and controlling clubroot mainly comprises the following steps: the first is to improve the soil. The pH value of the soil is adjusted mainly by a method of adjusting the pH value of the soil, and the adjustment is usually carried out by using hydrated lime, acid fertilizer, calcium salt and the like. Although the hydrated lime, the acid fertilizer and the calcium salt have certain control effect, the control duration is not long, and the long-term use can change the normal structure of the soil, and the cost is higher, so the hydrated lime, the acid fertilizer and the calcium salt are not suitable for long-term use. Secondly, chemical prevention and cure. This is a commonly used control method. The commonly used control medicines at present comprise carbendazim, dacoming, fushuaide, chlorothalonil, quintozene, cyazofamid, fluazinam, thiophanate methyl, carbendazim and the like which can inhibit plasmodiophora. However, in consideration of human health and environmental safety, many countries have prohibited or limited the use of some pesticide components, such as methyl bromide, formaldehyde, ethylene dibromide, mercuric chloride, etc., and it is the highly toxic components, which can cure the clubroot, thus limiting the development process of special pesticides for the clubroot. And thirdly, biological control. Mainly there is the new bacterial of introduction to parasitize the plasmodiophora root germ, reduces plasmodiophora root quantity, and the reduction disease takes place, perhaps adopts the method of planting the bait plant, makes the germ concentrate on the bait plant, reduces the loss to main cultivar, but the biological fungus prevents can introducing new microorganism, changes soil flora structure, destroys original ecological balance, can lead to the emergence of new plant diseases and insect pests, and the bait plant can only the reduction loss of certain degree, and the disease takes place still seriously, and loses soil fertility. Fourthly, selecting disease-resistant varieties. The breeding of new disease-resistant varieties is the basis for preventing and treating clubroot. The method for breeding disease-resistant varieties is the most economical, safe and effective method. However, due to the evolution and regional distribution differences of the physiological races of the Chinese cabbage clubroot pathogenic bacteria, the disease-resistant materials also have regional differences and cannot simultaneously generate resistance to all the physiological races.

At present, there are two main methods for identifying clubroot resistance in Chinese cabbage breeding: one is to plant the material in the affected area, and identify the resistance and the plant performance in the whole period; but has the following defects: firstly, carrying out experiments in affected areas with environmental uncertainty such as climate and the like; secondly, the scientific research institutions in the non-affected areas need affected areas for field tests, the process is complicated, the material growth process is long, and real-time observation cannot be achieved in the period. The other method is laboratory identification, which mainly comprises an injection method, a root dipping method and a bacterial soil method, and the seedling stage is usually identified; the disadvantages are that: laboratory identification has great limitation on the growth of the Chinese cabbage, cannot simulate the growth conditions of the whole field, can only carry out seedling stage identification at present, and cannot effectively observe the whole growth cycle of the Chinese cabbage.

At present, some documents exist for the research on the clubroot of the Chinese cabbage, including the research on the identification of the clubroot resistance of the Chinese cabbage variety in 2011 of the modern agricultural science and technology of horticulture, five traditional research methods such as a soil culture method, an injection method, a bud soaking method, a root dipping method and an insertion method are adopted in the research, but in the local research of southern Fujian, only the seedling stage is identified in a laboratory, and the resistance of the mature stage of the Chinese cabbage cannot be identified. Journal of Hunan university of agriculture, 3: 40, 2014CN201110458726.6 discloses an identification method of clubroot resistance of Brassica plants, which comprises sowing seeds in grids of a freezing box, sowing 1 seed in each grid, arranging the freezing box in a porcelain dish, preparing a 1/2MS culture medium and smashed clubroot rhizomes of Brassica oleracea with pH of 5.5 and germ spore content of 1 × 10⁸Inoculating more than one bacterium/ml of bacterial liquid; injecting the inoculated bacterial liquid into a porcelain plate, placing the porcelain plate into an artificial climate box for culture, and identifying the resistance of the variety to be identified to clubroot according to the investigated disease index; the method is also limited to traditional laboratory methods and can only identify seedling stage resistance. CN201610248555.7 discloses a method for obtaining clubroot-resistant germplasm resources of non-clubroot Chinese cabbages, which transfers resistance genes of the non-clubroot Chinese cabbages into the non-clubroot cabbages through bud-stage hybridization and backcross transfer technology, is simple and easy to implement, eliminates biological risks caused by other modes such as transgenosis, genetic engineering technology and the like, but only provides a method for obtaining germplasm resources, wherein the related identification method is also only a seedling-stage artificial inoculation identification method, and only can identify seedling-stage resistance.

In conclusion, the existing method for identifying the clubroot resistance of the Chinese cabbage is complicated, long in time, low in efficiency, large in field experiment area limitation and low in accuracy rate; namely, the cabbage clubroot disease of the whole period can not be accurately identified in the northern alkaline earth environment.

Disclosure of Invention

The invention aims to provide a seed production method of a Chinese cabbage with an anti-clubroot disease.

The seed production method of the clubroot-resistant Chinese cabbage provided by the invention comprises the following steps:

1) crossing with the special selfed progeny of Kanggen 51-1 as female parent and the special selfed progeny of Baogen Wang No. 1 as male parent to obtain hybrid progeny; carrying out clubroot resistance identification on the hybrid filial generation, and selecting the clubroot-resistant hybrid filial generation;

2) selfing the clubroot-resistant hybrid progeny to obtain selfed progeny F1, performing clubroot resistance identification on the selfed progeny F1, and selecting clubroot-resistant selfed progeny F1; selfing the clubroot-resistant selfing progeny F1 continuously to obtain selfing progeny F2, carrying out clubroot resistance identification on the selfing progeny F2, and selecting clubroot-resistant selfing progeny F2; repeating the steps, and continuously selfing for multiple generations to obtain a homozygous clubroot-resistant selfing line;

3) and (3) carrying out clubroot resistance identification on the homozygous clubroot-resistant inbred line, and selecting the clubroot-resistant inbred line to obtain the clubroot-resistant Chinese cabbage germplasm.

In the above method, in the step 1), the preparation method of the special selfed progeny of the cabbages Kanggen 51-1 and the special selfed progeny of the cabbages Baogen Wang No. 1 comprises the following steps: selfing the variety Kanggen 51-1 of Chinese cabbage variety or Baogouwang No. 1 of Chinese cabbage variety to obtain selfed progeny F1, carrying out club disease resistance identification on the selfed progeny F1, and selecting club disease resistant selfed progeny F1; selfing the clubroot-resistant selfing progeny F1 continuously to obtain selfing progeny F2, carrying out clubroot resistance identification on the selfing progeny F2, selecting clubroot-resistant selfing progeny F2, repeating the steps, and selfing continuously for multiple generations to obtain a homozygous clubroot-resistant selfing line;

the variety of the Chinese cabbage Kanggen 51-1 is Kanggen 51-1 with greener outer leaves, white petioles, overlapped leaf balls, straight cylindrical leaf balls, light yellow inner leaves and longer big roots;

the variety of the Chinese cabbage Baogouwang No. 1 is a Baogouwang No. 1 plant which has greener outer leaves, white petioles, closed ball centers, dark yellow inner leaves and a small root line phenotype;

the selfing frequency can be specifically 7 times;

and identifying the clubroot resistance as the clubroot resistance in the seedling stage.

