CN110004207B - SCD (SCD) plasmodiophora radicis physiological race identification system, identification method and application - Google Patents
SCD (SCD) plasmodiophora radicis physiological race identification system, identification method and application Download PDFInfo
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Abstract
The invention belongs to the technical field of identification of physiological races of brassica plasmodiophora, and particularly relates to an SCD (substation configuration description) plasmodiophora physiological race identification system and an identification method. The SCD plasmodiophora physiological race identification system comprises 9 host materials and 14 physiological races. The clubroot bacteria to be detected are inoculated into 9 host materials, and the physiological small type of the clubroot bacteria to be detected can be judged according to the disease resistance of the host materials. The SCD identification system and the corresponding identification method are beneficial to classification, distribution, variation, popularity and prevention research of the physiological races of the clubroot, and greatly accelerate the breeding process of Chinese cabbages and other brassica crops resistant to the clubroot.
Description
Technical Field
The invention belongs to the technical field of identification of physiological races of brassica plasmodiophora, and particularly relates to an SCD (substation configuration description) plasmodiophora physiological race identification system, an identification method and application.
Background
Rapid transmission of clubroot caused by the soil-borne obligate parasitic plant pathogen clubroot has become one of the most serious diseases in cruciferous crops worldwide. Infection by plasmodiophora radicis causes proliferation and hypertrophy of host root cells, which inhibits nutrient and water transport causing plant wilting, reducing plant yield. Clubroot can affect almost all brassica species, including chinese cabbage, canola, and radish. Crop rotation and chemical treatments such as fluazinam and cyazofamid have been shown to be effective in the control of clubroot. However, crop rotation will take a long time because the rhizomes can survive in the soil for more than 15 years; the chemical treatment method may cause environmental pollution. Thus, disease-resistant breeding is the most effective and environmentally friendly strategy for controlling clubroot.
Genetic variation of pathogenic bacteria causes resistance loss of clubroot disease-resistant varieties, and physiological race classification becomes more complex. Because of genetic variation of the clubroot or variation of the main pathotype of the clubroot in the soil, the clubroot resistant variety of one or two disease resistant genes which result in transformation will lose resistance within a few years, requiring re-breeding again. The key to successful breeding of disease-resistant varieties is the identification of physiological races of the plasmodiophora radiata. Based on investigation of the disease conditions of different brassica hosts over the past few decades, several physiological race identification systems for brassica plasmodiophora have been developed: (1) Williams identification systems (reference (1), williams, P.H. 1966), comprising four host Badger shifters, jersey Queen, laurentian and Wilhelmsburger, theoretically classify the physiological races of Rhizopus into 16. (2) ECD authentication systems (ref (2), buczacki et al, 1975), including Brassica campestris, brassica napus and Brassica oleracea; williams and ECD identification systems have been widely used for physiological race identification of brassica clubroot, although some of these hosts show intermediate or unstable disease indices. (3) Som e et al (ref (3), som e et al, 1996) developed a brassica plasmodiophora classification system consisting of three brassica lines, including those developed by Nevin, wilhelmsburger and Brutor, som e et al, to divide 20 plasmodiophora into 5 different physiological races. (4) Strelkov et al report (reference (4), strelkov et al 2018) a Canadian Clubroot Differential (CCD) system containing 13 hosts, dividing 106 plasmodiophora samples into 17 physiological races in Canada.
In China, the crucifer crops have large planting area and great difference of climate conditions, which provides favorable conditions for the variation of the plasmodiophora radiata bacterial flora, and the plasmodiophora radiata physiological races have different characteristics from other regions of China. The above-mentioned Williams, ECD, som e et al and CCD systems still have some limitations for classification of physiological races of the Rhizopus in China. For example, hosts used in these physiological race classification systems exhibit unstable disease resistance or disease-causing types to the national plasmodiophora. This will seriously affect the accuracy of the identification of physiological races of the plasmodiophora radiata, thereby affecting the breeding of disease-resistant varieties. Therefore, there is a need to develop a physiological race identification system suitable for China and having stable identification ability.
