CN113179898B - Resistance identification method for improving infection efficiency of soybean cyst nematodes - Google Patents
Resistance identification method for improving infection efficiency of soybean cyst nematodes Download PDFInfo
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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
- A01G22/00—Cultivation of specific crops or plants not otherwise provided for
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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
- A01G7/00—Botany in general
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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
- A01G7/00—Botany in general
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Abstract
The invention relates to a resistance identification method for improving infection efficiency of soybean cyst nematodes, which effectively improves the growth environment of soybean plants for resistance identification by improving the steps of culture conditions, a counting method, an inoculation method and the like, ensures that the number of inoculated eggs is better and more accurate, improves the egg infection efficiency, ensures that the data difference between different repetitions of the soybean cyst nematode resistance identification result is not obvious and the data is more accurate and reliable, reduces the workload caused by repeated times and has great application value for developing the soybean cyst nematode resistance identification work.
Description
Technical Field
The invention relates to a resistance identification method for improving infection efficiency of soybean cyst nematodes, belonging to the field of identification of disease resistance of crops.
Background
The soybean is one of the main economic crops in China, the demand of the soybean in China is increased rapidly along with the development of social economy and the improvement of living standard of people, and the serious yield reduction of the soybean is caused by the upgrade of dominant physiological race pathogenicity of soybean cyst nematode, the discovery of new race X12 of the soybean cyst nematode with super pathogenicity and the occurrence of diseases such as disease green and root rot in China in recent years in domestic and foreign soybean production areas, so that the urgent need for cultivating disease-resistant, high-quality and high-yield soybean varieties is provided. The soybean cyst nematode disease is a worldwide soybean disease and occurs in all countries of the world in soybean production areas. Production practices prove that planting soybean cyst nematode-resistant varieties is the most economic and effective measure for resisting the diseases, screening resistant germplasm resources is a prerequisite condition for cultivating the soybean cyst nematode-resistant varieties, and people divide the resistance level of the germplasm resources by taking cyst indexes (FI) as indexes.
The screening of soybean cyst nematode resistant germplasm resources mainly depends on the identification of an internal table type in a greenhouse. The phenotype identification work is time-consuming, labor-consuming and error-prone, and the main reasons for this are as follows: 1. because the soybean cysts growing on the root system are small in size and large in number, although the counting method is greatly improved, the accurate numerical value is still difficult to obtain; 2. although resistance identification is mostly carried out indoors, nematodes parasitizing in root systems are easily influenced by various factors such as temperature, humidity, irregular inoculation operation, inconsistent inoculation concentration, interference of activity of ova during inoculation and the like, and even though experiments in the same batch are carried out, the difference of the number of cysts on the root systems of plants to be identified is still large among multiple repetitions of the same variety, so that resistance identification needs to be repeated for multiple times in different time. Therefore, the phenotypic resistance identification which is developed in a greenhouse and looks simple is very labor-intensive and costly to operate due to the influence of a plurality of factors, and the screening efficiency of resistance resources is always low. Although a series of measures are mostly adopted in a greenhouse for controlling temperature and humidity, the existing measures have great defects in the resistance identification work of the soybean cyst nematodes due to the sensitivity of the soybean cyst nematodes to the environmental temperature and humidity. In particular, seedlings at the initial stage of soybean cyst nematode infection need to be subjected to accurate greenhouse control, and meanwhile, effective measures need to be taken to improve the soybean cyst nematode infection efficiency, ensure the infection consistency and improve the accuracy of resistance identification.
At present, more and more reports show that pathogenic capability of dominant physiological races of soybean cyst nematodes is upgraded, for example, in 2016, in the soybean main producing area-Huang-Huai area in China and the soybean producing area in Soy in America, the dominant physiological races are upgraded from No. 1 physiological races to No. 2 physiological races; in China, a new microspecies X12 with super pathogenicity is also found in the early stage, and the soybean cyst nematode still spreads. Under such circumstances, there is an urgent need for a screening method for stably identifying soybean cyst nematode resistance resources and for effectively utilizing the screened resistance resources in production practice.
Disclosure of Invention
Aiming at the defects of the prior art, the invention aims to provide a resistance identification method for improving the infection efficiency of soybean cyst nematodes.