In the above method, in the step 2), the number of selfing may be specifically 7;

In the method, in the step 1) and the step 2), the method for identifying the resistance of the clubroot disease in the seedling stage comprises the steps of sowing seeds obtained by selfing in a 72-hole seedling tray filled with a matrix, when the plant grows to 2 true leaves, injuring the root at a position 1cm away from the plant by using a knife, and pouring 3ml of bacterial liquid of No. 7 physiological microspecies of the clubroot pathogen into the wounded root, wherein the spore concentration of the bacterial liquid is 2.5 × 10⁸Per ml; after the root is injured, the soil temperature is controlled to be above 25 ℃, the humidity is controlled to be 80% -90%, the pH is controlled to be 4.5-5.5, and the plant which is normal in overground part growth and has no root tumor at the root is selected as the clubroot disease resistant material.

In the above method, in the step 3), the club root disease resistance is identified as club root disease resistance for the whole growth cycle in an alkaline soil environment.

Further, the method for identifying clubroot resistance of the whole growth cycle in the alkaline soil environment comprises the following steps:

a, selecting an area with alkaline soil as a test area, wherein the test area is provided with an arched shed, and a nursery garden is arranged in the arched shed; the nursery is divided into three equal-area cells; vertically digging isolation ditches around the nursery garden and between each small area; a waterproof plate is arranged in the isolation trench;

step B, preparing clubroot bacterium solutions of No. 2, No. 4 and No. 7 physiological races respectively;

c, fertilizing the soil in the nursery garden to obtain the soil after fertilization;

step D, inoculating the plasmodiophora tumefaciens liquid of the No. 2, No. 4 and No. 7 physiological races into the fertilized soil in the three communities respectively to obtain inoculated soil;

step E, fertilizing the inoculated soil to obtain soil to be sown;

and F, sowing the Chinese cabbage seeds to be detected in the soil to be sown, culturing, investigating clubroot during the culture period, and identifying the clubroot resistance of the Chinese cabbage to be detected according to a Williams system.

In the step A, the test area is 15m wide and 60m long, and the pH of the soil in the test area is 8.1. The arched shed that the test area was equipped with is steel frame construction, and its span is 12m, and the minimum height is 1.8m, and the maximum height is 2.5m, and is 60m long.

A nursery garden is arranged in the arched shed, and the specification of the nursery garden is specifically 10.5m multiplied by 58.5 m; the nursery is divided into 3 equal-area cells which are respectively used for identifying clubroot of different physiological races. And vertically digging isolation ditches with the depth of 0.7m around the nursery garden and among the communities, wherein the waterproof board arranged in the isolation ditches is made of polyester fiber reinforced polyvinyl chloride (pvc), and the waterproof board is 0.85m in height and 0.5cm in thickness. And (4) digging the isolation ditch downwards, backfilling soil into the isolation ditch to be 20cm lower than the ground, and then bricking to be 15cm higher than the ground to serve as isolation ridges and corridors.

A tool room and a disinfection pool are arranged in the arched shed, and the tools must pass through the disinfection pool when entering a nursery; the length of the disinfection pool is 1m, the width is 0.5m, the height is 0.15m, quicklime is arranged in the disinfection pool, the thickness of the quicklime is 0.08m, and the disinfection pool can disinfect soles of people who get in and out. The arched shed is covered by an insect-proof net, and the mesh size of the insect-proof net can be 40 meshes.

In order to effectively prevent the salinization of the soil, increase the soil fertility and stabilize the soil environment, the step A further comprises the step of applying an organic fertilizer into the soil in the nursery, wherein the application amount of the organic fertilizer is 2000kg, and the pH value of the soil is adjusted to be 5.0. The organic fertilizer is a product obtained by fermenting residues and vinasse obtained after starch is extracted from corn as main raw materials; the mass ratio of the residues to the vinasse is 2: 1. The specific preparation method of the organic fertilizer comprises the following steps: spraying water to residues obtained after starch extraction of corn, covering a plastic film, keeping the residues at a high temperature and high humidity state for 3 days, adding sucrose, lactic acid bacteria, saccharomycetes, bacillus and acetic acid (the mass ratio of the residues, the sucrose, the lactic acid bacteria, the saccharomycetes, the bacillus and the acetic acid is 1500:10:2:2:1:2), uniformly mixing, transferring into a fermentation tank, turning over every 2 days, mixing the fermented corn residues and bean pulp according to the mass ratio of 10:1 after 20 days, adding 1kg of urea every 2 cubic meters after mixing, spraying 1kg of EM (effective microorganisms) bacteria starter, pushing into the fermentation tank, keeping the temperature of the stack to 65 ℃ for 12 hours, turning over the stack, turning over every 2 days later, turning over every week when the temperature is reduced to 55 ℃, finishing fermentation when the temperature reaches room temperature, and preparing the special organic fertilizer.

In the step B, the concentration of the clubroot bacterium liquid is 2.5 × 10⁸L^-1(ii) a The preparation method of the clubroot bacterium liquid of the No. 2, No. 4 and No. 7 physiological races comprises the following steps:

B1) crushing Chinese cabbage root with clubroot by using a stirrer to obtain a mixture;

B2) filtering the mixture by 2 layers and 4 layers of gauze in sequence to obtain filtrate;

B3) centrifuging the filtrate at 4000r/min for 10min, removing the supernatant, and collecting the precipitate;

B4) suspending the precipitate with distilled water, and repeating the steps B2) -B3) for 3 times to obtain precipitate of plasmodiophora brassicae;

B5) diluting the obtained 3 strains with distilled water under microscope to obtain bacterial solutions of No. 2, No. 4, and No. 7 physiological microspecies with spore concentration of 2.5 × 10⁸L^-1。

And C, ploughing after the fertilization, and turning the fertilizer into a soil layer with the depth of 30 cm.

In the step D, the inoculation method is to spray 11mL of the solution with the concentration of 2.5 × 10 per square meter⁸L^-1The clubroot bacterium solution.

And E, after the fertilization, the step E of raking the fertilizer into a soil layer of 8cm is further included.

The fertilizer applied in the step C and the step E is obtained by uniformly mixing the matrix, the rice hull manure, the nitrogen-phosphorus-potassium compound fertilizer (the mass percentage content is 25%) and urea; the mixture ratio of the substrate, the rice hull manure, the nitrogen-phosphorus-potassium compound fertilizer and the urea is 3m³：4m³: (40-50) kg: 15 kg. The matrix is prepared from radix Codonopsis, bagasse, semen Ricini, crushed corn stalk, mushroom residue, fructus Lycii, chicken manure and Zaocys by fermenting. The specific preparation method of the matrix is as follows: radix Codonopsis, bagasse, semen Ricini, and crushed semen MaydisRespectively pulverizing stem, mushroom residue, fructus Lycii, chicken manure and Zaocys, mixing at volume ratio of 1:2:2:1:1:2:1:2, air drying until water content is 60%, and drying at a rate of 1kg/m³Spraying EM (effective microorganisms) starter, pushing into a fermentation tank for fermentation after spraying, keeping for 12 hours when the temperature of a pile reaches 65 ℃, turning over the pile once every 2 days, turning over once every week when the temperature of the pile is reduced to 55 ℃, and finishing the fermentation when the temperature is reduced to room temperature. And the mass ratio of the fertilizer application amount in the step C to the fertilizer application amount in the step E is 6: 4.

And a step of ridging and ridging is further included between the step E and the step F.