Disclosure of Invention
The invention provides an SCD (SCD) plasmodiophora radicis physiological race identification system, an identification method and application thereof, and provides a physiological race identification system which is applicable to China and has stable identification capability.
The first object of the invention is to provide an SCD plasmodiophora radicis physiological race identification system, as shown in the following table:
among them, pb1-Pb14 is of a physiologically small type.
The second object of the invention is to provide a method for identifying the physiological small type of the brassica plasmodiophora by using the SCD plasmodiophora radicis physiological small-seed identification system, which comprises the following steps:
s1, preparing a rhizomatous bacterial liquid
Grinding all brassica plasmodiophora brassicae to be detected, filtering with gauze to obtain bacterial liquid, and diluting the bacterial liquid to 10 7 Spores/ml for use;
s2, inoculating the rhizomatous bacteria
9 host material seeds in the physiological micro-seed identification system of the plasmodiophora radiata are respectively sown, and the cultivation and management conditions after sowing are as follows: the temperature is 20-25 ℃, and the illumination is carried out for 16 hours; inoculating bacteria 2 weeks after sowing, and investigating disease resistance of different host materials 6 weeks after sowing;
if the disease resistance of 9 host materials corresponding to a certain physiological small type is completely the same, dividing brassica plasmodiophora brassicae to be detected into the physiological small type; for example, after the brassica plasmodiophora to be detected is inoculated to H08, H03, H01, H04, H02, H05, H06 and H07, the disease resistance of the host is "-", and the disease resistance of the host after the brassica plasmodiophora to be detected is "+", the brassica plasmodiophora to be detected is classified into the physiological small type of Pb 1.
Preferably, in the above method, the inoculation procedure is as follows: applying the bacterial liquid diluted by S1 to the root of the plant seedling, wherein the diluted bacterial liquid isThe concentration of the liquid is 1 multiplied by 10 7 Spores/ml.
Preferably, in the above method, the morbidity is classified as follows: 0, no symptoms; 1, a plurality of small root nodules are arranged on lateral roots; 2, small root nodules are arranged on the main root or larger root nodules are arranged on the lateral heel; 3, the main root and the lateral root are provided with large nodules;
disease incidence index (DI) is calculated according to the formula di=Σ [ nw ] ×100/3T, where n is the number of plants in each incidence, w is the incidence (0-3), T is the statistical total number of plants;
disease resistance is defined as disease resistance '-', when DI is below 20%, otherwise as disease susceptibility '+'.
Preferably, in the above method, if a new type different from the physiological small type of P1-P14 in claim 1 appears, the brassica plasmodiophora to be detected is a new physiological small type.
The third object of the invention is to provide an application of the SCD plasmodiophora radicis physiological race identification system in the identification of parasitic plasmodiophora radicis physiological races in cruciferae crops.
The fourth object of the invention is to provide an application of the method for identifying the physiological small type of the brassica clubroot by utilizing the SCD clubroot physiological small seed identification system in cruciferae crop clubroot resistance breeding.
Compared with the prior art, the SCD plasmodiophora radicis physiological race identification system, the identification method and the application provided by the invention have at least the following beneficial effects:
the invention develops an SCD plasmodiophora physiological race identification system (SCD identification system for short) by using 9 Chinese cabbage inbred lines. Exhibits uniform resistance or susceptibility to 90 brassica plasmodiophora brassicae from china, korea and canada. According to the SCD identification system, 90 brassica plasmodiophora brassicae are classified into 14 physiological micro-species. The SCD identification system is simple, accurate and stable, and has high identification capacity.
The SCD identification system is a simple and effective identification system for physiological races of the brassica plasmodiophora brassicae, and is suitable for the physiological races identification of the Chinese plasmodiophora brassicae. The hosts used in SCD identification systems have identified the 14 physiological races (Pb 1-Pb 14) currently existing in China. It should be noted that, because the SCD identification system adopts 9 hosts, and the physiological population may have variation along with the transition of time, if a new host infection disease-resistant reaction type is found, our SCD identification system can continue to expand, and can theoretically expand to 256 physiological micro-types, that is, can differentiate up to 256 physiological micro-types of plasmodiophora.