In order to achieve the purpose, the technical scheme adopted by the invention is as follows:
a resistance identification method for improving infection efficiency of soybean cyst nematodes comprises the following steps:
(1) Selecting disease soil infected by soybean cyst nematode and sterile fine sand to be uniformly mixed according to the volume ratio of 3: 0.5-1.0 respectively to prepare test disease soil; transplanting disease-sensitive control soybean Lee plants which germinate for 3-5 days and are in the cotyledon emergence period into test cups filled with test disease soil, transplanting 1 plant into each test cup, and culturing in a plastic basket with a 4cm high-bottom filter screen at the bottom;
(2) After the soybean Lee plants are cultured for 25d, pouring the soybean Lee plants out of the soil with diseases, separating soybean cysts from root systems by adopting an elutriation-screening method, and collecting the soybean cysts washed from the root systems to a 100-mesh screen by using a 25-60-100-mesh screen in sequence; after purifying by a cyst breaking and sucrose gradient centrifugation method, counting the soybean cyst nematode eggs under a microscope for subsequent inoculation;
(3) Transplanting the materials to be identified, which germinate for 3-5 days, into sterile soil, repeating the steps for 4 times for each material, growing new roots after 3-5 days, pricking holes next to the root system of the materials to be identified by using a screwdriver with the diameter of 3 multiplied by 75mm, then injecting the inoculation liquid obtained in the step (2) into the vicinity of the root system through the holes by means of the assistance of a 1mL gun head, wherein the inoculation is carried out twice, one hole is injected each time, 1500 ova/plants are inoculated at one time, and the interval of the two inoculations is 24 hours;
(4) Inoculating insect eggs to a material to be identified, culturing in a greenhouse for 25 days, pouring out a plant of the material to be identified from test disease soil, separating cysts from a root system by adopting an elutriation-screening method, and counting the number of soybean cysts on the root system;
(5) And calculating a cyst index FI according to the average number of soybean cysts on each root system of the material to be identified, and determining the disease-resistant grade.
In the disease soil infected by the soybean cyst nematodes, the soybean cyst nematodes are selected from one or more of physiological races No. 1, no. 2, no. 3, no. 4 or No. 5.
The specification of the experimental cup is as follows: upper orifice diameter × lower orifice diameter × height =70mm × 50mm × 90mm; the internal dimensions of the plastic basket are: length × width × height =530mm × 341mm × 70mm.
The 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; the humidity is 65-70%; wherein water with the height of 0.3cm-0.5cm is injected into the plastic basket in daytime, and the bottom of the plastic basket is kept dry in night.
The purification treatment comprises the following specific steps: crushing soybean cyst with a rubber stopper, releasing worm eggs, sequentially sieving the worm eggs through a sieve of 100-200-500 meshes, finally collecting the worm eggs on the sieve of 500 meshes, transferring the worm eggs to a 50mL centrifuge tube by using a wash bottle and a funnel, 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;
if layering occurs, taking a middle layer of worm eggs, discarding the supernatant on the upper part and the sediment on the bottom, filtering on a 600-mesh screen, washing with clear water to remove sucrose, washing the worm eggs to a beaker with the assistance of a washing bottle and a funnel, diluting, taking 10 mu L of the diluted worm eggs, counting the number of the soybean cyst nematode worm eggs under a microscope, and using the number for subsequent inoculation;
if not, sucking the eggs in the supernatant, discarding the bottom precipitate, filtering the sucked supernatant on a 600-mesh sieve, washing with clear water to remove sucrose, and leaving the eggs on the 600-mesh sieve; and (4) washing the eggs to a beaker with the assistance of a washing bottle and a funnel, diluting, and counting the number of the soybean cyst nematode eggs by taking 10 mu L of the diluted eggs under a microscope for subsequent inoculation.
If the number of soybean cysts on the root system of the susceptible control Lee single plant for the identification test is more than or equal to 100, the test data is determined to be effective; if the number of soybean cysts on the root system of the individual plant of the susceptible control Lee for the identification test is less than 100, repeating the steps (1) and (2), utilizing the susceptible control Lee to expand the number of the cysts in the diseased soil again until the number of worm eggs enough for inoculation identification is propagated, and then carrying out resistance identification.
The cyst index FI = average number of cysts on the root system of the plant to be identified/number of cysts on the susceptible control Lee plant x 100.
