CN113249429A - Large-scale rice blast resistance identification method suitable for breeding application - Google Patents
Large-scale rice blast resistance identification method suitable for breeding application Download PDFInfo
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Abstract
The invention discloses a large-scale rice blast resistance identification method suitable for breeding application, which comprises the following steps: step 1, performing non-toxic gene sequencing analysis by using a plurality of rice blast strains collected at multiple points in a target ecological area, performing cluster analysis according to results to screen out representative physiological races in the target ecological area, culturing the screened physiological races and preparing a mixed spore suspension; step 2, sowing the identification material, inoculating the mixed spore suspension liquid in the 3-4 leaf stage of the plant, and investigating and grading the completely-attacked disease of the disease control; step 3, transplanting the screened seedling pest resistant material to a field and carrying out field management; inoculating mixed spore liquid at the early stage of booting ears; the disease condition is investigated and graded at the early yellow maturity of rice. According to the invention, a large amount of materials are identified in a small space, the identification efficiency and the result reliability are improved, and the screened resistant materials can give consideration to the resistance of the rice blast and the panicle blast, and have important significance for screening of rice disease-resistant germplasm resources and research of disease-resistant genetic breeding.
Description
Technical Field
The invention relates to the technical field of agriculture, in particular to a large-scale rice blast resistance identification method suitable for breeding application.
Background
The rice blast disease caused by the fungus Magnaporthe oryzae is one of the most serious diseases that are harmful to rice production. The rice blast can occur in the whole growth period of rice, and has various expression forms according to different infection parts, wherein seedling plague and panicle stem plague are the most common and have larger harm. Long-term production practice proves that breeding and utilizing disease-resistant varieties are the most safe and effective method for preventing and treating rice blast. However, the data show that most of the new varieties examined in countries and provinces in recent years are highly sensitive to rice blast and resistant varieties are few. Moreover, due to the complexity of the physiological race composition of the rice blast fungus and the rapid variation of the avirulence genes, the resistance of the cultivated rice varieties with rice blast resistance is gradually weakened or lost after the rice varieties are continuously planted for 3-5 years. Therefore, the physiological race composition and the non-toxic gene distribution condition of the rice blast fungus groups in each ecological area can be accurately known and mastered in real time; the rapid and large-scale identification of germplasm resources and the development of intermediate materials are of great significance for exploring local rice blast resistance germplasm resources and cultivating resistant varieties.
The rapid and effective resistance identification is one of the prerequisites for breeding disease-resistant varieties. At present, the resistance identification aiming at rice blast mainly comprises seedling stage resistance identification and adult stage panicle blast resistance identification in the identification period. Most of the existing seedling plague inoculation identification adopts a disc sowing method, when seedlings grow to 3-4 leaf stages, the seedlings are transferred to an inoculation box for single strain spraying inoculation, and although the inoculation box single strain inoculation method has the advantages of high repeatability, less influence of environmental factors and the like, the following defects also exist: 1) two parts of materials are required to be prepared simultaneously, one part is used for field planting, the other part is used for rice blast inoculation identification, the time and the labor are consumed, and the test field is wasted; 2) in order to improve the reliability of inoculation in the seedling stage, multiple strains need to be inoculated by single spore, but the inoculation is limited by an inoculation box and an inoculation field, the quantity of materials for single inoculation is small, and the resistance identification requirement of large-scale breeding materials cannot be met; 3) the plastic tray with the seedlings needs to be transferred to the inoculation box from the culture greenhouse before inoculation, the plastic tray needs to be transferred to the greenhouse from the inoculation box after inoculation for 24 hours, root damage is easily caused in the transportation process, the plastic tray is also easily subjected to external severe temperature difference change, the seedlings are easily dried due to water loss, and the identification result is influenced. In addition, the consistency of the identification result of the seedling stage and the identification result of the adult plant stage is different, and the comprehensive analysis needs to be carried out by combining the identification result of the adult plant stage of the field. The evaluation rule of the natural disease garden in the field plant growing period is greatly influenced by the field environment, and the repeatability and the accuracy of the evaluation result are poor. Therefore, establishing a set of high-efficiency and large-scale rice blast resistance identification system has important significance for improving the screening efficiency of rice blast resistance materials and cultivating resistant varieties; how to overcome the defects of the prior art is a problem to be solved urgently in the technical field of agricultural breeding at present.
Disclosure of Invention
The invention aims to solve the defects of the prior art and provides a large-scale rice blast resistance identification method suitable for breeding application, the method can be used for large-scale identification of a large amount of materials in a smaller space, the identification efficiency and the reliability of results are improved, the test field and the cost are saved, and the screened resistant materials can give consideration to seedling plague and panicle plague resistance, and have important significance for screening of rice disease-resistant germplasm resources and research on disease-resistant genetic breeding.