The identification method provided by the invention is simple and easy to implement, and the identification result can be obtained about 8 weeks. The SCD identification system and the corresponding identification method are beneficial to classification, distribution, variation, popularity and prevention research of the physiological races of the plasmodiophora radiata, and greatly accelerate the breeding process of Chinese plasmodiophora radiata-resistant cabbages and other brassica crops.
Drawings
FIG. 1 is a physiological race partitioning result of 42 plasmodiophora samples after 12 host inoculation identifications of Table 1;
FIG. 2 shows the proportion of different physiological races of 86 samples of the rhizomes collected from China.
Detailed Description
In order that those skilled in the art will better understand the technical scheme of the present invention, the present invention will be further described with reference to specific embodiments and drawings. The following examples, as well as the experimental procedures not identified in the above summary, were carried out in accordance with methods and conditions conventional in the art.
The 12 Chinese cabbage inbred host materials used in the present invention (Table 1, table 2) are all of Shenyang agricultural university and are available at any time from the molecular biology laboratory of the gardening university by way of gifting or exchange to those skilled in the art. Wherein H2 is disclosed in the article Transcriptome Analysis of Brassica rapa Near-lsogenic Lines Carrying Clubroot-resistance and-Susceptible Alleles in Response to Plasmodiophora brassicae during Early of Jingjin Chen et al, front Plant Sci,2016.
H1, H3, H4, H5, H6, H7 are disclosed in article Identification and Mapping of the Clubroot Resistance Gene CRd in Chinese Cabbage by Wenxing Pang et al (Brassica rapa ssp. H12 is disclosed in the article Genetic Detection of Clubroot Resistance Loci in a New Population of Brassica rapa. Hort. Environ. Biotechnol,55 (6): 540-547,2014 by Jingjin Chen et al.
In addition, host materials H8-H11 have been reported at the International conference on the following: international clubroot seminar (2018 International CLUBROOT Workshop) held in edmonton, canada, 8, 7-9; the host materials H8 to H11 mentioned in the report correspond to the host materials H8 to H11 in Table 2.
Table 3 90 brassica clubs numbered 1-90 were reported at the national academy of sciences, and the names of the brassica clubs mentioned in the report were consistent with the names mentioned in Table 3. Table 3, 90 brassica clubs numbered 1-90. Domestic meeting information is as follows: 11 months 19-22 in 2018 at 14 th academy of cruciferous vegetables held in martial arts. Those skilled in the art can also collect the brassica plasmodiophora in the corresponding disease areas of table 3.
The Badger shifter, jersey Queen, laurentian and Wilhelmsburger host materials are all overseas commercial varieties.
Example 1A SCD (Sinitic Clubroot Differentiation system) P1-P14 physiological race identification system (hereinafter abbreviated as SCD identification system) as shown in Table 1 was of a physiological race type in Table 1.
Table 1 SCD physiological race identification system for plasmodiophora
Example 2 acquisition pathway and validation of the SCD authentication System of example 1
12 Chinese cabbage inbred line hosts (codes H1-H12): including 11 anti-clubroot lines and 1 susceptible line used in this study, see table 2 for host information. The anti-clubroot Chinese cabbage inbred line (H1-H11) is used for screening a stable and accurate physiological race classification system host, and the susceptible line H12 is used as a positive control of a clubroot resistance test. Hosts of the Williams identification system: badger shifter, jersey Queen, laurentian and Wilhelmsburger were also used for physiological race classification in this study. We compared the difference in effect of the SCD identification system with the Williams identification system.
TABLE 2 12 self-bred host information of Chinese cabbage
(1) The name, collection place and identification of the test brassica plasmodiophora are divided into corresponding physiological small types, and the information of the test brassica plasmodiophora is shown in table 3. To facilitate comparison of the results after identification of different plasmodiophora, we write 90 physiological small-type information based on the 19 hosts of table 3 as shown in table 3.
Table 3 test of information on brassica plasmodiophora brassicae
Note that: "Unclear" means that the brassica plasmodiophora is from China, but the specific province is not clear.