The relationship between the cyst index FI and the disease resistance grade is as follows: FI =0, immune; 0 (all over) is FI less than or equal to 10, and disease resistance is achieved; 10-straw FI is less than or equal to 30, and resistance is achieved; 30-straw FI is less than or equal to 60, and feel; FI >60, susceptible to disease.
The invention has the beneficial effects that:
the invention provides a resistance identification method for improving infection efficiency of soybean cyst nematodes, which improves the steps of culture conditions, a counting method, an inoculation method and the like, so that the data difference between different repetitions of the soybean cyst nematode resistance identification result is not obvious, the data is more accurate and reliable, the workload caused by repeated use is reduced, and the method has great application value for developing the soybean cyst nematode resistance identification work, and is specifically analyzed as follows.
(1) The invention effectively controls the temperature and the humidity: a bottom filter screen is paved at the bottom of a container for resistance identification, water is added, the moisture required by the growth of the cyst nematodes is maintained by water evaporation, the high temperature caused by illumination is reduced, and the growth environment of the soybean plants for resistance identification is effectively improved in temperature and humidity.
(2) The invention improves the worm egg infection efficiency: the method has the advantages that the sucrose gradient centrifugation is utilized to purify the eggs for inoculation, the traditional one-time inoculation is changed into two times of inoculation with an interval of 24 hours, the perforation diameter during inoculation is reduced, the contact area between the eggs and the host is increased, the accurate number of the inoculated eggs is ensured, the hosts are found and infected as soon as possible after the eggs are hatched, and the egg infection efficiency is improved.
Drawings
FIG. 1 is a plastic basket used in the cultivation process;
wherein, the left is the invention; the right is the traditional method;
FIG. 2 is a schematic diagram showing the temperature and humidity differences between different temperature control measures in the same layer of cultivation shelves;
wherein, the left is the traditional method; the invention is shown on the right;
FIG. 3 is a schematic representation of soybean cyst nematode eggs before and after purification by sucrose gradient centrifugation;
wherein, the left is unpurified; the right is after purification;
FIG. 4 is a schematic diagram showing the comparison of the pore diameters of different perforation methods during inoculation;
wherein, the left part adopts a screwdriver with the diameter of 3 multiplied by 75mm to prick the hole; and the right part is punctured by adopting a 1mL gun head.
Detailed Description
The following examples further illustrate the embodiments of the present invention in detail.
Example 1
A resistance identification method for improving infection efficiency of soybean cyst nematodes comprises the following steps:
(1) According to different soil viscosities of disease soil infected by soybean cyst nematodes (disease soil identified as physiological races of soybean cyst nematodes No. 1, no. 2, no. 3, no. 4 or No. 5), uniformly mixing the disease soil and sterile fine sand in a volume ratio of 3: 0.5-1.0 to prepare test disease soil; transplanting soybean Lee plants (susceptible control) at the cotyledon emergence period (germination about 3-5 d) into test cups filled with test disease soil, and transplanting 1 plant in each test cup, wherein the specification of each test cup is as follows: upper mouth diameter, lower mouth diameter, height =70mm × 50mm × 90mm; putting the plastic basket into a plastic basket with the internal dimensions of length, width and height =530mm, 341mm and 70mm, wherein a bottom filter screen with the height of 4cm is padded at the bottom of the plastic basket; the culture conditions are as follows: day/night: 16h/8h; temperature: day at 27 +/-1 ℃ and night at 24 +/-1 ℃; the humidity is 65% -70%; and a small amount of water (0.3 cm-0.5cm high for maintaining humidity) is injected into the bottom of the plastic basket during the illumination time (daytime), and the bottom of the basket is kept dry during the night (so as to prevent the root system from passing through the Kong Changru water at the bottom of the cup).
(2) After the soybean Lee seedlings are cultured for about 25 days, pouring out the soybean Lee plants from the test disease soil, separating soybean cysts from root systems by adopting a washing-screening method, and collecting the soybean cysts from the root systems to a 100-mesh screen by using a 25-60-100-mesh screen in sequence; after purification treatment by cyst disruption and sucrose gradient centrifugation, soybean cyst nematode eggs are counted under a microscope for subsequent inoculation.