In order to achieve the purpose, the technical scheme adopted by the invention is as follows:
a method for large-scale identification of rice blast resistance suitable for breeding applications, the method comprising the steps of:
step 1, determining rice blast identification strains: firstly, carrying out non-toxic gene sequencing analysis by using a plurality of rice blast strains collected at multiple points in a target ecological area, carrying out cluster analysis according to a sequencing result to screen out representative physiological races of the target ecological area, and culturing the screened physiological races to prepare a mixed spore suspension;
step 2, identifying the plague resistance: sowing identification materials, inoculating the mixed spore suspension obtained in the step 1 in a 3-4 leaf stage of a plant, and investigating and grading the completely diseased contrast to be infected;
step 3, rice blast resistance identification: transplanting the screened seedling pest resistant material to a field according to the grading of the step 2, and performing field management; inoculating the mixed spore liquid obtained in the step 1 at the early stage of booting; in the early stage of yellow maturity of rice, the disease condition is investigated and graded.
Preferably, in the step 1, the specific steps of performing avirulence gene sequencing analysis by using a plurality of rice blast strains collected at multiple points in a target ecological area are as follows:
a) collecting and separating a large amount of rice blast monospore strains at multiple points in a target ecological area;
b) aiming at different collected monospore strains, 7 avirulence gene sequencing primers are used for sequencing analysis.
Preferably, the avirulence gene sequencing is AvrPi9, AvrPia, AvrPii, AvrPizt, AvrPik, AvrPita, AvrPib7 avirulence genes.
Preferably, in the step 1, the specific steps of performing cluster analysis according to the sequencing result to screen out the representative physiological race of the target ecological region are as follows:
a) performing clustering analysis by using PowerMarker V3.25 software according to the avirulence gene sequences of different rice blast strains;
b) aiming at the clustering result, 1-2 representative strains are selected from each subgroup for subsequent resistance inoculation identification.
Preferably, in the step 1, the preparation method of the mixed spore suspension comprises the following steps: culturing the selected physiological microspecies separately, washing spores on the surface of the culture medium with sterile water, and filtering off mycelia with gauze to obtain spore solution with concentration of 4 × 105Taking spore liquid of the same amount of physiological microspecies per mL, mixing uniformly, and adding 0.02% (v/v) of Tween-20 to prepare a mixed spore suspension.
Preferably, in the step 2, in the investigation and classification, 0-9 grade division and evaluation are adopted:
level 0: disease-free, immune;
level 1: needle-shaped large and small brown spots and high resistance;
and 2, stage: has large brown point, diameter less than 1mm, and good resistance
And 3, level: gray spots from circular to oval, brown edges, 1-2 mm in diameter and resistance;
4, level: typical fusiform lesion spots are 1 cm-2 cm long, are usually limited between two veins, are resistant when the area of injury is less than 2% of the area of the veins;
and 5, stage: typical fusiform disease spots, which account for 2% -10% of the area of the leaves and feel;
and 6, level: typical fusiform disease spots, which account for 10.1% -25% of the area of the leaves, and are felt;
and 7, stage: typical fusiform disease spots, which account for 25.1% -50% of the leaf area;
and 8, stage: typical fusiform disease spots, which account for 50.1% -75% of the area of the leaves; high feeling;
and 9, stage: typical fusiform disease spots, the damage area is more than 75.1 percent of the area of the leaf, and the feeling is high;
wherein, 4-9 grade materials of the Miao ethnicity are directly eliminated, and 0-3 grade materials are continuously identified.
Preferably, the material identified in step 2 refers to a medium and high generation stable material or natural germplasm resource with excellent comprehensive characters bred in the breeding process.
Preferably, in the step 2, germination is performed before sowing, after seeds emerge and sprout, the seeds are sequentially sown in the inoculation pools in a row, each inoculation pool is sown in two rows, the width of each row is 50cm, the width of each row is 20cm, the row spacing is 20cm, the row width of each row is 4-5 cm, the row spacing is 5cm, and each row is sown for about 120-150 grains, and then grains are pressed.
Preferably, in the step 3, the grading and evaluation criteria for disease condition investigation and grading are as follows:
level 0: no disease and high resistance;
level 1: small branch peduncles are attacked, the loss of each ear is less than or equal to 5 percent, and the resistance is achieved;
and 3, level: the disease of the principal axis or the neck of the ear is developed, the loss of each ear is less than 20 percent, and the resistance is moderate
And 5, stage: the main axis or the ear neck is attacked, grains are half shrunken, and the loss of each ear is less than 50 percent;
and 7, stage: the neck of the ear is attacked, most of the ears are shrunken, and the loss of each ear is less than 70 percent;
and 9, stage: the neck of the ear is attacked, the loss of each ear is more than or equal to 70 percent, and the ear is high;
wherein, the 0-3 grade material is the material which can be used for breeding, and the 5-9 grade material for rice blast is directly eliminated.
Preferably, step 3 adopts injection inoculation, and the specific steps are as follows: selecting rice husks which just break the chest in each cell at the early stage of booting, injecting the mixed spore suspension inoculum obtained in the step (1) into the rice husks in the rows 2-4 of each cell, and injecting 1ml into each ear; each line material was inoculated with 10 ears and the inoculated ears were marked.