In the step F, the sowing time is 8 ten days in the middle of the month, the row spacing of the sowing is 40cm, hole sowing is adopted in the sowing mode, the hole depth is 1.5cm, the hole spacing is 35-40 cm, 3-5 grains are planted in each hole, the soil is covered for 1.5cm, the step of scattering 1 layer of rice hulls or wheat bran on the holes for moisturizing is further included after the soil is covered, and irrigation is carried out in a spray irrigation mode after seedling emergence. Thinning is needed for 2 times in the seedling period of Chinese cabbage planting. The 1 st thinning time is about 5 days after seedling emergence, and the 2 nd thinning time is when the 4 th true leaf grows.

The method for supplementing the bacteria comprises the step of spraying the clubroot bacteria liquid to the root part of the Chinese cabbage by using a spray can, wherein the spray amount of the clubroot bacteria liquid is about 2ml per Chinese cabbage, and the concentration of the bacteria liquid is 2.5 × 10⁸L^-1And immediately watering after spraying the bacteria. The time and the quantity of the strain supplementation are important links for inducing clubroot, the strain supplementation is late, the quantity is small, the material is easy to cause difficult disease attack, and false resistance is generated; the early and large amount of the supplementary bacteria easily cause the infection of resistant materials or varieties, and the materials are discarded. At the moment, the fungus supplementation can not only cause the disease of the infected material, but also ensure that the resistant material is not infected. In practical application, the pathogenicity of No. 2 and No. 7 physiological races cannot be continued for 50 days, and the bacterium solution needs to be supplemented within 50 days after sowing, and the pathogenicity of No. 4 physiological races cannot be continued for 60 days, and the bacterium solution needs to be supplemented within 60 days after sowing. In the present invention, the fungus supplementation is carried out 15 days after the sowing.

The culture process also comprises the steps of conventional irrigation, additional fertilization and pest control. The method can be specifically carried out as follows:

F1) and (3) germination period: watering for the 1 st time during seeding, watering for the 2 nd time when seedlings push up soil, and watering for the 3 rd time when seedlings are discharged completely. Additional fertilizer is not generally needed in the germination period.

F2) And (3) seedling stage: watering for 4 th and 5 th times during thinning and final singling, wherein the watering times in the seedling stage are determined according to the weather and the concrete conditions of soil, the drought precipitation is small, once the furrows have a dry cracking phenomenon, the watering times are increased on the basis of no water, the watering and furrowing can be combined to apply urea or ammonium sulfate as a seedling raising fertilizer, and each 667m of the fertilizer is used²The dosage is 5kg, or the plant extract is prepared from LIANGFENGSU (containing P)₂O₅51％、K₂O₃4%) water-soluble fertilizer, etc. 100g is diluted by 300 times and applied to the leaf surface.

F3) A lotus throne stage: watering the lotus plumule for 1 time in the initial stage, then deep cultivating for 1 time, controlling water and squatting for about 10 days. The water is poured in small water service, and each 667m of water can be poured in combination²And applying 10-15 kg of ammonium sulfate or 10kg of ternary compound fertilizer.

F4) And (3) nodulation stage: from the rosette stage to the heading stage, the ridge surface can not crack, otherwise the side root can be broken, and the yield of the Chinese cabbage is influenced. And in drought weather, watering for 1 time every 5-6 days. The materials (Chinese cabbage) in the nursery are mainly used for identifying the clubroot resistance of the variety, so that the main pests are aphids, cabbage caterpillars and the like, and the 4.5 percent beta-cypermethrin solution with 1000-1500 times is used for preventing and treating.

It is another object of the present invention to provide the above novel use.

The invention provides application of the method in Chinese cabbage breeding.

It is still another object of the present invention to provide the above method for identifying clubroot resistance in a full growth cycle in an alkaline soil environment.

The application of the method for identifying the clubroot resistance in the whole growth cycle in the alkaline soil environment in the breeding of the clubroot-resistant Chinese cabbage also belongs to the protection scope of the invention.

The invention provides a method for creating a Chinese cabbage germplasm capable of resisting clubroot and a method for identifying clubroot resistance of the Chinese cabbage. According to the invention, Chinese cabbage germplasm with broad-spectrum resistance is created by character polymerization of different resistant materials and a method for identifying clubroot disease resistance by combining with a nursery. The method for identifying the clubroot resistance of the Chinese cabbage by the nursery garden overcomes the defect that the identification of the clubroot resistance of the Chinese cabbage in the whole growth cycle cannot be carried out in the alkaline soil environment in the prior art, realizes the real-time observation of the whole growth cycle of the identification of the clubroot resistance of the Chinese cabbage in the alkaline soil environment, can simultaneously identify the clubroot resistance of 3 physiological races, improves the accuracy and the identification efficiency of identification results, and lays a foundation for the breeding of clubroot-resistant Chinese cabbage varieties.

Detailed Description

The experimental procedures used in the following examples are all conventional procedures unless otherwise specified.

Materials, reagents and the like used in the following examples are commercially available unless otherwise specified.

In the quantitative tests in the following examples, three replicates were set up and the results averaged.

Example 1 Breeding method of clubroot-resistant Chinese cabbage

First, experimental material

In 1999, a CR (clubroot disease) -resistant chinese cabbage variety "kang gen 51-1" was introduced from korea; in 2000, the CR (clubroot disease) -resistant chinese cabbage "baogen wang No. 1" was introduced from japan. The Chinese cabbage varieties "kang Gen 51-1" and "Bao Gen Wang 1" are all described in the literature "molecular marker identification of gene loci CRa and CRb of clubroot resistance of Chinese cabbage [ J ]. North China agronomy, 2015,30(02): 87-92", of Yangfang City agricultural academy of sciences ".

Method for breeding Chinese cabbage with root swelling disease resistance

1. Screening and storing seed plants in field

(1) Planting each material in the same year of introduction, and screening field plants. Selecting 51-1 plants with greener outer leaves, white petioles, overlapped leaf balls, straight cylindrical leaf balls, light yellow inner leaves and longer big roots as 51-1 plants of Kanggen; the Baoguanwang No. 1 plant with greener outer leaf, white petiole, close shot shell shape of leaf ball, closed ball center, dark yellow inner leaf and small root system is selected to be marked by inserting rods to be used as the Baoguang No. 1 plant.

(2) Digging out the marked seed plants according to the root area of 10cm multiplied by 10 cm; cutting off outer leaves of the seed plants by a cutter to expose leaf balls; removing 40-60% of the upper part of the leaf ball to expose the tender leaf of the leaf ball, and inserting a label mark.

(3) Deep digging a deep groove with the length of 20m, the width of 1.2m and the height of 1.2m in an idle field area to be used as a storage groove for Chinese cabbage seed plants; the selected seed plants are placed in a deep groove and covered with a straw curtain and a plastic film.

2. Homozygous clubroot-resistant material prepared by seed plant selfing

In the spring of the next year, single plants of the stored Kanggen 51-1 plant and Baoguanwang No. 1 plant are respectively taken out and planted in open field, after blooming, the single plants are manually stripped and pollinated to carry out material selfing, selfing progeny F1 is obtained, the seeds of the harvested selfing progeny F1 are sowed in a clubroot identification nursery in the same year in autumn to carry out seedling-stage clubroot resistance identification, and a clubroot-resistant material is obtained by screening; selfing the obtained clubroot-resistant material continuously to obtain selfed progeny F2, sowing the seeds of the harvested selfed progeny F2 in a clubroot identification nursery in autumn in the same year to perform seedling-stage clubroot resistance identification, and screening to obtain the clubroot-resistant material; repeating the steps, selfing for 7 generations, and finally screening to obtain the homozygous clubroot-resistant material.