The purification treatment comprises the following specific steps: crushing soybean cyst with a rubber stopper, releasing worm eggs, sequentially passing the worm eggs through a sieve of 100-200-500 meshes, finally collecting the worm eggs on the sieve of 500 meshes, transferring the worm eggs to a 50mL centrifuge tube by using a wash bottle and a funnel, and controlling the volume within 20 mL; bringing the eggs back to a laboratory, adding 20mL of sucrose solution with the mass fraction of 40%, and slightly reversing and uniformly mixing; centrifuging at 2000rpm for 5min;
if layering occurs, sucking a middle layer of worm eggs to a 600-mesh sieve, and discarding the supernatant at the upper part and the sediment at the bottom; washing with clear water to remove sucrose, washing ovum with the help of a washing bottle and a funnel to a beaker, diluting, and counting the number of cysts under a microscope by 10 mu L for subsequent inoculation;
if not, sucking the worm eggs in the supernatant, discarding the bottom precipitate, filtering the sucked supernatant on a 600-mesh sieve, washing with clear water to remove sucrose, and leaving the worm eggs on the 600-mesh sieve; eggs were washed into a beaker with the aid of a wash bottle and funnel, and after dilution, 10. Mu.L were counted under a microscope for the number of cysts for subsequent inoculation.
(3) Transplanting the germinated material to be identified for 3d into sterile soil for 4d, pricking holes next to the root system of the material to be identified by using a screwdriver with the diameter of 3 multiplied by 75mm, then injecting the ova obtained in the step (2) into the positions near the root system through the holes by means of the assistance of a 1mL gun head, wherein the inoculation is carried out twice, one hole is injected each time, 1500 ova/plant are inoculated at one time, and the interval between the two inoculations is 24 hours. 5X 8 test cups can be placed in each plastic basket, and the same material is repeated for 4 times.
(4) Inoculating ova to a material to be identified, culturing for 25 days, pouring out a plant of the material to be identified from soil, separating cysts from a root system by adopting a elutriation-screening method, taking pictures by using a fluorescence microscope, identifying the cysts according to the shape and the size of the cysts by using phenotype data acquisition counting software, and automatically counting the number of soybean cysts on each picture by computer scanning and analyzing.
If the number of soybean cysts on the root system of the susceptible control Lee single plant for the identification test is more than or equal to 100, the test data is determined to be effective; if the number of soybean cysts on the root system of the individual plant of the susceptible control Lee for the identification test is less than 100, repeating the steps (1) and (2), utilizing the susceptible control Lee to expand the number of the cysts in the diseased soil again until the number of worm eggs enough for inoculation identification is propagated, and then carrying out resistance identification.
(5) Calculating the cyst index according to the average number of soybean cysts on each root system of the material to be identified, and determining the disease-resistant grade.
Cyst Index (felole Index, FI) = average number of cysts on root system of plant to be identified/number of cysts on susceptible control Lee x 100.
The relationship between cyst index and disease resistance grade is as follows: FI =0, immunization; 0 (all over) is FI less than or equal to 10, and disease resistance is achieved; 10-straw FI is less than or equal to 30, and resistance is achieved; 30-straw FI is less than or equal to 60, and feel; FI >60, affection.
The invention is compared with the traditional method:
1. the identification method of the invention is improved on the basis of the traditional method in the following three aspects:
(1) Improvement of plastic basket
The invention changes the plastic basket with the specification of 638mm multiplied by 420mm multiplied by 139mm (the internal dimension is long multiplied by width multiplied by height) into the plastic basket with the specification of 530mm multiplied by 341mm multiplied by 70mm (the internal dimension is long multiplied by width multiplied by height), reduces the height of the plastic basket, pads a bottom filter screen with the height of 4cm at the bottom of the plastic basket, injects 0.3cm-0.5cm of water into the bottom of the plastic basket during the illumination time during the culture, and keeps the bottom of the basket dry at night.
The height of the basket is reduced, so that the heat dissipation is facilitated, and the temperature consistency among the experimental cups is ensured; bedding filter screen and add a small amount of water in plastic basket bottom, also help the heat dissipation, can moisturize simultaneously, and because the water yield has been controlled, when the greenhouse was in the low temperature state evening, the water of basket bottom had basically evaporated, can effectively avoid the root system to extend to in the water through the hole of experiment cup bottom like this. The plastic baskets used before and after the modification are shown in figure 1.
