CN113678792B - Simple identification method and application of highly pathogenic soybean cyst nematode No. 4 physiological race, X12 and LY1 - Google Patents
Simple identification method and application of highly pathogenic soybean cyst nematode No. 4 physiological race, X12 and LY1 Download PDFInfo
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- 241000498254 Heterodera glycines Species 0.000 title claims abstract description 62
- 238000000034 method Methods 0.000 title claims abstract description 33
- 230000001717 pathogenic effect Effects 0.000 title claims abstract description 19
- 235000010469 Glycine max Nutrition 0.000 claims abstract description 34
- 244000068988 Glycine max Species 0.000 claims abstract description 34
- 230000007918 pathogenicity Effects 0.000 claims abstract description 14
- 238000012216 screening Methods 0.000 claims abstract description 6
- 108090000623 proteins and genes Proteins 0.000 claims abstract description 4
- 241000196324 Embryophyta Species 0.000 claims description 39
- 208000031513 cyst Diseases 0.000 claims description 36
- 239000002689 soil Substances 0.000 claims description 34
- 239000000463 material Substances 0.000 claims description 30
- 201000010099 disease Diseases 0.000 claims description 26
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 claims description 26
- 238000012360 testing method Methods 0.000 claims description 21
- 238000011081 inoculation Methods 0.000 claims description 20
- 206010011732 Cyst Diseases 0.000 claims description 19
- 235000013601 eggs Nutrition 0.000 claims description 19
- 208000035240 Disease Resistance Diseases 0.000 claims description 9
- 230000003071 parasitic effect Effects 0.000 claims description 9
- 239000007788 liquid Substances 0.000 claims description 8
- 238000012258 culturing Methods 0.000 claims description 6
- 238000000856 sucrose gradient centrifugation Methods 0.000 claims description 6
- 208000015181 infectious disease Diseases 0.000 claims description 4
- CZMRCDWAGMRECN-UGDNZRGBSA-N Sucrose Chemical compound O[C@H]1[C@H](O)[C@@H](CO)O[C@@]1(CO)O[C@@H]1[C@H](O)[C@@H](O)[C@H](O)[C@@H](CO)O1 CZMRCDWAGMRECN-UGDNZRGBSA-N 0.000 claims description 3
- 229930006000 Sucrose Natural products 0.000 claims description 3
- 239000004576 sand Substances 0.000 claims description 3
- 239000005720 sucrose Substances 0.000 claims description 3
- 239000002344 surface layer Substances 0.000 claims description 3
- 230000035784 germination Effects 0.000 claims description 2
- 230000002924 anti-infective effect Effects 0.000 claims 1
- 239000000427 antigen Substances 0.000 claims 1
- 108091007433 antigens Proteins 0.000 claims 1
- 102000036639 antigens Human genes 0.000 claims 1
- 230000002265 prevention Effects 0.000 claims 1
- 238000004519 manufacturing process Methods 0.000 abstract description 9
- 238000011160 research Methods 0.000 abstract description 8
- 230000007123 defense Effects 0.000 abstract description 2
- 238000009792 diffusion process Methods 0.000 abstract description 2
- 241000223198 Humicola Species 0.000 abstract 1
- 241000244206 Nematoda Species 0.000 description 4
- 230000004069 differentiation Effects 0.000 description 2
- 238000011835 investigation Methods 0.000 description 2
- 241001465321 Eremothecium Species 0.000 description 1
- 206010015535 Euphoric mood Diseases 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 230000007547 defect Effects 0.000 description 1
- 238000011161 development Methods 0.000 description 1
- 230000018109 developmental process Effects 0.000 description 1
- 238000009396 hybridization Methods 0.000 description 1
- 239000004033 plastic Substances 0.000 description 1
- 230000000644 propagated effect Effects 0.000 description 1
- 241000894007 species Species 0.000 description 1
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- 238000012795 verification Methods 0.000 description 1
- 238000004383 yellowing Methods 0.000 description 1
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01G—HORTICULTURE; CULTIVATION OF VEGETABLES, FLOWERS, RICE, FRUIT, VINES, HOPS OR SEAWEED; FORESTRY; WATERING
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Abstract
The invention provides a simple identification method and application of a strong pathogenic soybean cyst nematode No. 4 physiological race, X12 and LY1, wherein the three strong pathogenic soybean cyst nematode physiological races can be effectively distinguished by taking excellent Humicola cinerea (number: ZDD 2315) and PI567516C in China as discrimination hosts. The method can be used for screening the anti-source of the soybean cyst nematode with strong pathogenicity by distinguishing the physiological races of the soybean cyst nematode with strong pathogenicity, realizing the distribution and diffusion of the physiological races of the soybean cyst nematode with strong pathogenicity in a soybean production area, developing the research on pathogenic genes of the soybean cyst nematode, and adopting powerful defense measures for the soybean production area infected with the soybean cyst nematode with strong pathogenicity in a targeted manner, and has important significance.