The homozygous clubroot-resistant material obtained by selfing Kanggen 51-1 is recorded as a VB-KG series selfing line, and the phenotype is as follows: the external leaf is greener, the petiole is white, the leaf ball is folded and embraced, the leaf ball is straight and cylindrical, the internal leaf is light yellow, the big root is longer, and the root swelling disease is resisted. Finally obtaining 4 VB-KG type inbred lines.

The homozygous clubroot-resistant material obtained by selfing Baoguanwang No. 1 is recorded as a VB-BG series selfing line, and the phenotype is as follows: the outer leaf is greener, the petiole is white, the leaf ball is in a shape close to a cannonball, the ball center is closed, the inner leaf is dark yellow, the root system is small, and the clubroot disease is resisted. Finally 8 VB-BG type inbred lines are obtained.

The method for identifying the resistance of the clubroot at the seedling stage comprises the following steps: sowing seeds obtained by selfing in containersWhen the plant grows to 2 true leaves, the root of the plant is wounded by a knife at a position 1cm away from the plant, and 3ml of a bacterial liquid of a No. 7 physiological microspecies of plasmodiophora brassicae is poured into the wounded root, wherein the spore concentration of the bacterial liquid is 2.5 × 10⁸Per ml; after the root is damaged, the temperature of the soil environment for planting the Chinese cabbage is controlled to be above 25 ℃, the humidity is controlled to be 80-90%, and the pH is controlled to be 4.5-5.5. Selecting plants with normal overground part growth and root tumors as the clubroot-resistant materials.

3. Preparation of clubroot-resistant material by hybridization

And in spring 2008, respectively carrying out hybridization combination on the 4 VB-KG inbred lines and the 8 VB-BG inbred lines obtained in the step 2 (wherein VB-KG is used as a female parent and VB-BG is used as a male parent), namely respectively carrying out hybridization combination on each VB-KG inbred line and each VB-BG inbred line for 32 hybridization combinations. And (5) performing hybrid pollination in the bud period, and harvesting hybrid seeds.

In summer of 2008, the seeds obtained by 32 hybrid combinations are identified to be anti-clubroot disease in seedling stage, and the inoculation test adopts the identification of single inoculation of No. 2, No. 4 and No. 7 physiological races and mixed inoculation of 3 races.

The identification result shows that: the progeny obtained by VB08-CR06 hybrid combination has certain resistance to No. 2, No. 4 and No. 7 physiological races.

4. Inbreeding preparation of homozygous clubroot-resistant material

(1) Selfing progeny obtained by VB08-CR06 hybridization combination to obtain selfed progeny F1, sowing seeds of the harvested selfed progeny F1 in a clubroot disease identification nursery in autumn in the same year to perform seedling stage clubroot disease resistance identification, and screening to obtain a clubroot disease resistant material; selfing the obtained clubroot-resistant material continuously to obtain selfed progeny F2, sowing the seeds of the harvested selfed progeny F2 in a clubroot identification nursery in autumn in the same year to perform seedling-stage clubroot resistance identification, and screening to obtain the clubroot-resistant material; repeating the steps, selfing for 7 generations, and finally screening to obtain 12 homozygous clubroot-resistant inbred lines with regular agronomic characters: VB09-CR06-04, VB09-CR06-11, VB09-CR06-15, VB09-CR06-24, VB09-CR06-26, VB09-CR06-31, VB09-CR06-40, VB09-CR06-51, VB09-CR06-57, VB09-CR06-62, VB09-CR06-88 and VB09-CR 06-91.

(2) In 2014 and 2015, the acquired 12 strains are subjected to club root disease resistance identification by adopting a nursery identification method, and result verification is performed by combining a seedling stage identification method and a field identification method. The method comprises the following specific steps:

A. seedling stage identification

Judging the disease resistance/susceptibility degree according to the size and the quantity of the tumor at the root of the Chinese cabbage; r represents disease resistance to clubroot, and S represents disease susceptibility to clubroot. The specific method is the same as the identification of the clubroot resistance in the seedling stage in the step 2. The results of the evaluation are shown in Table 1.

Table 1, 12 inbred line seedling stage identification results

B. Identification in field

The identification of the field comprises respectively sowing the materials in infected fields of one year planting Chinese cabbage on Qingdao, New wild in Henan, Yuxi, etc., and determining the disease resistance/infection degree according to the tumor size and quantity of root of Chinese cabbage in harvest period; r represents disease resistance to clubroot, and S represents disease susceptibility to clubroot. The field identification results are shown in table 2.

Table 2, 12 inbred line field identification results

C. Nursery identification method

The nursery identification method of the invention can be used for identifying the resistance of the plants in the whole growth period. The method comprises the following specific steps:

step 1, land selection

The soil was subjected to pH measurement, and plots in which the soil was alkaline were selected as test areas.

Step 2, building a test area

Selecting an area with the width of 15m and the length of 60m as an experimental land in the land, and constructing an arched shed in a test area, wherein the arched shed is of a steel frame structure, the span is 12m, the height of the lowest part of the arched shed is 1.8m, the height of the highest part of the arched shed is 2.5m, and the length of the arched shed is 60 m; a nursery garden is built in the shed, and a tool room and a disinfection pool are attached; the tobacco leaves enter a nursery garden and must pass through a disinfection tank, the specification of the disinfection tank is that the length is 1m, the width is 0.5m, the height is 0.15m, quicklime is arranged in the disinfection tank, the thickness of the quicklime is 0.08m, and the tobacco leaves enter and exit the nursery garden and disinfect soles of people. The exterior of the arched shed is covered by an insect-proof net, and the mesh size of the insect-proof net is 40 meshes.

Step 3, nursery construction

1) Setting a nursery garden in the arched shed, wherein the size of the nursery garden is 10.5m multiplied by 58.5 m; the nursery is divided into 3 equal area cells. An isolation ditch with the depth of 0.7m is vertically dug at the periphery of a nursery garden and among cells, and a polyester fiber reinforced polyvinyl chloride (pvc) waterproof board is placed in the isolation ditch, wherein the height of the waterproof board is 0.85m, and the thickness of the waterproof board is 0.5 cm. And backfilling soil into the isolation ditch to be 20cm lower than the ground, and then bricking to be 15cm higher than the ground to serve as isolation ridges and corridors.

2) Measuring the pH value of the soil in the nursery by using a pH meter, wherein the pH value of the soil in the nursery is 8.1;

3) in order to effectively prevent the salinization of soil, increase the soil fertility and stabilize the soil environment, a special organic fertilizer is applied into a nursery, the application amount of the special organic fertilizer is 2000kg, and the pH value of the soil is adjusted to 5.0.

The special organic fertilizer is prepared by taking residues (Shandong Shouguangguan gold corn development Co., Ltd.) after starch is extracted from corn and vinasse (Jingzhi winery) as main raw materials (mass ratio is 2:1), and adding bean pulp, urea and microorganisms. The preparation method comprises the following steps: spraying water on the residue, covering a plastic film (with water content of 50%), keeping at high temperature and high humidity for 3 days, adding sucrose, lactobacillus, yeast, bacillus and acetic acid (the mass ratio of the residue, the sucrose, the lactobacillus, the yeast, the bacillus and the acetic acid is 1500:10:2:2:1:2), mixing uniformly, transferring into a fermentation tank, turning over every 2 days, mixing the fermented corn residue, vinasse and bean pulp according to the mass ratio of 10:1 after 20 days, adding 1kg of urea (union fertilizer) into every 2 cubic meters after mixing, spraying 1kg of EM (effective microorganism) starter (Shandong Junde Biotech Co., Ltd.), pushing into the fermentation tank, keeping for 12 hours after the stack temperature reaches 65 ℃, turning over the stack, turning over once every 2 days, turning over once a week when the temperature is reduced to 55 ℃, completing the fermentation when the temperature reaches room temperature, to prepare the special organic fertilizer.

