CN115362844A - Method for identifying soybean epidemic disease resistant resources, varieties and strains - Google Patents
Method for identifying soybean epidemic disease resistant resources, varieties and strains Download PDFInfo
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Abstract
The invention discloses a method for identifying soybean epidemic disease resistant resources, varieties and strains. The identification method for soybean epidemic disease resistant resources, varieties and strains, disclosed by the invention, has the advantages of high operation speed, simplicity, convenience, low cost, short period, less space inoculants required by tests, low requirement on environment, accurate and reliable identification result, capability of transplanting resistant plants to finish seed propagation after resistance identification is finished, and acceleration of homozygous and utilization of resistant materials. Therefore, the identification method of the invention is not only beneficial to the high-efficiency screening of soybean disease-resistant resources and the rapid breeding of disease-resistant varieties, but also can accelerate the resistance genetic research of soybean epidemic diseases and the related research of pathogenicity variation of soybean phytophthora. Has wide application prospect in soybean breeding and resistance heredity and disease resistance mechanism research.
Description
Technical Field
The invention belongs to the technical field of crop disease resistance identification, and particularly relates to an identification method of soybean epidemic disease resistant resources, varieties and strains.
Background
Phytophthora sojae Kaufmann & Gerdemann (Phytophthora sojae) caused soybean Phytophthora sojae is a devastating disease in soybean production. The disease is commonly caused in multiple provinces such as Uygur autonomous region in Xinjiang, heilongjiang province, anhui province, fujian province, jilin province and the like. Phytophthora sojae can infect in the whole growth period of soybean, causing root rot, stem rot, plant dwarfing, withering and death, generally reducing yield by 10-30% in field, reducing yield by 60-90% in disease-sensitive plot, and even causing dead production. The current worldwide economic loss due to phytophthora sojae is about $ 10 billion per year. The phytophthora sojae is a soil-borne pathogenic bacterium, and the oospore of the phytophthora sojae can survive in soil for a long time and is very difficult to control. The most economical, effective and environment-friendly method for preventing and treating soybean epidemic disease is to plant disease-resistant varieties.
At present, a large number of epidemic-resistant resources are identified through disease-resistant resource screening at home and abroad, and more than 30 epidemic-resistant genes are identified in the disease-resistant resources. So far, more than 10 soybean epidemic disease resistant genes have been identified in China. The phytophthora sojae colony has complicated structure and fast virulence variation. The service life of the soybean anti-epidemic disease gene is generally considered to be 10-15 years, and in order to continuously and effectively prevent the soybean epidemic disease, new anti-epidemic sources must be continuously excavated and utilized. Therefore, a method for rapidly and accurately identifying soybean epidemic disease resistance resources is needed, so that a new resistance source can be effectively obtained for disease resistance breeding. By utilizing the accumulation of a plurality of disease-resistant genes, a new soybean variety with durable and stable resistance is cultivated.
At present, most researchers adopt a hypocotyl wound inoculation method to identify and evaluate the soybean epidemic disease resistance. However, the operation of the inoculation process is complex, and the requirement on the environmental culture condition of the inoculated plants is high, so that soybean seedling stalks are easily broken when hypocotyls are wounded, and phytophthora sojae inoculants sometimes slide off from the hypocotyls in the transportation and moisture preservation processes, so that false positive results are caused. Therefore, there is a need to establish a new method for rapidly, simply, efficiently and accurately identifying the disease resistance of soybean phytophthora blight.
Disclosure of Invention
The invention aims to provide a novel method for rapidly, simply, efficiently and accurately identifying the disease resistance of soybean epidemic disease.
In order to achieve the above object, the present invention provides a novel method for identifying disease resistance of soybean phytophthora blight.