Description
Technical Field
The invention relates to a simple identification method and application of a strong pathogenic soybean cyst nematode No. 4 physiological race, X12 and LY1, belonging to the technical field of biology.
Background
Soybean Cyst Nematode (SCN) disease is a worldwide soybean disease that causes billions of dollars of economic losses to global soybean production throughout the year. Since SCN is a soil-borne disease, once a soybean-growing area is infected, it is extremely difficult to eradicate it. Although no official data statistics exists at present in China, the economic loss of SCN to Chinese soybean production every year is caused, investigation and research on Huang-Huai area in the early stage find that about 70% of Huang-Huai-Hai soybean production area in one of the two main soybean production areas in China has soybean cyst nematodes distributed, and the symptoms of 'burnt seedlings', plant dwarfing and yellowing appear in part of soybean fields in Shanxi province seriously infected by SCN.
In the investigation and research of 2012-2015, a new physiological race X12 (physiological race, hereinafter referred to as race) of soybean cyst nematode with super pathogenicity is also found, and the main resistance sources used at home and abroad at present cause diseases or high feelings to the new race X12 of the soybean cyst nematode. These include 4 of the Riggs identification patterns, 7 of the HG identification patterns, excellent Source resistance Huichun county Hui Picount in China (ZDD 2315), PI567516C, and the important source resistance PI437654 currently in use in the United states. The emergence of this race poses a great threat to soybean production. In addition, the more pathogenic nematodes include number 4 physiological races (distributed in shanxi province) and LY1 nematodes (a population of nematodes artificially synthesized by hybridization using number 2 and 3 physiological races), which are currently used only in SCN-related studies in the united states, and LY1 has not been reported in nature.
There are 2 internationally common methods for classifying physiological races of soybean cyst nematode: the soybean cyst nematodes were divided into 16 races by 4 discriminators (Pickett, peking, PI88788, PI 90763) established by Riggs and Schmitt in 1988, and into 128 races by 7 discriminators (PI 548402, PI88788, PI90763, PI437654, PI89772, PI209332, PI 548316) established by Niblack et al in 2002, both of which were used to differentiate populations of nematodes with different pathogenicity. The 2 division methods have suitable application ranges, but in China, riggs identification modes are mostly adopted by related research experts. With the emergence of the pathogenic X12 race, the Riggs identification pattern can not distinguish the No. 4 physiological race, X12 and LY1, because the three physiological races can successfully parasitize on the 4 identification hosts in the Riggs identification pattern, namely the 4 identification hosts are infected with the three physiological races; the HG type discrimination pattern did not distinguish between X12 and LY1 because both physiological races were able to successfully parasitize on 7 discriminatory hosts in the HG type discrimination pattern, i.e. the 7 discriminatory hosts were ill-behaved on both physiological races.