Step 4, collecting plasmodiophora

Collecting the susceptible white cabbage root of the physiological race of clubroot disease No. 2 from Qingdao of Shandong, collecting the susceptible white cabbage root of the physiological race of clubroot disease No. 4 from the new wild of Henan, and collecting the susceptible white cabbage root of the physiological race of clubroot disease No. 7 from Yunan Yuxi; the strains of physiological races of clubroot No. 2, No. 4 and No. 7 are respectively prepared according to the following method:

1) crushing Chinese cabbage root with clubroot by using a stirrer to obtain a mixture;

2) filtering the mixture by 2 layers and 4 layers of gauze in sequence to obtain filtrate;

3) centrifuging the filtrate at 4000r/min for 10min, removing the supernatant, and collecting the precipitate;

4) suspending the precipitate with distilled water, and repeating the steps 2) -3) for 3 times to obtain the precipitate of plasmodiophora brassicae;

5) diluting the obtained 3 strains with distilled water under microscope to obtain bacterial solutions of No. 2, No. 4, and No. 7 physiological microspecies with spore concentration of 2.5 × 10⁸L^-1And storing at 4 deg.C for use.

Step 5, soil preparation and inoculation

Soil preparation, inoculation and fertilization need to be carried out in a combined manner. The method comprises the following specific steps:

fertilizing for the first time: before soil preparation, fertilizer with the weight of 60% is uniformly scattered in a nursery garden to cultivate land, and the fertilizer is turned into a soil layer with the depth of 30 cm.

Inoculating bacteria: after the first fertilization, respectively inoculating the bacterial liquids of No. 2, No. 4 and No. 7 physiological races stored in the step 4 into three nursery plots, wherein the inoculation mode is as follows: uniformly spraying the soil after rotary tillage by using a spraying pot; spraying 11ml of bacteria per square meter;

fertilizing for the second time: after inoculation, the remaining 40% of the fertilizer by weight is scattered into a nursery garden and raked into a soil layer of 8cm, and land leveling is achieved after fine raking.

The fertilizer used in the two fertilization processes is prepared by using a special substrate of 3m³Rice husk and excrement (purchased in market) 4m³The mass percentage content is 2540-50 kg of nitrogen-phosphorus-potassium ternary compound fertilizer (Shidanli) and 15kg of urea (union fertilizer) are uniformly mixed to obtain the fertilizer. The preparation method of the special matrix comprises the following steps: respectively pulverizing radix Codonopsis, bagasse, semen Ricini, crushed corn stalk, mushroom residue, fructus Lycii, chicken manure and Zaocys, mixing at a volume ratio of 1:2:2:1:1:2:1:2, air drying until the water content is 60%, and drying at a rate of 1kg/m³Spraying EM (effective microorganisms) starter, pushing into a fermentation tank for fermentation after spraying, keeping for 12 hours when the temperature of a pile reaches 65 ℃, turning over the pile once every 2 days, turning over once every week when the temperature of the pile is reduced to 55 ℃, and finishing the fermentation when the temperature is reduced to room temperature.

Step 6, ridging and ridging

According to different varieties of the identified Chinese cabbages, different ridging and ridging modes are selected: the autumn cabbage is identified by adopting furrow making, and making a furrow after soil preparation, wherein the width of the furrow is 1.0 m.

Step 7, sowing seeds to be identified

Arranging and preparing Chinese cabbage seeds to be detected, and selecting proper sowing time according to different identified Chinese cabbage varieties: identifying autumn Chinese cabbage, wherein the sowing time is 8 middle ten days, the autumn Chinese cabbage is sowed in the furrow, and the row spacing is 40 cm.

The sowing mode adopts hole sowing, the hole depth is 1.5cm, the hole distance is 35-40 cm, each hole has 3-5 grains, the soil is covered by 1.5cm, 1 layer of rice husk or wheat bran is scattered on the holes after the soil is covered, the moisture is preserved, and the irrigation mode is adopted after the seedling emergence.

Step 8, thinning

Thinning is needed for 2 times in the seedling period of Chinese cabbage planting. The 1 st thinning time is about 5 days after seedling emergence, and the 2 nd thinning time is when the 4 th true leaf grows.

Step 9, replenishing bacteria liquid

The method for supplementing bacteria comprises spraying clubroot bacteria liquid to root of Chinese cabbage with spraying amount of about 2ml per Chinese cabbage, and the bacteria liquid concentration is 2.5 × 10⁸L^-1Immediately watering once after spraying the bacteria;

the time and the quantity of the supplemented bacteria are important links for inducing the occurrence of clubroot, and the late supplemented bacteria and the small quantity of the supplemented bacteria easily cause the material to have difficult morbidity and generate false resistance; the early and large amount of the supplementary bacteria easily cause the infection of resistant materials or varieties, and the materials are discarded. At the moment, the fungus supplementation can not only cause the disease of the infected material, but also ensure that the resistant material is not infected.

Step 10, irrigation, additional fertilization and pest control

1) And (3) germination period: watering for the 1 st time during seeding, watering for the 2 nd time when seedlings push up soil, and watering for the 3 rd time when seedlings are discharged completely. Additional fertilizer is not generally needed in the germination period.

2) And (3) seedling stage: watering for 4 th and 5 th times during thinning and final singling, wherein the watering times in the seedling stage are determined according to the weather and the concrete conditions of soil, the drought precipitation is small, once the furrows have a dry cracking phenomenon, the watering times are increased on the basis of no water, the watering and furrowing can be combined to apply urea or ammonium sulfate as a seedling raising fertilizer, and each 667m of the fertilizer is used²The dosage is 5kg, or the plant extract is prepared from LIANGFENGSU (containing P)₂O₅51％、K₂O₃4%) water-soluble fertilizer, etc. 100g is diluted by 300 times and applied to the leaf surface.

3) A lotus throne stage: watering the lotus plumule for 1 time in the initial stage, then deep cultivating for 1 time, controlling water and squatting for about 10 days. The water is poured in small water service, and each 667m of water can be poured in combination²And applying 10-15 kg of ammonium sulfate or 10kg of ternary compound fertilizer.

4) And (3) nodulation stage: from the rosette stage to the heading stage, the ridge surface can not crack, otherwise the side root can be broken, and the yield of the Chinese cabbage is influenced. And in drought weather, watering for 1 time every 5-6 days. The materials (Chinese cabbage) in the nursery are mainly used for identifying the clubroot resistance of the variety, so that the main pests are aphids, cabbage caterpillars and the like, and the 4.5 percent beta-cypermethrin solution with 1000-1500 times is used for preventing and treating.

Step 11, disease investigation

According to the maturity of different Chinese cabbage materials and the occurrence condition of diseases, clubroot is investigated timely. Disease conditions were investigated and counted according to the Williams system (Zhangxiao crucifer vegetable clubroot physiological race identification and resistance screening [ A ] Chinese horticulture institute [ A ] Abstract of academic annual meeting paper of Chinese horticulture institute 2015 [ C ] Chinese horticulture institute 2015: 1.). The results of the maturity resistance identification of the different chinese cabbage materials are shown in table 3.