The invention has the beneficial effects that:
1) the invention separates and collects a large number of rice blast monospore strains aiming at the target material to-be-popularized application area, has wide geographical representativeness, screens out identification strains through non-toxic gene sequencing for mixed strain inoculation, and obviously lightens the inoculation workload.
2) The invention is used for sowing, seedling raising and identification in an inoculation greenhouse. The controllability of the environment in the greenhouse is high, the repeatability is good, the quantity of materials identified at a time is large, and the large-scale rice blast identification can be realized in a small space. In addition, seedlings do not need to be transferred, the seedlings can be prevented from being damaged and dead due to root damage and external temperature difference change, and the quality of the seedlings and the reliability of identification results are improved.
3) The invention can realize two purposes of seedling culture and inoculation material preparation simultaneously by sowing in the inoculation greenhouse, in addition, a large amount of disease-sensitive materials can be eliminated after the inoculation in the seedling stage, and the disease-resistant materials are directly transplanted to a field identification garden, thereby obviously reducing the test field and the cost and saving labor and time.
4) The identification method provided by the invention can screen out the rice resistant material which is suitable for local and gives consideration to the resistance to the seedling blast and the panicle blast, avoids the loss caused by the occurrence of the rice blast, and has important significance for local rice production.
Drawings
FIG. 1 is a diagram of the collection of the sources and clusters of 106 rice blast strains in 20 regions of the entire region of Jiangsu province, A: distributing the strain collection site; b: cluster plot of 106 strains, gray triangles representing the selected identifying strains;
FIG. 2 is a representation of the seedling blast phenotype of a portion of germplasm resource material;
FIG. 3 is a spike blast phenotype diagram of germplasm resource material with partial resistance to seedling blast reaching grade 3 or more.
Detailed Description
In order to make the objects, technical solutions and advantages of the present invention more apparent, the technical solutions of the present invention will be clearly and completely described below with reference to the following examples. It should be understood, however, that the description herein of specific embodiments is only intended to illustrate the invention and not to limit the scope of the invention. It should be noted that the experimental methods used in the following examples are all conventional methods unless otherwise specified. The materials and reagents used in the following examples are commercially available unless otherwise specified.
A large-scale rice blast resistance identification method suitable for breeding application comprises the following steps:
(1) carrying out avirulence gene sequencing analysis by using a plurality of rice blast strains collected at multiple points in a target ecological area:
a) collecting and separating a large amount of rice blast single spore strains (more than 100) at multiple points in a target ecological area;
b) aiming at different collected monospore strains, 7 avirulence gene sequencing primers are used for sequencing analysis;
(2) and (3) performing cluster analysis according to the sequencing result to screen out representative physiological races of the target ecological region:
a) and (3) performing clustering analysis by using PowerMarker V3.25 software according to the avirulence gene sequences of different rice blast strains.
b) Aiming at the clustering result, 1-2 representative strains are selected from each subgroup for subsequent resistance inoculation identification.
(3) Preparation of mixed spore suspension: separately culturing the selected physiological microspecies in step (2), washing spores on the surface of the culture medium with sterile water, and filtering off mycelia with gauze to obtain spore solution with concentration of 4 × 105Taking spores of the same physiological race in each mLMixing the seed solution uniformly, and adding 0.02% (v/v) Tween-20 to prepare a mixed spore suspension;
(4) setting an inoculation pool in the greenhouse and sowing identification materials:
a) setting an inoculation pool in the greenhouse: the length of the inoculation pool in the greenhouse is 20m, the width of the inoculation pool is 1.2m, and the soil depth is 20 cm;
b) design of control material: setting disease-resistant and susceptible materials as a control group;
c) seed disinfection and seed soaking and germination acceleration: washing the seeds with clear water for multiple times, soaking the seeds for 10-20 s by using sodium hypochlorite with the concentration of 0.5%, then washing the seeds with clear water for multiple times, soaking the seeds for germination by adopting a 'three-soaking-accelerating' method, namely soaking the seeds for three days, accelerating the germination for one night, and sequentially drilling the seeds in an inoculation pool after the seeds emerge white and germinate;
(5) identifying and evaluating artificial resistance in seedling stage in greenhouse:
a) and (3) fertilizer and water management before inoculation: in the same field fertilizer and water management, heat preservation is carried out in the budding period, temperature control is carried out after the emergence of seedlings to prevent the seedlings from rising, urea is applied once two days before inoculation to keep the leaves green, and the disease attack is facilitated;