The above phenomena show that: the existing host adopted in the 2 sets of soybean cyst nematode physiological race identification modes can not effectively distinguish the No. 4 physiological race, X12 and LY1, thereby hindering the development of SCN related researches to a certain extent.
Disclosure of Invention
Aiming at the defects of the prior art, the invention aims to provide a simple identification method and application of the strong pathogenic soybean cyst nematode No. 4 physiological races, X12 and LY1, which can effectively distinguish the three strong pathogenic soybean cyst nematode physiological races by using the superior origin-resistant county ashbya (number: ZDD 2315) and PI567516C in China as differentiation hosts.
In order to achieve the purpose, the invention adopts the technical scheme that:
a simple identification method of the strong pathogenic soybean cyst nematode No. 4 physiological race, X12 and LY1 comprises the following steps:
(1) Collecting disease soil infected by soybean cyst nematode, uniformly mixing the disease soil and sterile fine sand according to the volume ratio of 3: 0.5-1.0 to prepare test disease soil, and subpackaging the test disease soil in a test cup for identifying the resistance of the soybean cyst nematode;
(2) Transplanting the Lee plants of the susceptible variety with germination time of 3-5 days and cotyledon emergence period into test cups filled with test disease soil, transplanting 1 plant in each test cup, and placing in a greenhouse for culturing;
(3) After culturing for 20-25 days, pouring out Lee plants from the soil with test diseases, and collecting soybean cysts separated from root systems to a 100-mesh sieve by adopting a elutriation-screening method;
(4) Crushing soybean cyst with rubber plug, releasing egg, collecting egg to 500 mesh sieve, and purifying with sucrose gradient centrifugation;
(5) Counting the purified soybean cyst nematode eggs under a microscope, preparing inoculation liquid for subsequent inoculation;
(6) Transplanting 2 Huishuhua county ash bark branches germinating for 3-5 d, PI567516C and susceptible control Lee plants into sterile soil, repeating each material for 4 times, and inoculating the inoculation liquid obtained in the step (5) when new roots grow out after 3-5 d;
(7) After the inoculated plants are cultured in a greenhouse for 25 days, the plants of the identification material and the control material are poured out from the test disease soil, and the number of soybean cysts on the root system is counted;
(8) Calculating the resistance according to the number of soybean cysts on each root system of the identified material, wherein the statistical standard is as follows: if the average cyst number on the root system of the differential host material is more than or equal to 10 percent of the parasitic cyst amount of the susceptible control Lee plant, the susceptible control Lee plant is expressed; if the average cyst number on the root system of the differential host material is less than 10 percent of the parasitic cyst amount of the susceptible control Lee plant, the disease resistance is shown;
(9) X12 physiological races if the average number of cysts on the susceptible control Lee plants is greater than or equal to 100 and 2 of the discriminatory host materials are all susceptible; no. 4 physiological race if Xingxian grey branch shows disease resistance and PI567516C shows infection; if PI567516C shows disease resistance, it is LY1 physiological race.
The collection method of the diseased soil comprises the following steps: in the soybean growth period, 35-40 days after seedling emergence, investigating the soybean root system in the field, and collecting the soil 2-6 cm below the surface layer of the soil in the soybean field infected by soybean cyst nematode for later use.
The greenhouse culture conditions are as follows: alternating 16h in the daytime and 8h in the night; temperature: day at 27 +/-1 deg.c and night at 24 +/-1 deg.c; humidity was 70% ± 5%.
The sucrose gradient centrifugation method comprises the following specific steps: transferring the worm eggs to a 50mL centrifuge tube, controlling the volume within 20mL, adding 20mL of sucrose solution with the mass fraction of 40%, slightly reversing and uniformly mixing; centrifuge at 2000rpm for 5min.
The specific method for inoculation is as follows: pricking holes close to root systems of the identification material and the control material by using a screwdriver head with the diameter multiplied by 3 multiplied by 75mm, and then injecting the inoculation liquid obtained in the step (5) into the vicinity of the root systems through the holes by means of the assistance of a 1mL pipette tip, wherein the inoculation is carried out twice, one hole is injected each time, 1500 worm eggs/plant are inoculated at one time, 3000 worm eggs/plant are inoculated in total, and the interval of the two inoculations is 24 hours.