Table 3, 12 inbred line nursery identification results

Through identification, the inbred line VB09-CR06-88 can show certain resistance to the No. 2, No. 4 and No. 7 physiological races (high resistance to the No. 7 physiological race and resistance to the No. 2 and No. 4 physiological races), and is named as VB15-CR 06. The VB15-CR06 phenotype is as follows: the plant is upward-flushing, the outer leaves are greener, the leaf surfaces are glossy and wrinkled, the leaves are thinner, the leaf stalks are white and wider, the leaf spheres are in a straight cylinder shape, the spherical top is sharp, the corm size is 26 cm multiplied by 19 cm, the core leaf is yellow, and the knot spheres are compact. The weight of the individual plant is 2.6kg, and the weight of the ball is 2.1 kg. The self-incompatibility index is more than or equal to 1. Against viral diseases and downy mildew.

In order to make the progeny material have the broad-spectrum clubroot resistance, VB15-CR06 can be selected as parent base material to be subjected to hybridization combination preparation.

Example 2 application of Chinese cabbage clubroot resistance identification method

Identifying the resistance of 11 varieties of Chinese cabbages including Chunxi, VB53, VB11, VB54, VB116, VB220, Qingyangchun No. 1 (CK), VB237, VB65, 10VB83 and 10VB83-1 by using the nursery identification method in the step two of the example 1, and meanwhile, setting up a control group which is respectively planted in a field with acid soil, a field with alkaline soil and a laboratory, wherein the field with acid soil is a field block infected with clubroot in the Qingdao area in the first year, the field with alkaline soil is a field block planted with Chinese cabbages in the Weifang area in the first year, and the two controls adopt a common Chinese cabbage planting method, the planting purpose in the laboratory is seedling stage identification, seedling stage sowing is adopted, roots are damaged when 5 leaves are adopted, and 2ml 2.5. 2.5 × 10 10.10.5⁸L^-1Spraying water on the bacterial liquid on time, and investigating after 1 month. Among them, Chunxi is known as a disease-resistant variety, and Qing Ming Chunbai No. 1 is known as an susceptible variety. Investigation of disease conditions statistics were performed according to the Williams system.

The results are shown in Table 4. As can be seen from the table: the resistance identification result of identifying the physiological races of the Chinese cabbage clubroot 2, 4 and 7 by adopting the nursery disclosed by the invention shows that: the seedling stage resistance is completely consistent with the laboratory identification result, and the mature stage resistance is completely consistent with the acid soil field identification result; therefore, the accuracy rate of identifying the clubroot of the Chinese cabbage by the method is 100%, the method is accurate and reliable, the property maintaining condition of the strain in the soil is stable, and the defects that the identification of an alkaline field is inaccurate, and the resistance of a mature period cannot be effectively identified in a laboratory are overcome.

The Chinese cabbage clubroot disease resistance is identified through the field, only according to the anti-infection performance of the mature materials, partial material identification results are different from laboratories and the nursery identification method, the field identification can only be performed in an acid soil field with high disease, and the experimental results are unavailable in an alkaline environment. Laboratory identification can only be carried out at the seedling stage, although the identification is more convenient, the later expression cannot be tracked, resistance expression data is not detailed enough, and some materials which show diseases at the seedling stage and show diseases at the mature stage cannot be identified correctly. The nursery identification method combines the advantages of field identification and laboratory identification, can identify the resistance of the material in the nursery stage, can track and investigate the resistance performance and growth condition in the whole growth period, and has obvious advantages.

TABLE 4 resistance Performance of different varieties of Chinese cabbage in the field, laboratory and nursery of the invention

Note: HS indicates high sensitivity, S indicates susceptibility, R indicates disease resistance, HR indicates high resistance, and nothing indicates failure to sense

Effective symptoms were observed.

Example 3 influence of germ isolation method on Leptoma bacteria diffusion in Nursery construction

The nursery identification method in step two, 4(2) C of example 1 was used to investigate the effect of the germ isolation method on the spread of plasmodiophora brassicae. The specific method comprises the following steps: inoculating No. 2 physiological microspecies plasmodiophora brassicae in a nursery, and not inoculating bacteria outside the nursery; planting a disease-resistant Chinese cabbage (Qing' e Chun Bai No. 1) inside and outside a nursery, performing an analysis experiment on a germ isolation method in the nursery construction in the step 3 of the nursery identification method, and dividing the method into 2 experimental groups according to the difference of the isolation method:

experimental group 1: only brick walls 35cm higher than the ground are used for isolation;

experimental group 2: the brick walls 35cm higher than the ground and the waterproof materials 85cm higher than the ground are combined to form isolation ridges, and the waterproof materials are buried 70cm underground and 15cm above the ground;

the experimental results are as follows: the Chinese cabbage planted outside the nursery in the experimental group 1 was partially infected; the transverse diffusion of the plasmodiophora is proved; the Chinese cabbage planted outside the nursery in experimental group 2 was not infected. The combination of the brick wall of 35cm and the waterproof material of 85cm can effectively prevent the transverse diffusion of the plasmodiophora. The brick wall and the waterproof material are combined to form the isolation ridge, so that the transverse diffusion of the plasmodiophora can be effectively prevented.

Example 4 Single-factor analysis of physiological races of Leptosphaeria maculans 2

Single-factor analysis experiment of physiological microspecies inoculation mode of Chinese cabbage plasmodiophora brassicae 2

Identifying clubroot resistance (to No. 2 physiological race) of the Chinese cabbage varieties of Chunxi, VB53, VB11, VB54, VB116 and Qing-Jiangchun No. 1 by adopting the nursery identification method in the step two of the embodiment 1 and the step 4(2) C, and carrying out the following analysis experiment on the No. 2 physiological race by adopting different inoculation modes;

group A, adopting the inoculation mode that 2ml of the seed with the concentration of 2.5 × 10 is added into a seedling pit when in direct seeding⁸L^-1The bacterial liquid of (a);

group B, the inoculation mode adopted is that the spraying concentration before direct seeding is 2.5 × 10⁸L^-120ml of the bacterial liquid per square meter;

c group, the inoculation mode is to spray 2.5 × 10 to the soil before direct seeding⁸L^-111ml per square meter of the bacterial liquid, and irrigating roots after emergence of seedlings (2.5 × 10)⁸L^-11ml of the bacterial suspension of (1);

control group: the clubroot resistance performance of the Chinese cabbage in the seedling stage is measured by a root injury method in a laboratory and is used as a reference standard;

and observing and counting the resistance performance of the Chinese cabbage in the seedling stage to the physiological race of plasmodiophora brassicae No. 2.

The results are shown in Table 5. As can be seen from Table 5, the clubroot resistance of the Chinese cabbage of group C was completely consistent with that of the control group, whereas the disease-susceptible material VB53 was not induced in A, B group. Therefore, the inoculation method of the No. 2 physiological race is preferably as follows: spraying bacteria liquid to soil before direct seeding and irrigating roots after seedling emergence.

TABLE 5 influence of different inoculation patterns on the clubroot resistance of Chinese cabbage

Note: HS indicates that the material is highly sensitive, S indicates that the material is susceptible, R indicates that the material is resistant, and HR indicates that the material is highly resistant.