The method for identifying the disease resistance of the soybean phytophthora blight provided by the invention comprises the following steps:
(1) And (3) culturing soybean inoculated plants: culturing seeds of soybean resources, varieties or strains to be detected to obtain soybean inoculated plants;
(2) Culturing the soybean phytophthora inoculum: culturing the soybean epidemic disease mold strain, preparing a mycelium block and obtaining a phytophthora inoculum;
(3) Soybean plant inoculation: aligning cotyledon nodes of soybean plants to be inoculated, trapping the cotyledon nodes into a bundle, putting the bundle into a container filled with water, adding a proper amount of water into the inoculation container, and then obtaining mycelium blocks of phytophthora inoculants to obtain inoculated plants;
(4) Inoculating plant culture and disease resistance evaluation: and culturing the inoculated plants, and evaluating the disease resistance of the soybean resources to be tested to the soybean phytophthora blight after culturing.
In the method, the step (1) is preceded by a step of cleaning soybean material seeds to be detected; the cleaning method can be used for selecting healthy seeds for sowing.
In the above method, in the step (1), the soybean material seeds to be tested may be sown in a container using vermiculite as a matrix.
The seeding container can be a seedling tray, a paper cup or a plastic cup made of various materials and having a depth of 6-13cm, and specifically can be a disposable paper cup (200 mL paper cup with the specification of 85mm x 70 mm) having a depth of 8 cm.
The temperature of the culture can be 20-30 deg.C (or 20-25 deg.C or 26-30 deg.C), specifically 20 deg.C or 21 deg.C or 22 deg.C or 23 deg.C or 24 deg.C or 25 deg.C or 26 deg.C or 27 deg.C or 28 deg.C or 29 deg.C or 30 deg.C, preferably 25 deg.C; the culture time may be 7-15 days (or 7-11 days or 12-15 days), specifically 7 days or 8 days or 9 days or 10 days or 11 days or 12 days or 13 days or 14 days or 15 days, preferably 10 days.
In the above method, in the step (2), the phytophthora sojae strain may be a common phytophthora sojae strain in the art, including strains with different virulence genotypes, such as Race1, JS2, BS7, BSD36, HN1, and the like. In a specific embodiment of the invention, the strain of phytophthora sojae is the Race1 strain.
The method for culturing can be inoculating the strain of the phytophthora sojae in the culture medium.
The medium may be V8 medium.
Wherein, the solvent of the V8 culture medium is distilled water, and the solutes and the concentrations are respectively as follows: v8 vegetable juice 200g/L, caCO 3 3g/L, agar 20g/L, pH5.8. The solvent of the CA culture medium is distilled water, and the solutes and the concentrations are respectively as follows: 200g/L carrot and 20g/L agar.
The temperature of the culture can be 20-28 deg.C (or 20-24 deg.C or 24-28 deg.C), specifically 20 deg.C or 21 deg.C or 22 deg.C or 23 deg.C or 24 deg.C or 25 deg.C or 26 deg.C or 27 deg.C or 28 deg.C, preferably 25 deg.C; the culture time may be 4-7 days (or 4-5 days or 5-7 days), specifically 4 days or 5 days or 6 days or 7 days, preferably 5 days.
The diameter of the mycelium block can be 5-8mm.
The method for preparing the mycelium block can be to punch the mycelium block by using a puncher with the aperture of 5-8mm. The perforator can be any conventional cylindrical article capable of making the pathogen culture into a disk with a diameter of 5-8mm, such as 200 μ l pipette tip, 1mL pipette tip, beverage pipette and other perforating punches, preferably 1mL pipette tip.
In the above method, before the step (3), the method further comprises the following steps: and (3) pulling the soybean plants out of the vermiculite, washing the vermiculite on the root system off by using clear water, and selecting healthy plants with consistent height and size.
In the above method, in the step (3), the cotyledon nodes of the plurality of soybean plants are aligned and tied into a bundle and placed into the inoculation container, and the cotyledon nodes of 5 to 10 soybean plants may be aligned and tied into a bundle, and a part of the root system is cut off to make the length from the cotyledon node to the root of each plant consistent, and then the bundle is placed into the inoculation container.
The inoculation container can be any conventional cup-shaped container, such as a disposable paper cup, a disposable plastic cup, a glass beaker, a plastic beaker, and the like, preferably an 800mL clear plastic cup.