The identification method is applied to anti-source screening of the soybean cyst nematode with strong pathogenicity.
The identification method is applied to the research of soybean cyst nematode pathogenic genes.
The identification method is applied to preventing soybean cyst nematodes which are strongly pathogenic from infecting soybean producing areas.
The invention has the beneficial effects that:
the identification method of the invention utilizes two excellent resistance sources in China as identification hosts: the identification method is simple and easy to operate, and can effectively distinguish the physiological race of the soybean cyst nematode with strong pathogenicity of No. 4 physiological race of the soybean cyst nematode, X12 physiological race and LY1 physiological race of the soybean cyst nematode. The problem that three soybean cyst nematode physiological races with strong pathogenicity, namely the soybean cyst nematode No. 4 physiological race, the soybean cyst nematode physiological race X12 and the soybean cyst nematode physiological race LY1, cannot be distinguished in the conventional Riggs identification mode and HG type identification mode is solved.
The identification method has important application value in the research aspect related to the soybean cyst nematode. By distinguishing the soybean cyst nematode physiological races with strong pathogenicity, the anti-source of the soybean cyst nematode with strong pathogenicity can be screened in a targeted manner, the distribution and the diffusion of the soybean cyst nematode physiological races with strong pathogenicity in a soybean production area can be realized, the research on the soybean cyst nematode pathogenic genes can be carried out, and strong defense measures can be adopted for the soybean production area infected with the soybean cyst nematode with strong pathogenicity, so that the method has important significance.
Detailed Description
The following examples further illustrate the embodiments of the present invention in detail.
Example 1
A simple identification method of the strong pathogenic soybean cyst nematode No. 4 physiological race, X12 and LY1 comprises the following steps:
(1) Collecting disease soil infected by soybean cyst nematode, uniformly mixing the disease soil and sterile fine sand according to the volume ratio of 3: 0.5-1.0 to prepare test disease soil, and subpackaging the test disease soil in a disposable plastic test cup for identifying the resistance of the soybean cyst nematode;
the collection method of the diseased soil comprises the following steps: in the soybean growth period, 35-40 days after seedling emergence, investigating the root system of field soybeans, and collecting the soil 2-6 cm below the surface layer of the soil in a soybean field infected by soybean cyst nematodes for later use;
(2) Transplanting the Lee plants of the susceptible variety which germinate for 3-5 days and have cotyledon emergence period into experimental cups filled with experimental disease soil, transplanting 1 plant into each experimental cup, and placing the experimental cups in a greenhouse for culturing; greenhouse culture conditions: alternating 16h in the daytime and 8h in the night; temperature: day at 27 +/-1 deg.c and night at 24 +/-1 deg.c; humidity 70% +/-5%;
(3) After culturing for 20-25 days, pouring the Lee plants out of the test disease soil, and collecting soybean cysts separated from root systems to a 100-mesh screen by adopting a elutriation-screening method;
(4) Crushing soybean cyst with rubber plug, releasing egg, collecting egg to 500 mesh sieve, and purifying with sucrose gradient centrifugation;
the sucrose gradient centrifugation method comprises the following specific steps: transferring the worm eggs to a 50mL centrifuge tube, controlling the volume within 20mL, adding 20mL of sucrose solution with the mass fraction of 40%, slightly reversing and uniformly mixing; centrifuging at 2000rpm for 5min;
(5) Counting the purified soybean cyst nematode eggs under a microscope, preparing inoculation liquid for subsequent inoculation;