Single-factor analysis experiment for inoculation concentration of No. 2 physiological microspecies of clubroot bacteria of Chinese cabbage

The evaluation of clubroot resistance (to physiological race 2) of the chinese cabbage varieties spring happiness, VB53, VB11, VB54, VB116 and qing grind spring white 1 was carried out by the nursery evaluation method in step two, step 4(2) C of example 1, and the following analysis experiment was carried out by changing the inoculation concentration of only physiological race 2:

group a, the inoculation concentration of No. 2 physiological race is 1 × 10⁷L^-1；

Group b, the inoculation concentration of No. 2 physiological race is 0.5 × 10⁸L^-1；

Group c, the inoculation concentration of the No. 2 physiological race adopted is 2.5 × 10⁸L^-1；

Group d, the inoculation concentration of No. 2 physiological race adopted is 5 × 10⁸L^-1；

Observing and counting the resistance performance of the 4 groups of Chinese cabbages to the physiological race of plasmodiophora brassicae No. 2.

The results are shown in Table 6, from Table 6 it can be seen that 1 × 10 was used⁷L^-1The concentration of the Chinese cabbage does not play a role in all varieties of Chinese cabbage materials and cannot induce clubroot, 0.5 × 10⁸L^-1The concentration of (A) can induce partial material to generate diseases, but partial susceptible material does not generate diseases, 2.5 × 10⁸L^-1Inducing disease in susceptible material and producing no disease in resistant material 5 × 10⁸L^-1The inoculation concentration of the No. 2 physiological race is preferably 2.5 × 10⁸L^-1。

TABLE 6 influence of different inoculation concentrations on the clubroot resistance Performance of Chinese cabbage

Experiment for analyzing pathogenicity persistence of No. 2 physiological races of Chinese cabbage plasmodiophora brassicae

The nursery identification method in the step two, 4 and 2, C, of the embodiment 1 is adopted to identify the clubroot disease resistance (to No. 2 physiological races) of the Chinese cabbage variety Chunxi and Qingyanchun white No. 1, and the experiment is carried out by changing the inoculation method into a seedling stage root injury method, and the specific method is as follows: after the nursery garden is built, 10 batches of Chinese cabbages with damaged roots are planted in a fixed planting mode, and one batch of Chinese cabbages is sowed every 10 days.

The results are shown in Table 7. As can be seen from Table 7, in the experiment, the susceptible material "Qing Jiang Chun Bai No. 1" of the first 5 batches had diseases, and the susceptible material "Qing Jiang Chun Bai No. 1" of the last 5 batches had no diseases, so the pathogenicity of No. 2 physiological race could not be extended to 50 days, and the bacterial liquid was required to be supplemented within 50 days after sowing.

TABLE 7 Chinese cabbage infection at different sowing periods

Example 5 physiological microspecies single factor analysis of Brassica rapa 4

Single-factor analysis experiment of Chinese cabbage plasmodiophora brassicae 4 physiological microspecies inoculation mode

The nursery identification method in step two, step 4(2) C, of example 1 is used for identifying the clubroot disease resistance of the celery cabbage varieties spring happiness, VB53, VB11, VB54, VB116 and qing grind spring white No. 1 (for No. 4 physiological races), and the following analysis experiments are carried out on the No. 4 physiological race by adopting different inoculation modes:

and observing and counting the resistance performance of the Chinese cabbages in the seedling stage to the physiological race of plasmodiophora brassicae 4.

The results are shown in Table 8. As can be seen from Table 8, the clubroot resistance of the Chinese cabbage in group C was completely consistent with that of the control group, while group A failed to induce the onset of the disease-susceptible material VB116, and group B failed to induce the onset of the disease-susceptible material VB 53. Therefore, the inoculation method of the No. 4 physiological race is preferably as follows: spraying bacteria liquid to soil before direct seeding and irrigating roots after seedling emergence.

TABLE 8 influence of different inoculation patterns on the clubroot resistance of Chinese cabbage

Second, Chinese cabbage clubroot 4 physiological microspecies inoculation concentration single factor analysis experiment

The evaluation of clubroot resistance (to physiological race 4) of the chinese cabbage varieties spring happiness, VB53, VB11, VB54, VB116 and qing grind spring white 1 was carried out by the nursery evaluation method in step two, step 4(2) C of example 1, and the following analysis experiment was carried out by changing the inoculation concentration of only physiological race 4:

group a, the inoculation concentration of No. 4 physiological race is 1 × 10⁷L^-1；

Group b, the inoculation concentration of No. 4 physiological race is 0.5 × 10⁸L^-1；

Group c, the inoculation concentration of No. 4 physiological race adopted is 2.5 × 10⁸L^-1；

Group d, the inoculation concentration of No. 4 physiological race is 5 × 10⁸L^-1；

Observing and counting the resistance performance of the 4 groups of Chinese cabbages to the physiological race of plasmodiophora brassicae No. 4.

The results are shown in Table 9, from Table 9, it can be seen that 1 × 10 was used⁷L^-1、0.5×10⁸L^-1The concentration of (A) can only induce partial material to generate diseases, but partial susceptible material does not generate diseases, 2.5 × 10⁸L^-1Inducing disease in susceptible material and producing no disease in resistant material 5 × 10⁸L^-1The concentration of (A) causes the formation of clubroot disease in the partially resistant material, therefore, the inoculation concentration of No. 4 physiological race is preferably 2.5 × 10⁸L^-1。

TABLE 9 Effect of different inoculum concentrations on the Chinese cabbage clubroot resistance Performance

Third, cabbage clubroot 4 physiological race pathogenicity persistence analysis experiment

The nursery identification method in step two, 4(2) C, in the embodiment 1, is adopted to identify the clubroot disease resistance of the Chinese cabbage variety Chunxi and Qingyangchun No. 1 (to No. 4 physiological races), and the experiment is carried out by changing the inoculation method into a seedling stage root injury method, and the specific method is as follows: after the nursery garden is built, 10 batches of Chinese cabbages with damaged roots are planted in a fixed mode, and one batch of Chinese cabbages are sowed every 10 days.

The results are shown in Table 10. As can be seen from Table 10, in the experiment, the susceptible material "Qing Jiang Chun Bai No. 1" of the first 6 batches had a disease, and the susceptible material "Qing Jiang Chun Bai No. 1" of the last 4 batches had no disease, so the pathogenicity of No. 4 physiological races did not exceed 60 days, and the bacterial liquid needs to be supplemented within 60 days after sowing.

TABLE 10 Chinese cabbage infection at different sowing periods

Example 6 physiological microspecies single factor analysis of Brassica rapa Ledeb 7

Single-factor analysis experiment of physiological microspecies inoculation mode of Chinese cabbage plasmodiophora brassicae 7

The nursery identification method in step two, step 4(2) C, of example 1 is used for identifying the clubroot disease resistance of the celery cabbage varieties spring happiness, VB53, VB11, VB54, VB116 and Qing-grind spring white No. 1 (for No. 7 physiological races), and the following analysis experiments are carried out on the No. 7 physiological race by adopting different inoculation modes:

and observing and counting the resistance performance of the Chinese cabbage in the seedling stage to the physiological race of plasmodiophora brassicae 7.

The results are shown in Table 11. As can be seen from Table 11, the clubroot resistance of the Chinese cabbage of group C was completely consistent with that of the control group, while the disease-susceptible material VB116 was not induced in A, B group; therefore, the inoculation method of the No. 7 physiological race is preferably as follows: spraying bacteria liquid to soil before direct seeding and irrigating roots after seedling emergence.