Each inoculation container can be used for containing multiple portions of soybean materials to be tested. Specifically, 5-10 parts of the soybean material to be tested can be placed in a 800mL transparent plastic cup. Preferably, 8 parts of the soybean material to be tested are placed in a 800mL disposable paper cup.
The adding of a proper amount of water into the inoculation container can be adding water into the inoculation container to a position 0.5-1cm below the cotyledonary node of the plant. When the inoculation vessel is a 800mL clear plastic cup, 700mL sterile water is preferably added to the inoculation vessel.
When the inoculation containers are 800mL clear plastic cups and 700mL sterile water is added to each inoculation container, 10-20 pieces of mycelium, preferably 15 pieces of mycelium, can be inoculated into each inoculation container.
In the above method, in the step (4), the temperature of the culture may be 22 to 28 ℃ (or 22 to 25 ℃ or 25 to 28 ℃), specifically 22 ℃ or 23 ℃ or 24 ℃ or 25 ℃ or 26 ℃ or 27 ℃ or 28 ℃, preferably 25 ℃; the culture time may be 3-7 days, specifically 3 days, 4 days, 5 days, 6 days, 7 days, and preferably 5 days.
In the above method, in the step (4), the criterion for evaluating the disease resistance of the soybean material to be tested against phytophthora rot of soybean is one or two of the following criteria:
the first criterion is as follows: if the plant survives and the plant grows normally, the soybean material to be detected is resistant to soybean phytophthora blight (R); if the plant dies, the soybean material to be detected is susceptible to soybean epidemic disease (S).
The second criterion is as follows: if the plant mortality is less than or equal to 30%, the soybean material to be detected is resistant to soybean phytophthora blight (R), if the plant mortality is more than or equal to 70%, the soybean material to be detected is susceptible to soybean phytophthora blight (S), and if the plant mortality is more than 30% and less than 70%, the soybean material to be detected is intermediate in type (I) to soybean phytophthora blight.
In practical application, the single soybean plant pair is identifiedWhen resistance to epidemic diseases is desired, e.g. identification of hybridized F 1 And F 2 When a single soybean plant of a genetic group is used, evaluation of soybean epidemic disease resistance can be performed according to the death of the phenotype of the plant. In the identification of soybean varieties, resources or F 3 And when the high-generation strain has the resistance to the soybean epidemic disease, the disease resistance of the soybean to the epidemic disease can be evaluated according to the plant mortality, namely the standard two.
In any of the above methods, the culture environment may be a light temperature controllable culture room, such as a greenhouse, a light incubator, a plant culture room, etc., preferably a light incubator.
In order to achieve the above object, the present invention also provides a novel use of the above method.
The invention provides the application of the method in any one of the following a 1) to a 4):
a1 Identifying or evaluating disease resistance of soybean against soybean blight;
a2 Screening or auxiliary screening of soybean epidemic disease resistance resources, varieties and strains;
a3 Screening or auxiliary screening of soybean epidemic disease resources, varieties and strains;
a4 ) soybean breeding.
In any of the above methods or uses, the soybean comprises not only various soybean resources and related species, varieties, lines, breeding materials thereof, but also varieties, cultivars thereof, and breeding progeny thereof. In a specific embodiment of the invention, the soybean is at least one of the following varieties: williams, zhonghuang 13, jike bean No. 2, qicha bean No. 1, xiuyan 94-1, ludou bean No. 4, mutagenesis 30, precocious 18, williams82 and Wan bean 15.
The soybean breeding aims at cultivating soybean epidemic disease resistant varieties.
The invention provides a method for identifying the disease resistance of soybean phytophthora blight for the first time, the method has the advantages of high operation speed, simplicity, convenience, low cost, short period, less space inoculants required by tests, low requirement on environment, accurate and reliable identification result because pathogenic bacteria infection is the same as natural infection, and meanwhile, after the resistance identification is finished, resistant plants can be transplanted to finish the propagation of seeds, thereby accelerating the homozygous utilization of resistant materials. Therefore, the identification method of the invention is not only beneficial to the efficient screening of soybean disease-resistant resources and the rapid breeding of disease-resistant varieties, but also can accelerate the resistance genetic research of soybean phytophthora blight and the related research of pathogenicity variation of soybean phytophthora. Has wide application prospect in soybean breeding and resistance heredity and disease resistance mechanism research.