Inoculating 1-42 brassica rhizomes in the side 3 to 12 Chinese cabbage inbred lines (H1-H12) and a host Badger shifter of a Williams system, jersey Queen, laurentian and Wilhelmsburger; each host was inoculated with brassica plasmodiophora brassicae No. 1-42 shown in table 3; three replicates were performed for each host; spring in 2016, spring in 2017 and autumn.
48 plasmodiophora rhizomes numbered 43-90 of Table 3 were inoculated into each of the H1-H12 hosts of the SCD identification system of Table 1 and identified in autumn 2018.
In this step, 90 species of plasmodiophora are collected in total from chinese, canadian and korean infected chinese cabbage, canola, broccoli and wild mustard. Details of 90 types of clubroot are listed in table 3. Of the 90 species of plasmodiophora, 86 were from china, 1 from canada, and 3 from korea.
(2) Preparation of a plasmodiophora radicis bacterial solution
Grinding all collected brassica plasmodiophora with a refiner, filtering with 8 layers of gauze to obtain bacterial liquid, calculating bacterial liquid concentration under microscope with a blood cell counting plate, and finally diluting bacterial liquid to 1×10 7 Spores/ml for use.
(3) Root swelling fungus inoculation
All plant inoculation experiments are carried out from spring 2016 to autumn 2018, the places are Shenyang agricultural university, seeds of a brassica plasmodiophora brassicae host material to be detected are respectively sown, each repetition contains 24 seeds, and all plants are planted in a 72-hole tray at the temperature of 20-25 ℃ and grow in a 16-hour photoperiod; inoculation is carried out 2 weeks after sowing, and the inoculation procedure is as follows: applying 1mL of the brassica plasmodiophora fluid diluted in the step 2 (2) to the root of the plant seedling, wherein the concentration of the fluid is 1 multiplied by 10 7 Spores/ml; disease resistance was identified 6 weeks after inoculation.
Step 3, disease investigation and physiological race classification
The disease was investigated 6 weeks after inoculation of the bacterial liquid. Investigation of the onset was slightly modified according to Pang et al (Pang, w., liang, s., li, x., li, p., yu, s., and Yong, p.l., et al 2014.genetic detection of clubroot resistance loci in a new population of Brassica rapa.hortic environ.biotechnol.55 (6), 540-547). The morbidity grade is divided as follows: 0, no symptoms; 1, a plurality of small root nodules are arranged on lateral roots; 2, small root nodules are arranged on the main root or larger root nodules are arranged on the lateral heel; and 3, large root nodules are arranged on the main root and the lateral root.
Disease incidence index (DI) is calculated according to the formula di=Σ [ nw ] ×100/3T, where n is the number of plants in each incidence, w is the incidence (0-3), and T is the statistical total number of plants. When the DI value is lower than 20%, the disease resistance is defined as disease resistance '-', otherwise, the disease susceptibility is defined as '+'.
The 12 cabbage inbred lines (H1-H12) were carefully compared, based on the infectivity of 42 plasmodiophora radicis of Table 3 numbers 1-42, and the highly similar hosts were removed. Finally, the selected hosts are used to construct an SCD authentication system. An additional 48 samples of plasmodiophora were then identified for physiological race using the constructed SCD identification system. Based on the maximum composite model, the geographical distribution of the main physiological races in China is analyzed by using the MEGA version 5 for the phylogenetic tree.
(1) Identification of physiological race results using Williams identification System
The 42 brassica rhizomes numbered 1-42 of table 3 were inoculated into the bar shifter, jersey Queen, laurentian and Wilhelmsburger and repeated three times in the spring 2016, the spring 2017 and the autumn. For some plasmodiophora, among the hosts of the Williams identification system, some show unstable disease resistance or disease response. Based on the DI values mentioned in step 3, the responses of brassica clubroot to the host were recorded as disease resistance '-' and disease susceptibility '+'. The results of the Williams identification system are shown in Table 4 for a total of 4 physiological micro-organisms, no. 2, no. 4, no. 7 and No. 11, respectively. The results show that 6 strains are No. 2 and occupy 14 percent; 33 strains were No. 4, with a 79% ratio; 2 strains are No. 7, and the ratio is 5%;1 strain is 11 # and accounts for 2%; most brassica plasmodiophora brassicae was identified as number 4.