TABLE 11 inoculation method for No. 7 physiological race

Second, Chinese cabbage clubroot germ No. 7 physiological microspecies inoculation concentration single factor analysis experiment

The evaluation of clubroot resistance (to physiological race 7) of the chinese cabbage varieties spring happiness, VB53, VB11, VB54, VB116 and qing grind spring white 1 was carried out by the nursery evaluation method in step two, 4(2) C of example 1, and the following analysis experiment was carried out by changing the inoculation concentration of only physiological race 7:

group a, the inoculation concentration of No. 7 physiological race adopted is 1 × 10⁷L^-1；

Group b, the inoculation concentration of No. 7 physiological race is 0.5 × 10⁸L^-1；

And c, group: number 7 physiological races adoptedThe inoculation concentration of (A) is 2.5 × 10⁸L^-1；

Group d, the inoculation concentration of No. 7 physiological race adopted is 5 × 10⁸L^-1；

And observing and counting the resistance performance of the 4 groups of Chinese cabbages to the physiological race of plasmodiophora clubmoss 7.

The results are shown in Table 12, from Table 12 it can be seen that 1 × 10 was used⁷L^-1The concentration of the Chinese cabbage does not play a role in all varieties of Chinese cabbage materials and cannot induce clubroot, 0.5 × 10⁸L^-1The concentration of (A) can induce partial material to generate diseases, but partial susceptible material does not generate diseases, 2.5 × 10⁸L^-1Inducing disease in susceptible material and producing no disease in resistant material 5 × 10⁸L^-1The concentration of (A) causes the formation of clubroot disease in the partially resistant material, therefore, the inoculation concentration of No. 7 physiological race is preferably 2.5 × 10⁸L^-1。

TABLE 12 Effect of different inoculum concentrations on the Chinese cabbage clubroot resistance Performance

Experiment for analyzing pathogenicity persistence of No. 7 physiological races of Chinese cabbage plasmodiophora brassicae

The nursery identification method in the step two, 4 and 2, C, of the embodiment 1 is adopted to identify the clubroot disease resistance of the Chinese cabbage variety Chunxi and Qingyangchun No. 1 (to No. 7 physiological races), and the experiment is carried out by changing the inoculation method into a seedling stage root injury method, and the specific method is as follows: after the nursery garden is built, 10 batches of Chinese cabbages with damaged roots are planted in a fixed mode, and one batch of Chinese cabbages are sowed every 10 days.

The results are shown in Table 13. As can be seen from Table 13, in the experiment, the susceptible material "Qing Jiang Chun Bai No. 1" of the first 5 batches had a disease, and the susceptible material "Qing Jiang Chun Bai No. 1" of the last 5 batches had no disease, so the pathogenic ability of No. 7 physiological races did not exceed 50 days, and the bacterial liquid needs to be supplemented within 50 days after sowing.

TABLE 13 Chinese cabbage infection at different sowing periods

The foregoing is illustrative of the best mode of the invention and details not described herein are within the common general knowledge of a person of ordinary skill in the art. The scope of the present invention is defined by the appended claims, and any equivalent modifications based on the technical teaching of the present invention are also within the scope of the present invention.

Claims

1. A method for producing Chinese cabbage with clubroot resistance comprises the following steps:

1) hybridizing a Chinese cabbage variety Kanggen 51-1 serving as a female parent and a Chinese cabbage variety Baogouwang No. 1 serving as a male parent to obtain hybrid progeny; carrying out clubroot resistance identification on the hybrid filial generation, and selecting the clubroot-resistant hybrid filial generation;

3) carrying out clubroot resistance identification on the homozygous clubroot-resistant inbred line, and selecting the clubroot-resistant inbred line as clubroot-resistant Chinese cabbage germplasm;

identifying clubroot resistance in a full growth cycle in an alkaline soil environment;

the method for identifying clubroot resistance in the whole growth cycle in the alkaline soil environment comprises the following steps:

step E, fertilizing the inoculated soil to obtain soil to be sown;

step F, sowing the Chinese cabbage seeds to be detected in the soil to be sown, culturing, carrying out clubroot investigation during the culture period, and identifying the clubroot resistance of the Chinese cabbage to be detected according to a Williams system;

in the step C and the step E, the fertilizer used for fertilization is obtained by uniformly mixing the matrix, the rice hull manure, the nitrogen-phosphorus-potassium compound fertilizer and the urea; the matrix is prepared from radix Codonopsis, bagasse, semen Ricini, crushed corn stalk, mushroom residue, fructus Lycii, chicken manure and Zaocys by fermenting.

2. The method for producing Chinese cabbage with clubroot resistance as claimed in claim 1, which is characterized in that: in the step 1), the Chinese cabbage variety Kanggen 51-1 and the Chinese cabbage variety Baogouwang 1 both further comprise a step of screening a homozygous clubroot-resistant inbred line before hybridization;

the method for screening the homozygous clubroot-resistant inbred line comprises the following steps: selfing the Chinese cabbage variety Kanggen 51-1 or the Chinese cabbage variety Baogouwang No. 1 to obtain selfed progeny F1, carrying out club disease resistance identification on the selfed progeny F1, and selecting club disease resistant selfed progeny F1; selfing the clubroot-resistant selfing progeny F1 continuously to obtain selfing progeny F2, carrying out clubroot resistance identification on the selfing progeny F2, selecting clubroot-resistant selfing progeny F2, repeating the steps, and selfing continuously for multiple generations to obtain a homozygous clubroot-resistant selfing line;

the selfing times are 7 times;

3. The method for producing brassica rapa pekinensis seeds as claimed in claim 1 or 2, wherein the method comprises the following steps: in the step 2), the selfing times are 7 times;

4. The method for producing Chinese cabbage with clubroot resistance as claimed in claim 1, which is characterized in that:

the step A also comprises the steps of backfilling soil into the isolation trench and then bricking to a position higher than the ground;

the step A also comprises the step of applying organic fertilizer;

the organic fertilizer is a product obtained by fermenting residues and vinasse obtained after starch is extracted from corn as main raw materials;

the method also comprises a step of supplementing bacteria within 15-60 days after sowing.

5. The method for producing Chinese cabbage with clubroot resistance as claimed in claim 4, wherein the method comprises the following steps:

the mixture ratio of the substrate, the rice hull manure, the nitrogen-phosphorus-potassium compound fertilizer and the urea is 3m³：4m³：（40～50）kg：15kg；

The mass ratio of the residue to the vinasse is 2: 1.

6. the method for producing Chinese cabbage with clubroot resistance according to claim 4 or 5, characterized in that:

in the step A, backfilling soil into the isolation trench to be 20cm lower than the ground, and then bricking to be 15cm higher than the ground;

in the step B, the concentration of the clubroot bacterium liquid is 2.5 × 10⁸L^-1；

The mass ratio of the fertilizer application amount in the step C to the fertilizer application amount in the step E is 6: 4;

the method for supplementing the rhizomaea bacteria comprises the steps of inoculating a plasmodiophora bacteria solution to the root part near the Chinese cabbage to be detected;

a tool room and a disinfection pool are also arranged in the arched shed;

quicklime is placed in the disinfection tank;

the exterior of the arched shed is covered by an insect-proof net;

the waterproof board material is polyester fiber.

7. Use of the method for producing Chinese cabbage against clubroot as claimed in any one of claims 1 to 6 in Chinese cabbage breeding.

8. The method of identifying clubroot resistance as recited in claim 1.

9. The use of the method of claim 8 for identifying clubroot resistance in breeding of clubroot-resistant Chinese cabbage varieties.