b) spray inoculation: inoculating the disease-resistant and disease-susceptible control and the material to be identified in the 3-4 leaf stage of the plant, humidifying the greenhouse, the disease-resistant and disease-susceptible control and the material to be identified by spraying water before inoculation, ensuring that the surface of the leaf is moist, keeping the humidity in the greenhouse between 90-100%, and performing spray inoculation on the disease-resistant and disease-susceptible control and the material to be identified by adopting the mixed spore suspension obtained in the step (3);
c) temperature and humidity setting after inoculation:
controlling the temperature in the greenhouse to be 26 ℃ within 24 hours after inoculation, keeping the humidity between 90 and 100 percent, and keeping the greenhouse in a dark condition after all the greenhouse is shielded by a sunshade net;
spraying 3-5 times with water every day 2-8 days after inoculation to ensure that the surface of the plant is moist, the humidity in the greenhouse is stably above 90%, the temperature in the greenhouse is controlled to be 26-28 ℃ in the daytime, the temperature at night is 20-24 ℃, and shading treatment is carried out to prevent direct sunlight;
d) disease condition investigation and grading:
after 7 days of inoculation, investigation and evaluation are carried out on the complete morbidity of the disease-susceptible control;
grading the disease condition: investigating the phenotype of the seedling leaf with the heaviest disease in the seedlings, and adopting 0-9 grade division and evaluation:
level 0: disease-free, immune;
level 1: needle-shaped large and small brown spots and high resistance;
and 2, stage: has large brown point, diameter less than 1mm, and good resistance
And 3, level: gray spots from circular to oval, brown edges, 1-2 mm in diameter and resistance;
4, level: typical fusiform lesion spots are 1 cm-2 cm long, are usually limited between two veins, are resistant when the area of injury is less than 2% of the area of the veins;
and 5, stage: typical fusiform disease spots, which account for 2% -10% of the area of the leaves and feel;
and 6, level: typical fusiform disease spots, which account for 10.1% -25% of the area of the leaves, and are felt;
and 7, stage: typical fusiform disease spots, which account for 25.1% -50% of the leaf area;
and 8, stage: typical fusiform disease spots, which account for 50.1% -75% of the area of the leaves; high feeling;
and 9, stage: typical fusiform disease spots, the damage area is more than 75.1 percent of the area of the leaf, and the feeling is high;
(6) transplanting seedling pest resistant materials and performing field management:
a) transplanting the seedling pest 0-3 grade material to a field for planting in an identification garden, and directly eliminating the seedling pest 4-9 grade material;
b) field management: the fertilizer and water operation is equivalent to the local production level, and the insect prevention and the fungal disease prevention are realized;
(7) identification and evaluation of artificial resistance of field panicle blast:
a) injection and inoculation: and (4) selecting rice husks which just break the chest (the rice husks are not exposed) in each cell at the early stage of booting, injecting the mixed spore suspension inoculum obtained in the step (3) into the rice husks in the 2 nd to 4 th rows of each cell, and injecting 1ml of the mixed spore suspension inoculum into each ear. Inoculating 10 ears on each strain material, and marking the inoculated rice ears;
b) disease condition investigation and grading:
investigation period: the panicle blast survey is carried out at the early stage of yellow maturity of rice (when 80% of the grains at the tips of rice ears are mature), and 10 ears are surveyed for each material;
grading and evaluating standard:
level 0: no disease and high resistance;
level 1: small branch peduncles are attacked, the loss of each ear is less than or equal to 5 percent, and the resistance is achieved;
and 3, level: the disease of the principal axis or the neck of the ear is developed, the loss of each ear is less than 20 percent, and the resistance is moderate
And 5, stage: the main axis or the ear neck is attacked, grains are half shrunken, and the loss of each ear is less than 50 percent;
and 7, stage: the neck of the ear is attacked, most of the ears are shrunken, and the loss of each ear is less than 70 percent;
and 9, stage: the neck of the ear is attacked, the loss of each ear is more than or equal to 70 percent, and the ear is high;
wherein the panicle blast grade 0-3 material is a breeding available material, and the panicle blast grade 5-9 material is eliminated;
further, the greenhouse in step (4) has an automatic temperature and humidity control system, automatic sun-shading devices are installed above and around the greenhouse, and 10-20 inoculation pools are arranged inside the greenhouse according to the size of the greenhouse.
Furthermore, the material identified in the step (4) refers to a medium and high generation stable material with excellent comprehensive properties or natural germplasm resources bred in a breeding process.
Further, in the step (6), the seedling plague 0-3 grade materials are transplanted to a field and planted in an identification garden, 10 rows of materials are planted, 10 plants are planted in each row, a single seedling is planted, the row spacing is 13.3cm multiplied by 20cm, and the inoculated materials are arranged in sequence.
Furthermore, the breeding material in the step (7) can be used for breeding, and the breeding material has the advantages of high resistance to panicle blast, moderate growth period and good agronomic characters and yield characters.
Example 1
Determination of Rice blast-identifying Strain
1. Collection of disease samples of Magnaporthe grisea
Collecting rice blast disease ears at 20 places around the universe of Jiangsu province in China, sampling at multiple points around disease nursery in each target area, and allowing ears and neck nodes of the selected disease ears to wither and hyphae to be obviously visible in the neck nodes. And (3) collecting more than 10 ear of disease samples in each place, marking the information of collection date, the place and the like, and bringing the samples back to the laboratory for separation.