The temperature and the humidity in the plastic basket during the plant cultivation process on the same layer of cultivation frame are respectively measured in the same environment, and the results show that: before improvement, the temperature in the plastic basket without the bottom filter screen is 31.4-32.9 ℃, and the humidity is 45-67%; reduce the height of basket after improving, put at the bottom of the filter screen and in the watered plastic basket: the temperature and the humidity in the plastic basket are 26.6-28.2 ℃ and 65-68 percent respectively; the temperature of the culture shelf is 41.3-42.2 deg.C, and the humidity is 25-28%, as shown in FIG. 2 and Table 1. It can be seen that the improved conditions are closer to the optimal growth conditions of the cysts (the optimal growth conditions of the cysts in the illumination period are that the temperature is 27 +/-1 ℃ and the humidity is 65-70%).
TABLE 1 comparison of temperature and humidity before and after improvement of the present invention and conventional methods
(2) Increase in purification treatment after cyst isolation
The traditional counting method comprises the following steps: without purification, after collecting cysts, the cysts were broken by gentle grinding with a mortar until white cysts were not visible, and then the released eggs were washed into a beaker, and the eggs were suspended with clear water, and 10. Mu.L of them were counted under a microscope for subsequent inoculation. The cysts obtained by separation are purified by a sucrose gradient centrifugation method and then counted under a microscope.
Because the traditional counting method does not undergo a purification step, the worm egg solution contains residues such as sand, vermiculite, root systems and the like, and the inoculum is dirty, so that the counting accuracy is poor. And the purified eggs are subjected to sucrose gradient centrifugation, so that visible residues under a microscope are removed, the eggs are clean and relatively accurate in counting, and the number consistency of the eggs inoculated into the material to be identified is higher. A comparison of the results before and after egg purification is shown in FIG. 3.
(3) Improvements in or relating to vaccination methods
The traditional method for pricking holes by adopting a 1mL gun head is changed into the method for pricking holes by adopting a screwdriver with the diameter of 3 multiplied by 75 mm. The diameter of the improved hole is about 1/3-1/6 of the original diameter, and the hole diameter is compared and shown in figure 4.
The traditional method is used for inoculation, one hole is drilled, 3000 eggs are injected into one hole, after the hole diameter is reduced, the inoculation liquid is injected into the hole twice, and one hole is injected each time, so that experimental errors caused by artificial missed inoculation when the activity of the eggs is poor or the identification quantity is large at one time are effectively avoided. The contact space of ova and root systems is effectively reduced after the aperture is reduced, the hatched larvae find hosts more easily, the phenomenon of cracks around the root systems is effectively reduced when holes are punctured, and the inoculated ova are gathered around the root systems more easily.
2. Soybean cyst nematode resistance identification and analysis
The traditional method and the method of the invention are respectively adopted, different resistance resources are utilized to compare the soybean cyst nematode resistance identification data (cyst number) before and after improvement, wherein the variety 1 is a susceptible control (soybean Lee seedling plant), the varieties 2-8 are to-be-identified material plants, and the results are shown in the following table 2.
TABLE 2 comparison of soybean cyst nematode resistance identification data on partial soybean resources using the present invention and conventional methods
Description of the drawings: the disease resistance grade in the table is divided into the following standard: FI =0, immune; 0< -FI is less than or equal to 10, and disease resistance (R); 10-straw FI is less than or equal to 30, and is resistant to (MR); 30-straw FI is less than or equal to 60, and is felt (MS); FI >60, susceptible (S).
As can be seen from Table 2, first, the method of the present invention shows a better uniformity of the number of cysts and a reduction in the standard deviation for each replicate. For example, the control variety Lee, traditional method, standard deviation 133.9, the range of values between replicates 35-347; the standard deviation of the method is 25.1, and the numerical range between the repetitions is 102-166. Secondly, in the identification process, the identification method of the traditional method is used, the variety which is initially identified as the neutral or neutral variety is encountered, but the phenomenon that the variety is susceptible or neutral after repeated identification is respectively detected, in short, the traditional method needs to repeat 2-4 times for identifying the neutral or neutral variety, and needs to repeat 4 times or more for identifying the immune and disease-resistant materials, so that the conclusion can be drawn and the data can be submitted. The method of the invention has relatively stable identification result, does not repeat the identified material with neutral feeling generally, and can draw a conclusion and submit data when repeating the identified material with immunity, disease resistance and resistance for 1-2 times according to the test requirements.