(6) Transplanting 2 germinated 2 identifying hosts Xingxian gray bark branches (number: ZDD 2315) and PI567516C and disease control Lee plants into sterile soil, repeating each material for 4 times, and when new roots grow after 3-5 days, pricking holes beside root systems of the identifying materials and the control materials by using a screwdriver bit with the diameter multiplied by 3 multiplied by 75mm, then injecting the inoculation liquid obtained in the step (5) to the vicinity of the root systems through the holes by using a 1mL pipette head, wherein the inoculation is performed twice, one hole is injected each time, 1500 worm eggs/plants are inoculated at one time, 3000 worm eggs/plants are inoculated in total, and the interval between the two times of inoculation is 24 hours;
(7) After the inoculated plants are cultured in a greenhouse for 25 days, the plants of the identification material and the control material are poured out from the test disease soil, and the number of soybean cysts on the root system is counted;
(8) Calculating the resistance according to the number of soybean cysts on each root system of the identified material, wherein the statistical standard is as follows: if the average cyst number on the root system of the differential host material is more than or equal to 10 percent of the parasitic cyst amount of the susceptible control Lee plant, the susceptible control Lee plant is expressed; if the average cyst number on the root system of the differential host material is less than 10 percent of the parasitic cyst amount of the susceptible control Lee plant, the disease resistance is shown;
(9) X12 physiological races if the average number of cysts on the susceptible control Lee plants is greater than or equal to 100 and 2 of the discriminatory host materials are all susceptible; no. 4 physiological race if Xingxian grey branch shows disease resistance and PI567516C shows infection; if PI567516C shows disease resistance, it is LY1 physiological race.
And (5) if the average cyst number on the susceptible control Lee plant is less than 100, repeating the steps (1) to (5), and expanding the cyst number in the diseased soil again by using the susceptible control Lee plant until the quantity of worm eggs enough for inoculation identification is propagated, and then carrying out identification.
Application example 1
The identification mode and the Riggs identification mode are respectively adopted to identify the No. 4 physiological race, the X12 and the LY1 of the soybean cyst nematode, the results are shown in the following table 1, the host used in the Riggs identification mode shows that the No. 4 physiological race, the X12 and the LY1 are all susceptible (the average cyst number on the root system of the host material is more than or equal to 10 percent of the Lee parasitic cyst amount), and the No. 4 physiological race, the X12 and the LY1 cannot be effectively distinguished. In the identification mode, the No. 4 physiological race, the X12 and the LY1 all show different resistances, so that the effective differentiation of the three physiological races can be realized.
Table 1 identification pattern and results for differentiating strong pathogenic soybean cyst nematode populations
| Identifying host | X12 | No. 4 physiological race | LY1 |
| Pickett * | + | + | + |
| Peking * | + | + | + |
| PI88788 * | + | + | + |
| PI90763 * | + | + | + |
| ZDD2315 ** | + | - | / |
| PI567516C ** | + | + | - |
Note: * Representing a discriminatory host in the Riggs discriminatory mode; * Denotes newly added discrimination hosts; "+" indicates that the average number of cysts on the root system of the host material is more than or equal to 10% of the amount of Lee parasitic cysts; "-" indicates the average number of cysts on the host material root system < 10% of the amount of Lee parasitic cysts; "/" indicates that resistance identification was not performed. LY1 is a strong pathogenic SCN physiological race cultivated in the United states, and is not reported in China, and ZDD2315 is a specific resistance source in China.
Application example 2
The identification mode of the application is utilized to identify the No. 4 physiological race and the X12 physiological race of known species, and the results show that: all the physiological races were identified without errors, and the identification results are shown in the following table 2.
TABLE 2 verification of No. 4 and X12 physiological races with newly added hosts
Note that the mean number of cysts on Lee, which is greater than 100, indicates that the identification data is valid.