2. Isolation and culture of pathogenic bacteria
Stripping off branches and small ears of the ears with rice blast diseases, only leaving ear stems which are about 10cm and contain ear necks, washing the ear stems for 2-3 times by using sterile water, and placing the ear stems into a test tube. The tube was filled with sterile water and covered with a plug of silica gel and soaked at 26 ℃ for 24 hours. And (3) soaking a little absorbent cotton in sterile water, placing the absorbent cotton at the bottom of a new test tube, pouring the sterile water in the test tube filled with the ear stems, taking out the ear stems by using forceps, placing the ear stems in the new test tube, and culturing for 48 hours at 26 ℃. The ear stems were observed under a microscope at a suitable field (10X 10), and single spores of Pyricularia oryzae were picked with a pick needle and transferred to a slant of PDA medium, and the test tubes were incubated at 26 ℃ in the dark after the number of the strain was noted and the date of isolation.
3. Morphological identification of pathogenic bacteria
The size, morphology, color and the like of colonies at the observation period after the isolated strains were cultured on a PDA medium for 5 days, and the size, morphological characteristics and the like of hyphal morphological conidia were observed under a microscope, and finally 106 monospore strains with good hypha growth and normal spore morphology were selected (Table 1).
TABLE 1106 Single spore isolation sites and Strain names
4. DNA extraction of rice blast bacteria
Activating and culturing the separated and purified monospore strain of rice blast fungus on a PDA solid culture medium, selecting a proper amount of mycelium blocks, inoculating the mycelium blocks into a yeast liquid culture medium, performing shaking culture for 3-5 days at 28 ℃ and 120r/min, collecting the mycelium, subpackaging in 1.5mL centrifuge tubes, and extracting the genomic DNA of the rice blast fungus by using a CDAB method.
5. Avirulence gene detection and sequencing analysis
Based on the DNA sequence of the known avirulence gene, gene amplification primers (Table 2) synthesized by the Anhui general biosystems, Inc. were designed using Primer 5.0 software. Amplification system and reaction procedure: PCR system was 20. mu.l, MgCl2(25mmol L-1) 2.0. mu.L, 10 XPCR buffer 2.0. mu.L, primers (upstream and downstream primers, 10. mu. mol L)-1)1.5μL，dNTP(10mmol L-1) 0.4. mu.L, Taq DNA polymerase (5U. mu.L)-1) 0.2. mu.L, template DNA 2.0. mu.L, ddH2O11.9. mu.L. The PCR reaction program is pre-denaturation at 94 ℃ for 5min, denaturation at 94 ℃ for 45s, annealing at 55-58 ℃ (depending on different primers) for 45s, extension at 72 ℃ for 1min, 32 cycles, and final extension at 72 ℃ for 10 min.
And (4) detecting a result: detecting the DNA amplification product by gel electrophoresis.
And (3) sequence determination: and (3) sending the amplification product with clear electrophoresis bands to Anhui general biosystems, Inc. for sequencing, and comparing the sequencing result with a GeneBank database.
TABLE 2 avirulence genes and amplification primer information thereof
6. Cluster analysis and selection of strains for inoculation
According to the non-toxic gene sequence in the physiological race of rice blast fungus, the PowerMarker V3.25(http:// www.powermarker.net) software is used for cluster analysis, and the result shows that 106 strains can be divided into 7 subgroups. According to the "selection triple principle", i.e.the selected strain must encompass all subgroups; the strains selected should have the widest genetic variation among them; the selected strains should have wide geographical representativeness of the target ecological region, and 1 representative strain with large spore yield is selected for resistance inoculation identification in each subgroup.
Example 2
1196 parts of japonica rice seed for identifying blast resistance
1. Preparation of inoculum
And transferring the selected 7 representative strains into a culture dish filled with corn flour and rice straw juice culture medium on an ultra-clean workbench, and culturing for 7-10 days at 26 ℃ under the condition of full darkness. And (3) brushing the aerial hyphae on the surfaces of the bacterial colonies of the culture dishes by using a sterile brush pen respectively, airing the surfaces of the culture mediums, and then placing the culture mediums under a fluorescent lamp at 26 ℃ for 3-4 days until a large number of conidia are generated. Washing conidia on the surface of the culture medium with sterile water, filtering with double-layer gauze, and making into the final product with concentration of 4 × 105Taking spore liquid of the same amount of physiological microspecies, uniformly mixing, adding 0.02% (v/v) of Tween-20, and preparing into a mixed spore suspension for later use.