That is, according to the resistance identification conditions of the conventional methods, since the numerical difference between the reference material repeats is large, it is often necessary to repeat the test several times to conclude the resistance identification. The possible reasons for the large difference in the numerical value between the repeats are presumed as follows: the results are very common in experiments when the inoculation concentration is inconsistent or overinoculation is carried out between the same varieties and the temperature is far from the optimum growth temperature of cysts in the identification of resistance for many years. Therefore, in order to ensure the accuracy of the identification result, the identification of each variety can be repeated for more than 3 times, particularly, when resistant materials are identified, repeated identification needs to be carried out for multiple times at different time to draw a conclusion, which is one of the reasons for slow progress of the resistance identification work of the soybean cyst nematode, and through the convenient optimization of temperature control, cyst purification, inoculation modes, inoculation holes and the like, the resistance identification inoculation efficiency is effectively improved, so that the numerical difference between the repetitions is obviously reduced. The improvement of the technology has great promotion effect on promoting the research on the soybean cyst nematode, including resistant resource screening, physiological race identification of the soybean cyst nematode, disease-resistant gene excavation of the soybean cyst nematode, disease-resistant variety cultivation of the soybean cyst nematode and interaction research of the soybean cyst nematode.
Claims (7)
1. A resistance identification method for improving infection efficiency of soybean cyst nematodes is characterized by comprising the following steps:
(1) Selecting disease soil infected by soybean cyst nematodes and sterile fine sand to be uniformly mixed according to the volume ratio of 3: 0.5 to 1.0 respectively, and preparing test disease soil; transplanting disease-sensitive control soybean Lee plants which germinate for 3-5 days and have cotyledon emergence period into test cups filled with test disease soil, transplanting 1 plant into each test cup, and culturing in a plastic basket with a 4cm high-bottom filter screen at the bottom;
the culture conditions were: alternating 16h in the daytime and 8h in the night; temperature: day at 27 +/-1 ℃ and night at 24 +/-1 ℃; the humidity is 65-70%; wherein, water with the height of 0.3cm to 0.5cm is injected into the plastic basket in daytime, and the bottom of the plastic basket is kept dry at night;
(2) After the soybean Lee plants are cultured to 25d, the soybean Lee plants are poured out of the test disease soil, soybean cysts are separated from root systems by adopting an elutriation-screening method, and the soybean cysts washed from the root systems are collected to a 100-mesh screen by using a 25-60-100-mesh screen in sequence; after purifying by a cyst breaking and sucrose gradient centrifugation method, counting the soybean cyst nematode eggs under a microscope for subsequent inoculation;
(3) Transplanting the materials to be identified, which germinate for 3-5d, into sterile soil, repeating the steps for 4 times for each material, growing new roots after 3-5d, pricking holes next to the root system of the materials to be identified by using a screwdriver with the diameter of 3 multiplied by 75mm, then injecting the inoculation liquid obtained in the step (2) into the vicinity of the root system through the holes by means of the assistance of a 1mL gun head, wherein the inoculation is carried out twice, one hole is injected each time, 1500 worm eggs/plant are inoculated once, and the interval of the two inoculations is 24 hours;
(4) Inoculating insect eggs to a material to be identified, culturing in a greenhouse for 25 days, pouring out a plant of the material to be identified from test disease soil, separating cysts from a root system by adopting an elutriation-screening method, and counting the number of soybean cysts on the root system;
(5) And calculating a cyst index FI according to the average number of soybean cysts on each root system of the material to be identified, and determining the disease-resistant grade.
2. The method according to claim 1, wherein the soil infected with soybean cyst nematode is selected from one or more of the race 1, 2, 3, 4, or 5 species of soybean cyst nematode.
3. The method of claim 1, wherein the test cup is of the following specification: diameter of upper port x diameter of lower port x height =70mm x 50mm x 90mm; the internal dimensions of the plastic basket are: length × width × height =530mm × 341mm × 70mm.