Claims (5)
1. A simple identification method for the number 4 physiological race, X12 and LY1 of the soybean cyst nematode with strong pathogenicity is characterized by comprising the following steps:
(1) Collecting disease soil infected by soybean cyst nematodes, uniformly mixing the disease soil and sterile fine sand according to the volume ratio of 3: 0.5-1.0 to prepare test disease soil, and subpackaging the test disease soil in a test cup for identifying the resistance of the soybean cyst nematodes;
(2) Transplanting the susceptible variety Lee plants with germination of 3-5d and cotyledon emergence period into test cups filled with test disease soil, transplanting 1 plant in each test cup, and placing in a greenhouse for culturing;
the greenhouse culture conditions are as follows: alternating 16h in the daytime and 8h in the night; temperature: day at 27 +/-1 deg.c and night at 24 +/-1 deg.c; humidity is 70% +/-5%;
(3) After culturing for 20 to 25d, pouring the Lee plant out of the test disease soil, and collecting soybean cysts separated from the root system to a 100-mesh screen by adopting an elutriation-screening method;
(4) Crushing soybean cyst with rubber plug, releasing egg, collecting egg to 500 mesh sieve, and purifying with sucrose gradient centrifugation;
the sucrose gradient centrifugation method comprises the following specific steps: transferring the worm eggs to a 50mL centrifuge tube, controlling the volume within 20mL, adding 20mL of sucrose solution with the mass fraction of 40%, and slightly inverting and uniformly mixing; centrifuging at 2000rpm for 5min;
(5) Counting the purified soybean cyst nematode eggs under a microscope, preparing inoculation liquid for subsequent inoculation;
(6) Transplanting 2 of the germinated 2 differential hosts Xingxian grey bark branches and PI567516C and susceptible control Lee plants into sterile soil, repeating the operation for 4 times for each material, and inoculating the inoculation liquid obtained in the step (5) when new roots grow out after 3-5 d;
the specific method for inoculation is as follows: pricking holes close to root systems of the identification material and the comparison material by using a screwdriver head with the diameter multiplied by 75mm, and then injecting the inoculation liquid obtained in the step (5) into the vicinity of the root systems through the holes by means of the assistance of a 1mL pipette head, wherein the inoculation is carried out twice, one hole is injected each time, 1500 worm eggs/plant are inoculated at one time, 3000 worm eggs/plant are inoculated in total, and the interval between the two inoculations is 24 hours;
(7) After the inoculated plants are cultured in a greenhouse for 25 days, the plants of the identification material and the control material are poured out from the test disease soil, and the number of soybean cysts on the root system is counted;
(8) The anti-infection condition is calculated according to the number of soybean cysts on each root system of the identified material, and the statistical standard is as follows: if the average cyst number on the root system of the differential host material is more than or equal to 10 percent of the parasitic cyst amount of the susceptible control Lee plant, the plant is expressed as susceptible; if the average cyst number on the root system of the differential host material is less than 10 percent of the parasitic cyst amount of the susceptible control Lee plant, the disease resistance is shown;
(9) X12 physiological races if the average number of cysts on the susceptible control Lee plants is greater than or equal to 100 and 2 of the discriminatory host materials are all susceptible; no. 4 physiological race if Xingxian grey branch shows disease resistance and PI567516C shows infection; if PI567516C shows disease resistance, it is LY1 physiological race.
2. The method of claim 1, wherein the diseased soil is collected by a method comprising: in the soybean growth period, after the seedlings emerge for 35 to 40 days, investigating the soybean root systems in the fields, and collecting the soil between 2cm and 6cm below the surface layer of the soil in the soybean fields infected by soybean cyst nematodes for later use.
3. Use of the identification method according to claim 1 or 2 for screening against a virulent soybean cyst nematode antigen.
4. Use of the identification method according to claim 1 or 2 for the study of pathogenic genes of soybean cyst nematodes.
5. Use of the identification method according to claim 1 or 2 for the prevention of infection of soybean producing areas with strongly pathogenic soybean cyst nematodes.
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| US20060206946A1 (en) * | 2005-02-15 | 2006-09-14 | University Of Maryland, College Park | Method of disrupting heme transport in nematodes and of modelling and evaluating eukaryotic heme transport |
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