CN113957119A - Indoor bioassay method for wheat rust prevention and control effect of bactericide - Google Patents
Indoor bioassay method for wheat rust prevention and control effect of bactericide Download PDFInfo
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- 241000209140 Triticum Species 0.000 title claims abstract description 92
- 235000021307 Triticum Nutrition 0.000 title claims abstract description 92
- 230000000844 anti-bacterial effect Effects 0.000 title claims abstract description 70
- JEIPFZHSYJVQDO-UHFFFAOYSA-N iron(III) oxide Inorganic materials O=[Fe]O[Fe]=O JEIPFZHSYJVQDO-UHFFFAOYSA-N 0.000 title claims abstract description 70
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- FFSJPOPLSWBGQY-UHFFFAOYSA-N triazol-4-one Chemical compound O=C1C=NN=N1 FFSJPOPLSWBGQY-UHFFFAOYSA-N 0.000 claims description 6
- 229920001817 Agar Polymers 0.000 claims description 4
- OKTJSMMVPCPJKN-UHFFFAOYSA-N Carbon Chemical compound [C] OKTJSMMVPCPJKN-UHFFFAOYSA-N 0.000 claims description 4
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- 230000001580 bacterial effect Effects 0.000 claims description 3
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Abstract
The invention provides an indoor bioassay method for the control effect of a bactericide on wheat rust, which comprises the steps of quantitatively inoculating a spore suspension prepared from wheat rust summer spores into a leaf segment interval of 4cm in the middle of a first leaf after seedling cultivation, preparing a culture medium with different concentrations of active ingredients of the bactericide, cutting off a marked leaf segment after the rust flower spots of the inoculated wheat seedling grow completely, placing the leaf segment in a culture dish with a medicine with the leaf surface facing upwards, carrying out in-vitro culture under alternating illumination and darkness, measuring the pixel values of the leaf segment to be photographed and the summer spores by using Photoshop CS6 software, calculating the proportion of the disease area, calculating a toxicity regression equation by using DPS7.05 software, and obtaining the EC of a strain to be measured50The strains to be tested are classified according to their level of resistance to the fungicide. The method is finished indoors, the rust fungus summer spores are quantitatively inoculated, the disease area is measured through a software system after symptoms are revealed, the experimental conditions are strictly controllable, and a foundation is laid for carrying out large-scale monitoring work of the resistance of the wheat rust fungus to the bactericide by biological measurement of the indoor resistance of the wheat rust fungus to the bactericide.
Description
Technical Field
The invention belongs to the technical field of wheat rust prevention and control effect measurement, and particularly relates to an indoor bioassay method for wheat rust prevention and control effects of a bactericide.
Background
Wheat has three rust diseases, namely wheat stripe rust, wheat leaf rust and wheat stem rust. Wheat rust is wide in distribution, rapid in spread, large in damage area and frequent in disease prevalence, disastrous loss is caused to wheat production in the period of disease, and wheat production safety is seriously threatened.
The chemical control is one of the main measures for the comprehensive treatment of the wheat rust. In areas and years where the disease-resistant varieties lose rust resistance and rust outbreaks and disasters occur, the use of the bactericide becomes the most important means for controlling the prevalence of rust in a large area. The use of fungicides is a necessary supplement to rust control measures even in areas where rust-resistant gene layouts are implemented.
The chemical control of wheat rust has more varieties of selectable bactericides, and the quality of the control effect of the bactericides needs to be determined through biological tests. Since wheat rust is a strict obligate parasitic fungus and can only grow on living wheat, an experiment for determining the control effect of the bactericide on the diseases is usually carried out on potted wheat or field wheat. Traditional potting or field experiments are not only time consuming, labor intensive, land occupied, but also the experimental results exhibit significant errors due to differences in environmental conditions.
Disclosure of Invention
The invention aims to solve the technical problem that the defects of the prior art are overcome, and the indoor bioassay method for the wheat rust prevention and control effect of the bactericide is provided.