TABLE 4 identification results of Williams identification System
(2) Acquisition of SCD authentication System
42 brassica plasmodiophora brassicae of table 3 nos. 1-42 were inoculated into 12 hosts of table 1 and DI values were calculated. In order to improve the accuracy and stability of the physiological race identification system of the plasmodiophora, the disease resistance '-' is recorded when the DI value is lower than 10%, and the disease feeling '+' is determined when the DI value is higher than 60%. The disease-sensitive control Chinese cabbage inbred line H12 is severely infected by 42 brassica clubroot bacteria, and the other 11 anti-clubroot Chinese cabbage inbred lines show different responses to the 42 brassica clubroot bacteria. Host H05 had similar resistance to H09, H10 and H11, we selected H05 instead of H09, H10 and H11. The resistance of 9 hosts (H09, H10 and H11 removed) was used for 42 P.tumefaciens phylogenetic tree analysis. The results show that 42 samples of plasmodiophora can be divided into 14 different physiological races, see figure 1. The 14 physiologically small types are designated Pb1-Pb14. FIG. 2 shows the proportion of different physiological races of 86 samples of the rhizomes collected from China. Finally, an SCD identification system was constructed, comprising 8 clubroot resistant hosts H1 to H8 and 1 susceptible host H12, see table 1. All hosts showed a high degree of resistance or susceptibility, with a sensitivity higher than that of the Williams identification system, i.e.we constructed SCD identification systems with higher accuracy than the Williams identification system.
(3) Verification and application of SCD authentication system
To verify the SCD identification system, we applied the SCD identification system to 48 brassica plasmodiophora brassicae numbered 43-90 of table 3 for physiological race identification. The results are shown in Table 6, and the test results show that the SCD identification system identifies the 48 types of the clubroot bacteria as Pb1, pb3, pb5, pb6 and Pb12, which can be found in Table 1, and the results are shown in Table 6, and the results are analyzed, so that the SCD identification system constructed by us has good stability and application potential, and is suitable for the identification of all the clubroot bacteria physiological small types in the current China area. If a new type different from the P1-P14 physiological types in the table 1 appears, the brassica plasmodiophora to be detected is a new physiological type, the naming of the new physiological type is kept by the existing naming rule Pb (14+n), n is a positive integer, and the type numbers of the physiological types are sequentially increased.
Table 6 identification results of 48 physiological races of brassica plasmodiophora brassicae with numbers 43-90
Example 3 comparison and advantages of the SCD authentication System of the present invention with the Prior Art
(1) The existing foreign identification system is briefly described: the economic losses caused by cruciferous crop clubroot are well known. The accurate identification of physiological races of the plasmodiophora radiata is one of the preconditions for breeding disease-resistant varieties. Scientists from around the world have made great efforts to develop physiological micro-differentiation systems of brassica plasmodiophora brassicae with remarkable results. However, none of the previously developed Williams authentication systems, ECD systems and authentication systems developed by Som e et al, distinguish between some physiological races of Canada, korea, japan and China (references (4) - (6), kuginuki et al 1999; kim et al 2016; strelkov et al 2018). The host used in the Williams identification system and the identification system of Som e et al (reference (3)) did not show a uniform resistance or susceptibility to some brassica plasmodiophora brassicae (Kuginuki et al 1999). In korea, 4 disease-resistant chinese cabbage F was reported by Kim et al (reference (6)) 1 The generation variety divides 12 plasmodiophora radicis into 4 physiological micro-species. However, 4 kinds of F 1 The generation variety and the Williams identification system have the same limitation, namely do not have rich identification capability. In addition, due to F 1 The market for commodity seeds is atrophic or even vanishing within a few years, F 1 Seeds of the generation variety will be difficult to obtain.