2. Seeding and management of germplasm resource materials
Respectively taking the resistant material Yangjing 7311 and the susceptible material 07GY31 as disease-resistant and susceptible controls, and sequencing 1196 germplasm resources in sequence. Sowing in 5-middle ten days of 2020, washing the seeds with clear water for multiple times, soaking the seeds for 10-20 s with sodium hypochlorite with the concentration of 0.5%, then washing the seeds with clear water for multiple times, performing seed soaking and germination acceleration by adopting a 'three-seed soaking and one-accelerating' method, namely soaking the seeds for three days and accelerating germination for one night, performing double-width sowing in a seed receiving room in sequence after the seeds emerge white and germinate, wherein the width of each seed receiving room is 50cm, the width of each seed receiving room is 20cm, the row spacing is 20cm, the row sowing is performed in sequence, the width of each seed is 4-5 cm, the row spacing is 5cm, each sowing material is about 120-150 grains, and then pressing the grains. The inoculation pools were set to be 20m long, 1.2m wide and 20cm deep, so that 400 parts of material could be sown per inoculation pool.
And (3) fertilizer and water management before inoculation: with field fertilizer and water management, heat preservation is paid attention to during germination, temperature control is paid attention to after emergence of seedlings to prevent seedlings from rising, urea is applied once every two days before inoculation to keep leaves green, and disease attack is facilitated.
3. Spray inoculation
And (3) inoculating the disease-resistant and disease-susceptible control and the material to be identified in the 3-4 leaf stage of the plant, humidifying the greenhouse, the disease-resistant and disease-susceptible control and the material to be identified by spraying water before inoculation, ensuring that the surface of the leaf is moist, keeping the humidity in the greenhouse between 90-100%, and spraying and inoculating the mixed spore suspension on the rice leaf by adopting a high-pressure spraying method for the disease-resistant and disease-susceptible control and the material to be identified. And (3) controlling the temperature in the greenhouse to be 26 ℃ within 24 hours after inoculation, keeping the humidity between 90 and 100 percent, and keeping the greenhouse in a dark condition after being shielded by a sunshade net. And (3) spraying with water for 2-8 days after inoculation for 5 times every day to ensure that the surface of the plant is wet, the humidity in the greenhouse is stably above 90%, the temperature in the greenhouse is controlled to be 26-28 ℃ in the daytime, the temperature at night is 20-24 ℃, and shading treatment is carried out to prevent direct sunlight.
4. Statistical investigation of disease incidence
7 days after inoculation, after the disease control 07GY31 is completely attacked, investigation is carried out, the phenotype of the seedling leaf with the heaviest attack in the seedlings is investigated, and 0-9 grade division and evaluation are adopted:
level 0: disease-free, immune;
level 1: needle-shaped large and small brown spots and high resistance;
and 2, stage: has large brown point, diameter less than 1mm, and good resistance
And 3, level: gray spots from circular to oval, brown edges, 1-2 mm in diameter and resistance;
4, level: typical fusiform lesion spots are 1 cm-2 cm long, are usually limited between two veins, are resistant when the area of injury is less than 2% of the area of the veins;
and 5, stage: typical fusiform disease spots, which account for 2% -10% of the area of the leaves and feel;
and 6, level: typical fusiform disease spots, which account for 10.1% -25% of the area of the leaves, and are felt;
and 7, stage: typical fusiform disease spots, which account for 25.1% -50% of the leaf area;
and 8, stage: typical fusiform disease spots, which account for 50.1% -75% of the area of the leaves; high feeling;
and 9, stage: typical fusiform lesion with lesion area larger than 75.1% of leaf area and high feeling
And (4) conclusion: after 7 days of inoculation, the disease-affected materials are fully developed, and the feasibility of the method is fully shown. In 1196 parts of japonica rice material, the level of the seedling plague resistance is obviously different, wherein the resistance level is only 116 parts in 3-grade and above materials, and the proportion is 9.7% (table 3), which indicates that the whole seedling plague resistance level of the existing japonica rice material is low, and a new antigen material is urgently needed to be introduced.
Example 3
Panicle blast resistance identification of 116 parts of japonica rice varieties to rice blast
1. Transplanting seedling pest resistant materials and performing field management:
a) transplanting the seedling pest grade 0-3 material to a field for planting in an identification garden, and directly eliminating the seedling pest grade 4-9 material.
b) Field management: the fertilizer and water operation is equivalent to the local production level, and the insect prevention and the fungal disease prevention are realized;
2. identification and evaluation of artificial resistance of field panicle blast:
a) injection and inoculation: and (4) selecting rice husks which just break the chest (the rice husks are not exposed) in each cell at the early stage of booting, injecting the mixed spore suspension inoculum obtained in the step (3) into the rice husks in the 2 nd to 4 th rows of each cell, and injecting 1ml of the mixed spore suspension inoculum into each ear. Each line material was inoculated with 10 ears and the inoculated ears were marked.