4. The method according to claim 1, wherein the purification treatment comprises the following specific steps: crushing soybean cyst with a rubber stopper, releasing worm eggs, sequentially sieving the worm eggs through a sieve of 100-200-500 meshes, finally collecting the worm eggs on the sieve of 500 meshes, transferring the worm eggs to a 50mL centrifuge tube by using a wash bottle and a funnel, controlling the volume within 20mL, adding 20mL of sucrose solution with the mass fraction of 40%, slightly inverting and uniformly mixing; centrifuging at 2000rpm for 5min;
if layering occurs, taking a middle layer of worm eggs, discarding the supernatant on the upper part and the sediment on the bottom, filtering on a 600-mesh screen, washing with clear water to remove sucrose, washing the worm eggs to a beaker with the assistance of a washing bottle and a funnel, diluting, taking 10 mu L of the diluted worm eggs, counting the number of the soybean cyst nematode worm eggs under a microscope, and using the number for subsequent inoculation;
if not, sucking the eggs in the supernatant, discarding the bottom precipitate, filtering the sucked supernatant on a 600-mesh sieve, washing with clear water to remove sucrose, and leaving the eggs on the 600-mesh sieve; the eggs are washed into a beaker with the aid of a wash bottle and a funnel, and after dilution, 10 μ L of the eggs are taken and counted under a microscope for subsequent inoculation.
5. The method of claim 1, wherein the test data is validated if the number of soybean cysts in the root line of the disease control Lee individual used in the identification test is greater than or equal to 100; if the number of soybean cysts on the root system of the individual plant of the susceptible control Lee for the identification test is less than 100, repeating the steps (1) and (2), utilizing the susceptible control Lee to expand the number of the cysts in the diseased soil again until the number of worm eggs enough for inoculation identification is propagated, and then carrying out resistance identification.
6. The method according to claim 1, wherein the cyst index FI = average number of cysts on the root system of the plant to be identified/number of cysts on the susceptible control Lee plant x 100.
7. The method of claim 6, wherein the cyst index, FI, is related to disease resistance rating: FI =0, immune; 0 (all over) is FI less than or equal to 10, and disease resistance is achieved; 10-straw FI is less than or equal to 30, and resistance is achieved; 30-straw FI is less than or equal to 60, and feel; FI >60, affection.
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Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CA2078986A1 (en) * | 1990-04-11 | 1991-10-12 | Robin A. Bedding | Apparatus and method for rearing nematodes, fungi, tissue cultures and the like, and for harvesting nematodes |
| CN102246662A (en) * | 2011-05-16 | 2011-11-23 | 河南省农业科学院 | Method for identifying resistance against heterodera glycines |
| CN103650844A (en) * | 2013-11-22 | 2014-03-26 | 山西省农业科学院农作物品种资源研究所 | Disease-resistant plant transplanting method in soybean cyst nematode resistance research |
Family Cites Families (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107875124B (en) * | 2016-09-30 | 2021-08-06 | 湖北盛齐安生物科技股份有限公司 | Method for extracting and purifying cell vesicles wrapping medicine from cell suspension |
| CN209215036U (en) * | 2018-10-12 | 2019-08-06 | 沈阳农业大学 | A kind of cyst nematode collection device |
-
2021
- 2021-05-28 CN CN202110588951.5A patent/CN113179898B/en active Active
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CA2078986A1 (en) * | 1990-04-11 | 1991-10-12 | Robin A. Bedding | Apparatus and method for rearing nematodes, fungi, tissue cultures and the like, and for harvesting nematodes |
| CN102246662A (en) * | 2011-05-16 | 2011-11-23 | 河南省农业科学院 | Method for identifying resistance against heterodera glycines |
| CN103650844A (en) * | 2013-11-22 | 2014-03-26 | 山西省农业科学院农作物品种资源研究所 | Disease-resistant plant transplanting method in soybean cyst nematode resistance research |
Non-Patent Citations (3)
| Title |
|---|
| 应县小黑豆对大豆胞囊线虫(SCN)1号生理小种的抗性遗传分析;王树峰等;《应县小黑豆对大豆胞囊线虫(SCN)1号生理小种的抗性遗传分析》;20040531;第29-31页 * |
| 影响大豆胞囊线虫生理小种鉴定因素探讨;练云等;《影响大豆胞囊线虫生理小种鉴定因素探讨》;20150628;第 1259-1264 页 * |
| 黑龙江省大豆胞囊线虫种群分布和卵密度研究;孙玉秋等;《黑龙江省大豆胞囊线虫种群分布和卵密度研究》;20110425;第250-253,258页 * |
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