(2) Comparison of the SCD authentication System of the invention with foreign Williams authentication System: according to the Williams identification system, 33 brassica plasmodiophora brassicae were classified as number 4. However, these 33 species of plasmodiophora have different disease resistance to some chinese cabbage inbred lines, suggesting that the Williams identification system is not suitable for classification of physiological races of plasmodiophora chinese. The same results are reported by Strelkov et al (reference (4)), nor did the Williams identification system differentiate between all brassica physiological races in canada. In this case, in China, the identification of physiological races of the brassica plasmodiophora brassicae, the distribution of physiological races and the popular research thereof become very difficult. Therefore, we need to establish a physiological race identification system of brassica plasmodiophora in China. With the construction of the SCD identification system, 33 brassica plasmodiophora brassicae identified as physiological race number 4 by the Williams identification system were separated into 14 different physiological races using the SCD identification system. Development of SCD identification system will greatly improve the identification, distribution, variation, prevalence and prevention of physiological races of clubroot in China and even other clubroot-causing countries and regions.
The physiological race identification of the rhizomatous bacteria is important for the positioning of the resistance genes of the rhizomatous bacteria and the breeding of disease-resistant varieties. So far, the SCD identification system has great potential for CR variety breeding of brassica plants, and once the SCD identification system is used for identifying the physiological brassica rhizopus of brassica, a host can be used for resistance breeding. In addition, the aggregation of disease resistance genes enables us to plant a broad spectrum of resistant varieties in most areas.
(4) Summary of the advantages of the invention: the invention develops an SCD plasmodiophora physiological race identification system (SCD identification system for short) by using 9 Chinese cabbage inbred lines. Exhibits uniform resistance or susceptibility to 90 brassica plasmodiophora brassicae from china, korea and canada. According to the SCD identification system, 90 brassica plasmodiophora brassicae were classified into 14 physiological small types. The SCD identification system is simple, accurate and stable, and has high identification capacity.
Our study found that 53 plasmodiophora radiata were identified as Pb1 in china, and all harvested brassica plants from southwest to northeast were detected, except for the provinces of the black dragon river and the province of the river. The results indicate that Pb1 is the main pathotype in China. If more brassica clubroot can be collected from both provinces, pb1 may also be present in Heilongjiang and Henan.
5 physiological races Pb1, pb6, pb7, pb11 and Pb12 were detected in Yunnan province, and the physiological races were the most. Pb7 and Pb11 are special physiological races of Yunnan province. The disease of clubroot in Yunnan is serious, and the result shows that the climate and the planted crop of the land are more suitable for the diffusion and the variation of the clubroot. Four physiological races Pb1, pb3, pb13 and Pb14 are detected in Hubei province, wherein Pb13 and Pb14 are special physiological races in Hubei province. Brassica crops are widely planted in southwest and Anhui, hubei and Chongqing areas, and the physiological species of the plasmodiophora are obviously more than those in northeast China. Research results show that the transmission of clubroot is closely related to the environment and the planted crops. Most importantly, the development and application of the SCD identification system are beneficial to the classification, distribution, variation, popularity and prevention research of clubroot physiological races, which can greatly accelerate the breeding process of Chinese clubroot resistant cabbages and other brassica crops.
Reference information for prior art physiological race identification systems used in the present invention:
(1)Williams,P.H.1966.A system for the determination of races of Plasmodiophora brassicae that infect cabbage and rutabaga.Phytopathology.56,624–626.
(2)Buczacki S T,Toxopeus H,Mattusch P,et al.Study of physiologic specialization in Plasmodiophora brassicae:proposals for attempted rationalization through an international approach.Trans.Br.Mycol.Soc.,1975,65(2):295-303.
(3)Some,A.,Manzanares,M.J.,Laurens,F.,Baron,F.,Thomas,G.,and Rouxel,F.1996.Variation for virulence on Brassica napus L.amongst Plasmodiophora brassicae collections from France and derived single-spore isolates.Plant Pathol.45(3),432-439.
(4)Strelkov,S.E.,Hwang,S.F.,Manolii,V.P.,Cao,T.,Freduaagyeman,R.,and Harding,M.W.,et al.2018.Virulence and pathotype classification of Plasmodiophora brassicae populations collected from clubroot resistant canola (brassica napus)in Canada.Can.J.Plant Pathol.40(2),07060661.2018.1459851.