b) Disease condition investigation and grading:
investigation period: the panicle blast survey is carried out at the early stage of yellow maturity of rice (when 80% of the grains at the tips of rice ears are mature), and 10 ears are surveyed for each material;
grading and evaluating standard:
level 0: no disease and high resistance;
level 1: small branch peduncles are attacked, the loss of each ear is less than or equal to 5 percent, and the resistance is achieved;
and 3, level: the disease of the principal axis or the neck of the ear is developed, the loss of each ear is less than 20 percent, and the resistance is moderate
And 5, stage: the main axis or the ear neck is attacked, grains are half shrunken, and the loss of each ear is less than 50 percent;
and 7, stage: the neck of the ear is attacked, most of the ears are shrunken, and the loss of each ear is less than 70 percent;
and 9, stage: the neck of the ear is attacked, the loss of each ear is more than or equal to 70 percent, and the ear is highly susceptible
Wherein the panicle blast grade 0-3 material is a breeding available material, and the panicle blast grade 5-9 material is eliminated;
the test identification results are shown in table 3:
TABLE 3 presentation of panicle blast resistance of material with more than grade 3 of Miao blast
As seen from Table 3, the resistance level of the panicle plague reaches 3 grades and is 52 parts in the 116 identified materials with the plague reaching 3 grades, and the percentage is 44.83%; in particular 12 parts of materials in Jiaxing area of Zhejiang, the spike blast can be fully resistant to more than the level, and the materials can be used for breeding resistant germplasm.
Although the present invention has been described in detail with reference to the above embodiments, it is only a part of the embodiments of the present invention, not all of the embodiments, and other embodiments can be obtained without inventive step according to the embodiments, and the embodiments are within the scope of the present invention.
Claims (10)
1. A large-scale rice blast resistance identification method suitable for breeding application is characterized by comprising the following steps:
step 1, determining rice blast identification strains: firstly, carrying out non-toxic gene sequencing analysis by using a plurality of rice blast strains collected at multiple points in a target ecological area, carrying out cluster analysis according to a sequencing result to screen out representative physiological races of the target ecological area, and culturing the screened physiological races to prepare a mixed spore suspension;
step 2, identifying the plague resistance: sowing identification materials, inoculating the mixed spore suspension obtained in the step 1 in a 3-4 leaf stage of a plant, and investigating and grading the completely diseased contrast to be infected;
step 3, rice blast resistance identification: transplanting the screened seedling pest resistant material to a field according to the grading of the step 2, and performing field management; inoculating the mixed spore liquid obtained in the step 1 at the early stage of booting; in the early stage of yellow maturity of rice, the disease condition is investigated and graded.
2. The method for identifying rice blast resistance in scale suitable for breeding applications as claimed in claim 1, wherein in step 1, the specific steps of performing avirulence gene sequencing analysis by using a plurality of rice blast strains collected at multiple points in a target ecological region are as follows:
a) collecting and separating a large amount of rice blast monospore strains at multiple points in a target ecological area;
b) aiming at different collected monospore strains, 7 avirulence gene sequencing primers are used for sequencing analysis.
3. The method for large-scale identification of rice blast resistance suitable for breeding applications as claimed in claim 2, wherein the avirulence gene sequencing is AvrPi9, AvrPia, AvrPii, AvrPizt, AvrPik, AvrPita, AvrPib7 avirulence genes.
4. The method for identifying resistance to rice blast in scale suitable for breeding application as claimed in claim 1, wherein in the step 1, the step of performing cluster analysis according to the sequencing result to screen out the representative physiological races of the target ecological region comprises the following steps:
a) performing clustering analysis by using PowerMarker V3.25 software according to the avirulence gene sequences of different rice blast strains;
b) aiming at the clustering result, 1-2 representative strains are selected from each subgroup for subsequent resistance inoculation identification.
5. The method for identifying resistance to rice blast in scale suitable for breeding applications as claimed in claim 1, wherein in step 1, the mixed spore suspension is prepared as follows: separately culturing the selected physiological microspecies, washing spores on the surface of a culture medium with sterile water, filtering hyphae with gauze, taking the spore liquid of the physiological microspecies with the same amount, uniformly mixing, and adding 0.02% (v/v) Tween-20 to prepare a mixed spore suspension.
6. The method for identifying resistance to rice blast suitable for breeding applications as claimed in claim 1, wherein in the step 2, classification and evaluation are performed by using 0-9 grades:
level 0: disease-free, immune;
level 1: needle-shaped large and small brown spots and high resistance;
and 2, stage: has large brown point, diameter less than 1mm, and good resistance
And 3, level: gray spots from circular to oval, brown edges, 1-2 mm in diameter and resistance;
4, level: typical fusiform lesion spots are 1 cm-2 cm long, are usually limited between two veins, are resistant when the area of injury is less than 2% of the area of the veins;
and 5, stage: typical fusiform disease spots, which account for 2% -10% of the area of the leaves and feel;
and 6, level: typical fusiform disease spots, which account for 10.1% -25% of the area of the leaves, and are felt;
and 7, stage: typical fusiform disease spots, which account for 25.1% -50% of the leaf area;
and 8, stage: typical fusiform disease spots, which account for 50.1% -75% of the area of the leaves; high feeling;
and 9, stage: typical fusiform disease spots, the damage area is more than 75.1 percent of the area of the leaf, and the feeling is high;
wherein, 4-9 grade materials of the Miao ethnicity are directly eliminated, and 0-3 grade materials are continuously identified.