Detailed Description
The present invention is described in further detail below with reference to specific embodiments, which are given for the purpose of illustration only and are not intended to limit the scope of the invention. The examples provided below serve as a guide for further modifications by a person skilled in the art and do not constitute a limitation of the invention in any way.
The experimental procedures in the following examples, unless otherwise indicated, are conventional and are carried out according to the techniques or conditions described in the literature in the field or according to the instructions of the products. Materials, reagents and the like used in the following examples are commercially available unless otherwise specified.
Williams, zhonghuang 13, jike bean No. 2, qicha bean No. 1, xiuyan 94-1, lu bean No. 4, mutation 30, precocity 18, williams82 and Wan bean 15 in the following examples are described in the literature "Zhu Sha Dong et al, molecular identification of a new gene against phytophthora sojae root rot. The article for crops, 2007,33 (1): 154-157; zuguiqing, evaluation of soybean resistance to epidemic diseases, excavation of disease-resistant genes and analysis of candidate genes [ D ]. Chinese academy of agricultural sciences, 2013; identification of phytophthora root rot resistant gene and analysis of candidate gene [ D ] of the academy of agricultural sciences of china, 2017; soyabean gene discovery and specific marker development [ D ] chinese academy of agricultural sciences, 2018; zhong et al, genetic mapping and Molecular characterization of a broad-spectrum photophthora sojae resistance gene in Chinese scientific Journal of Molecular Sciences,2019, 20.
Phytophthora sojae (Phytophthora sojae) strain, race1, in the examples below is described in "Yang et al, rats of Phytophthora sojae in Iowa soybeans fields. Plant diseases" 1996, 1418-1420. Reference numerals in FIGS.
The solvent of the V8 medium in the following examples is distilled water, and the solutes and concentrations are as follows: v8 vegetable juice 200g/L, caCO 3 3g/L, agar 20g/L, pH5.8.
Example 1 method for identifying disease resistance to Soybean epidemic disease (zoospore Natural infection method)
(1) And (3) culturing soybean plants: selecting a plurality of healthy and plump seeds from each part of soybean material, sowing the seeds in a disposable paper cup which takes vermiculite as a matrix and has the depth of 8cm, putting the paper cup into a greenhouse for culturing after sowing, and culturing for 7-15 days to obtain a soybean plant.
(2) Preparation of inoculum: inoculating Phytophthora sojae strain into culture medium, culturing for 4-8 days, and preparing hypha block with diameter of 5-8mm on the colony with a puncher to obtain inoculum.
(3) Inoculating soybean plants: firstly, pulling the soybean plant obtained in the step (1) out of vermiculite, and washing away the vermiculite on a root system by using clear water; then selecting a plurality of healthy plants with consistent height and size for each part of soybean material, tying the plants into a bundle after the cotyledon nodes are aligned, putting the bundle into an inoculation container, and adding water into the inoculation container until the position 0.5-1cm below the cotyledon nodes of the plants; and (3) finally, beating the fungus cake of the inoculum prepared in the step (2) and putting the fungus cake into an inoculation container.
(4) Inoculating plant culture and resistance evaluation: and (3) culturing the inoculated soybean plants in a greenhouse or a plant growth chamber for 3-6 days, and then carrying out disease investigation, and carrying out resistance identification and evaluation according to the following first standard or second standard.
The first standard: if the plant grows normally, the soybean material to be detected is resistant to phytophthora sojae (R); if the plant withers and dies, the soybean material to be detected is susceptible to phytophthora sojae (susceptable, S).
And a second standard: if the plant mortality is less than or equal to 30%, the soybean material to be detected is resistant to soybean epidemic disease (R), if the plant mortality is more than or equal to 70%, the soybean material to be detected is susceptible to soybean epidemic disease (S), and if the plant mortality is more than 30% and less than 70%, the soybean material to be detected is intermediate type (intermediate, I) to soybean epidemic disease.