(5)Kuginuki,Y.,Yoshigawa,H.,and Hirai,M.1999.Variation in virulence of Plasmodiophora brassicae in Japan tested with clubroot resistant cultivars of Chinese cabbage.Eur.J.Plant Pathol.105,327-332.doi:10.1023/A:1008705413127.
(6)Kim,H.,Jo,E.J.,Yong,H.C.,Jang,K.S.,and Choi,G.J.(2016).Pathotype classification of plasmodiophora brassicae isolates using clubroot-resistant cultivars of Chinese cabbage.Plant Pathol.Journal.32(5),423-430.
it should be noted that, when reference is made to a range of values in the claims of the present invention, it is understood that both the end points of each range of values and any value between the end points are optional, and the preferred embodiments are described for preventing redundancy, but other variations and modifications can be made to these embodiments by one skilled in the art once the basic inventive concept is known. It is therefore intended that the following claims be interpreted as including the preferred embodiments and all such alterations and modifications as fall within the scope of the invention. It will be apparent to those skilled in the art that various modifications and variations can be made to the present invention without departing from the spirit or scope of the invention. Thus, it is intended that the present invention also include such modifications and alterations insofar as they come within the scope of the appended claims or the equivalents thereof.
Claims (3)
1. A method for identifying physiological small types of brassica plasmodiophora by utilizing an SCD plasmodiophora physiological small-species identification system is characterized in that the SCD plasmodiophora physiological small-species identification system is shown in the following table:
wherein, the brassica plasmodiophora brassicae Pb1-Pb14 is of a physiological small type;
the method is characterized in that the host material for identifying the physiological small type consists of 9 Chinese cabbage inbred lines, wherein codes of the 9 Chinese cabbage inbred lines are H01, H02, H03, H04, H05, H06, H07, H08 and H12, the H01 is CR-26, the H02 is CRBJN3-2, the H03 is CR-20, the H04 is CR-77, the H05 is CR-75, the H06 is 85-74, the H07 is CR-73, the H08 is CR-096, and the H12 is BJN3-1;
the morbidity grade is divided as follows: 0, no symptoms; 1, a plurality of small root nodules are arranged on lateral roots; 2, small root nodules are arranged on the main root or larger root nodules are arranged on the lateral root; 3, the main root and the lateral root are provided with large nodules;
disease incidence index DI is calculated according to the formula di=Σ [ nw ] ×100/3T, where n is the number of plants in each incidence, w is the incidence 0-3, T is the total number of plants counted;
disease resistance is defined as disease resistance '-', when DI is below 10%, otherwise as disease susceptibility '+';
the method for identifying the physiological small type of the brassica plasmodiophora by utilizing the SCD plasmodiophora minor physiological seed identification system comprises the following steps of:
s1, preparing a rhizomatous bacterial liquid
Grinding the brassica plasmodiophora brassicae to be detected, filtering with gauze to obtain bacterial liquid, and diluting the bacterial liquid to 10 7 Spores/ml for use;
s2, inoculating the rhizomatous bacteria
9 host material seeds in the physiological micro-seed identification system of the plasmodiophora radiata are respectively sown, and the cultivation and management conditions after sowing are as follows: the temperature is 20-25 ℃, and the illumination is carried out for 16 hours; inoculating bacteria 2 weeks after sowing, and investigating disease resistance of different host materials 6 weeks after sowing;
if the disease resistance of 9 host materials corresponding to a certain physiological small type is completely the same, the brassica plasmodiophora to be detected is classified into the physiological small type.
2. The method of claim 1, wherein the inoculation procedure is as follows: applying the bacterial liquid diluted by S1 to the root of the plant seedling, wherein the concentration of the bacterial liquid after dilution is 1 multiplied by 10 7 Spores/ml.
3. The method according to claim 1, wherein the brassica plasmodiophora brassicae to be tested is a new physiological race if a new type different from the physiological race of Pb1-Pb14 in claim 1 occurs.
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