7. The method for identifying rice blast resistance on a large scale suitable for breeding applications as claimed in claim 1, wherein the material identified in step 2 refers to medium and high generation stable materials or natural germplasm resources with excellent comprehensive properties developed in breeding process.
8. The method as claimed in claim 1, wherein in step 2, germination is performed before seeding, after seeds emerge and sprout, the seeds are sowed in the inoculation pools in sequence, each inoculation pool is sowed in double rows with the width of 50cm and the distance between the rows of 20cm, the seeds are sowed in sequence with the width of 4 cm-5 cm and the distance between the rows of 5cm, and each row is sowed with 150 grains of 120-150 grains of each material, and then the grains are pressed.
9. The method for identifying rice blast resistance in large scale suitable for breeding use according to claim 1, wherein in the step 3, the grading and evaluation criteria for disease condition investigation and grading are as follows:
level 0: no disease and high resistance;
level 1: small branch peduncles are attacked, the loss of each ear is less than or equal to 5 percent, and the resistance is achieved;
and 3, level: the disease of the principal axis or the neck of the ear is developed, the loss of each ear is less than 20 percent, and the resistance is moderate
And 5, stage: the main axis or the ear neck is attacked, grains are half shrunken, and the loss of each ear is less than 50 percent;
and 7, stage: the neck of the ear is attacked, most of the ears are shrunken, and the loss of each ear is less than 70 percent;
and 9, stage: the neck of the ear is attacked, the loss of each ear is more than or equal to 70 percent, and the ear is high;
wherein, the 0-3 grade material is the material which can be used for breeding, and the 5-9 grade material for rice blast is directly eliminated.
10. The method for identifying the rice blast resistance in scale suitable for breeding application as claimed in claim 1, wherein injection inoculation is adopted in the step 3, and the specific steps are as follows: selecting rice husks which just break the chest in each cell at the early stage of booting, injecting the mixed spore suspension inoculum obtained in the step (1) into the rice husks in the rows 2-4 of each cell, and injecting 1ml into each ear; each line material was inoculated with 10 ears and the inoculated ears were marked.
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN114711109A (en) * | 2022-04-12 | 2022-07-08 | 海南省农业科学院粮食作物研究所 | Identification method for rice blast resistance |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20040083501A1 (en) * | 2000-10-20 | 2004-04-29 | Leong Sally A. | Plant genes that confer resistance to strains of Magnaporthe grisea having AVR CO39 cultivar specificity gene |
| CN105210749A (en) * | 2015-10-12 | 2016-01-06 | 江苏里下河地区农业科学研究所 | A kind of method improving rice blast neck blast resistance qualification accuracy |
-
2021
- 2021-04-15 CN CN202110404922.9A patent/CN113249429A/en active Pending
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20040083501A1 (en) * | 2000-10-20 | 2004-04-29 | Leong Sally A. | Plant genes that confer resistance to strains of Magnaporthe grisea having AVR CO39 cultivar specificity gene |
| CN105210749A (en) * | 2015-10-12 | 2016-01-06 | 江苏里下河地区农业科学研究所 | A kind of method improving rice blast neck blast resistance qualification accuracy |
Non-Patent Citations (7)
| Title |
|---|
| 朱业宝;方珊茹;沈伟峰;陈立?;江川;王金英;: "国外引进水稻种质资源的稻瘟病抗性基因检测与评价", 植物遗传资源学报, no. 02 * |
| 李刚;王健;曹奎荣;李军;孙祥良;程保山;罗伯祥;杨子博;徐卫军;唐久友;储成才;袁彩勇;: "江苏水稻主导品种的稻瘟病抗性及抗性基因遗传多样性", 湖南农业大学学报(自然科学版), no. 03 * |
| 李旭升;向小娇;申聪聪;杨隆维;陈凯;王小文;邱先进;朱小源;邢丹英;徐建龙;: "水稻重测序核心种质资源的稻瘟病抗性鉴定与评价", 作物学报, no. 06 * |
| 李育红等: "杂交籼稻稻瘟病抗性分子标记辅助育种的探索与实践", 《扬州大学学报（农业与生命科学版）》, vol. 41, no. 6, pages 23 * |
| 王妍;郑文静;王辉;张丽霞;王世维;赵家铭;刘志恒;: "辽宁省主栽水稻品种抗稻瘟病基因的鉴定及分析", 植物遗传资源学报, no. 03 * |
| 袁筱萍;魏兴华;徐群;王一平;余汉勇;王彩红;: "中国主栽品种对7个白叶枯病菌菌株的抗性分析", 植物保护, no. 04 * |
| 马作斌;王丽丽;顾爽;付亮;王昌华;张丽颖;郑文静;: "辽宁地区粳稻品种抗稻瘟病基因Pita的等位基因类型及其功能研究", 辽宁农业科学, no. 04 * |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN114711109A (en) * | 2022-04-12 | 2022-07-08 | 海南省农业科学院粮食作物研究所 | Identification method for rice blast resistance |
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