Example 2 application of method for identifying disease resistance of soybean phytophthora
1. Test material
The 10 parts of soybean germplasm test materials are as follows: williams, zhonghuang 13, jike bean No. 2, qicha bean No. 1, xiuyan 94-1, ludou bean No. 4, mutagenesis 30, precocious 18, williams82 and Wan bean 15.
2. Identification of disease resistance of soybean phytophthora blight
The evaluation of the phytophthora sojae resistance of the test materials was carried out in the same manner as in example 1. The method comprises the following specific steps:
(1) And (3) culturing soybean plants: selecting 15 healthy and plump seeds from each part of soybean material, sowing the seeds in a disposable paper cup of 200mL taking vermiculite as a matrix, sowing one cup of soybean material in each part of the soybean material, putting the soybean material into a greenhouse at 25 ℃ after sowing, and culturing for 10 days under the conditions of 12-hour illumination/12-hour dark to obtain soybean plants.
(2) Preparation of inoculum: inoculating Phytophthora sojae (Phytophthora sojae) strain Race1 into V8 culture medium, culturing at 25 deg.C for 5 days, and preparing hypha block with diameter of 8mm on bacterial colony with 1mL pipette tip end to obtain inoculum.
(3) Inoculating soybean plants: firstly, pulling the soybean plant obtained in the step (1) out of vermiculite, and washing away the vermiculite on a root system by using clear water; then selecting 10 healthy plants with the same size for each part of soybean material, tying the leaves with plastic tying silk into a bundle after aligning the leaf nodes, and cutting off part of root systems to ensure that the length from the leaf nodes to the roots of each plant is the same; the material was then placed into 800mL clear plastic cups, 10 parts of material were placed in each cup, and then about 700mL of sterile water was added to the cups; and (3) finally, inoculating the mycelium blocks of the inoculum prepared in the step (2) into transparent plastic cups, and inoculating 15 mycelium blocks in each cup.
(4) Inoculated plant culture and resistance evaluation: the inoculated soybean plants were placed in a 25 ℃ plant growth chamber and cultured under 12 hours of light/12 hours of darkness. Disease investigation was performed after 5 days of culture, and resistance identification and evaluation were performed according to the following criteria: in each part of soybean material, if the plant mortality is less than or equal to 30%, the soybean material to be detected is resistant to phytophthora sojae (resistance, R), if the plant mortality is more than or equal to 70%, the soybean material to be detected is susceptible to the phytophthora sojae (susceptable, S), and if the plant mortality is more than 30% and less than 70%, the soybean material to be detected is intermediate in type to the phytophthora sojae (intermediate, I).
The following embryo axis wound inoculation method was also used as a control. The hypocotyl wound inoculation method refers to a document 'Zhongchao, soybean anti-epidemic disease gene discovery and specific marker development [ D ]. Chinese academy of agricultural sciences, 2018', and specific steps are detailed in Table 2.
3. Results of the evaluation
The results are shown in Table 1. The identification result of the soybean disease resistance of 10 parts of soybean germplasm materials by using the hypocotyl wound inoculation method shows that: of the 10 parts of soybean test material, 7 parts of the test material showed disease resistance, and 3 parts of the test material showed infection. The identification result of the zoospore natural infection method is consistent with the identification result, and compared with the hypocotyl wound inoculation method, the zoospore natural infection method also has the following advantages: the operation speed is high, the operation is simple and convenient, the cost is low, the test period is short, the space and the inoculum required by the test are less, the requirement on the environment is not high, the manual scratching is not needed, the infection of pathogenic bacteria is the same as the natural infection, the identification result is accurate and reliable, meanwhile, after the resistance identification is finished, the resistant plants can be transplanted to finish the propagation of seeds, the homozygosis and the utilization of resistant materials are accelerated, and the detailed comparison conditions are shown in table 2.
TABLE 1 evaluation results of two evaluation methods on Phytophthora sojae disease resistance
Table 2 shows the comparison between the zoospore inoculation method and the hypocotyl wound inoculation method
The present invention has been described in detail above. It will be apparent to those skilled in the art that the invention can be practiced in a wide range of equivalent parameters, concentrations, and conditions without departing from the spirit and scope of the invention and without undue experimentation. While the invention has been described with reference to specific examples, it will be appreciated that the invention may be further modified. In general, this application is intended to cover any variations, uses, or adaptations of the invention following, in general, the principles of the invention and including such departures from the present disclosure as come within known or customary practice within the art to which the invention pertains. The use of some of the essential features is possible within the scope of the claims attached below.
Claims (10)
1. A method for identifying disease resistance of soybean phytophthora blight comprises the following steps:
(1) And (3) culturing soybean plants: culturing soybean material seeds to be detected to obtain soybean plants;
(2) Culturing of the inoculum: culturing the soybean epidemic mold strains, preparing mycelium blocks and obtaining an inoculum;
(3) Soybean plant inoculation: firstly, aligning a plurality of cotyledon nodes of the soybean plants, putting the soybean plants into an inoculation container in a bundle, adding water into the inoculation container, and adding the inoculum into the inoculation container to obtain an inoculated plant;
(4) Inoculating plant culture and resistance evaluation: and culturing the inoculated plant, and evaluating the disease resistance of the soybean material to be tested to the phytophthora sojae after culturing.
2. The method of claim 1, wherein: the step (1) is also preceded by a step of cleaning soybean seeds to be detected; the cleaning method is to select healthy seeds for sowing.
3. The method according to claim 1 or 2, characterized in that: in the step (1), the soybean material seeds to be tested are sowed in a sowing container with vermiculite as a matrix;
and/or the seeding container is a disposable paper cup with the depth of 8 cm;
and/or the temperature of the culture is 20-30 ℃; the culture time is 7-15 days.
4. A method according to any one of claims 1 to 3, wherein: in the step (2), the cultivation method is to inoculate the phytophthora sojae strain in the culture medium;
and/or the phytophthora sojae strain is a Race1 strain;
and/or the culture medium is a V8 culture medium;
and/or the culture temperature is 20-28 ℃; the culture time is 4-7 days;
and/or the diameter of the mycelium block is 5-8mm.
5. The method according to any one of claims 1 to 4, wherein: the method also comprises the following steps before the step (3): and (3) pulling the soybean plants out of the vermiculite, washing the vermiculite on the root system off by using clear water, and selecting healthy plants with consistent height and size.
6. The method according to any one of claims 1 to 5, wherein: in the step (3), 10 soybean plant cotyledonary nodes are aligned and tied into a bundle, and part of root systems are cut off to enable the length from the cotyledonary node to the root of each plant to be consistent;
and/or, adding water into the inoculation container to a position 0.5-1cm below the cotyledon node of the plant.
7. The method according to any one of claims 1 to 6, wherein in the step (4), the temperature of the culture is 20 to 28 ℃ and the culture time is 4 to 7 days.
8. The method as claimed in any one of claims 1 to 7, wherein in the step (4), the criteria for evaluating the disease resistance of the soybean material to be tested against phytophthora sojae are as follows: if the plant grows normally, the soybean material to be detected is resistant to phytophthora sojae; if the plant dies, the soybean material to be detected is infected with the phytophthora sojae.
9. The method according to any one of claims 1 to 7, wherein in the step (4), the criterion for evaluating the disease resistance of the soybean material to be tested against soybean blight is: if the plant mortality is less than or equal to 30 percent, the soybean material to be detected is resistant to phytophthora sojae; if the plant death rate is more than or equal to 70 percent, the soybean material to be detected is infected by phytophthora sojae; if the plant mortality is more than 30% and less than 70%, the soybean material to be detected is of an intermediate type to the soybean phytophthora.
10. Use of the method of any one of claims 1 to 9 in any one of a 1) to a 4) as follows:
a1 Identifying or evaluating disease resistance of soybeans to phytophthora sojae;
a2 Screening or auxiliary screening of soybean epidemic disease resistant resources, varieties and strains;
a3 Screening or auxiliary screening of soybean epidemic disease susceptible resources, varieties and strains;
a4 ) soybean breeding.
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