In order to solve the technical problems, the invention adopts the technical scheme that: an indoor bioassay method for the control effect of a bactericide on wheat rust comprises the following steps:
s1, seedling culture:
selecting and selecting large and full wheat seeds, planting the wheat seeds in a flowerpot, culturing the wheat seeds for 10 to 12 days under the greenhouse condition of alternating illumination and darkness, and selecting strong seedlings and keeping the strong seedlings when the first leaves of the wheat are completely unfolded to obtain wheat seedlings to be tested;
s2, quantitatively inoculating wheat rust:
adding the wheat rust fungus summer spores into the electronic fluoridation liquid, and uniformly mixing to prepare a spore suspension with the concentration of 2 g/L; quantitatively inoculating the spore suspension to the middle 4cm leaf segment region of the first leaf of the tested wheat seedling obtained in S1 to obtain an inoculated wheat seedling; marking the middle 4cm leaf segment of the first leaf in advance;
s3, preparation of a culture medium:
dissolving 6-BA by using NaOH solution with the concentration of 1mol/L, and adding distilled water to dilute to obtain mother liquor a with the concentration of 6-BA of 1000 mg/L;
dissolving the original pesticide of the bactericide by adding acetone, and diluting with distilled water to prepare a mother solution b with the concentration of the active ingredient of the bactericide being 100 mug/mL;
adding the obtained mother liquor a and the mother liquor b with different masses into sterilized water agar solution with the mass fraction of 0.5% to obtain culture media with the 6-BA concentration of 50 mu g/mL and different concentrations of active ingredients of the bactericide, and naming the culture media as culture media a to culture media h, respectively placing the culture media a to culture media h into culture dishes to obtain 8 culture dishes with medicines, and naming the culture media as the culture dishes with medicines a to culture dishes h in sequence, wherein the concentrations of the active ingredients of the bactericide in the culture media a to culture media h are 0 mu g/mL, 0.03 mu g/mL, 0.09 mu g/mL, 0.27 mu g/mL, 0.81 mu g/mL, 2.43 mu g/mL, 7.29 mu g/mL and 21.87 mu g/mL in sequence;
s4, leaf section shearing and culturing:
culturing the wheat seedlings inoculated in the S2 for 5-6 days until wheat rust spots completely grow out, cutting off the marked leaf sections according to a marking line, parallelly placing the wheat seedlings in 8 culture dishes with medicines obtained in S3 with the leaf surfaces facing upwards, attaching 5 leaf sections to each dish to obtain 8 groups of leaves, pressing two ends of each leaf section by using a transparent plastic film to ensure that the leaf sections are fully contacted with a culture medium, carrying out in-vitro culture, culturing each culture dish with the leaf sections in an incubator alternately carrying out illumination and darkness for 6-7 days until the leaf sections in the culture dish a with the medicines serving as a control group are fully attacked, and obtaining 8 groups of leaf sections to be photographed;
s5, photographing the 8 groups of leaf segments to be photographed obtained in the S4, respectively measuring the pixel values of the leaf segments to be photographed and the summer spores by using Photoshop CS6 software, calculating the proportion of the disease area, and then calculating a toxicity regression equation by using DPS7.05 software to obtain the EC of the strain to be photographed50Value using said EC50The resistance of the test strain to the bactericide is classified.
Preferably, the greenhouse conditions in S1 are: culturing at 16 deg.C for 17h in light, and culturing at 13 deg.C for 7h in dark, with light and dark being alternated.
Preferably, the Puccinia triticina in S2 is Puccinia striiformis f.sp.tritici or Puccinia recondita f.sp.tritici.
Preferably, the spore suspension in S2 is inoculated in an amount of 2.5. mu.L per leaf.
Preferably, the raw pesticide of the bactericide in S3 is 97.5% of raw pesticide of triazolone by mass fraction.
Preferably, the conditions for cultivation in the incubator of S4 are: culturing at 16 deg.C for 17h in light, and culturing at 13 deg.C for 7h in dark, with light and dark being alternated.
Preferably, the EC described in S550Values classify the resistance of the test strain to the fungicide as:
when the ratio of 0 mu g/mL to EC is less than or equal to50When the concentration is less than 0.75 mu g/mL, the resistance coefficient RI is more than or equal to 0 and less than 5 times, and the resistance coefficient RI is a bacterial strain sensitive to the bactericide by the rust fungi;
when the concentration of the active carbon is less than or equal to 0.75 mu g/mL50When the concentration is less than 1.50 mu g/mL, the resistance coefficient RI is less than 10 times and is not more than 5 times, and the strain is a low-resistance strain of rust bacteria to the bactericide;
when the ratio of 1.50 mu g/mL is less than or equal to EC50When the concentration is less than 6.00 mu g/mL, the resistance coefficient RI is less than 40 times and is equal to or more than 10 times, and the obtained product is an anti-bacterial strain of rust bacteria to a bactericide;
when EC50When the concentration is more than or equal to 6.00 mu g/mL, the resistance coefficient RI is more than or equal to 40 times, which is a high-resistance strain of rust fungi to the bactericide.
Compared with the prior art, the invention has the following advantages:
1. all the operations of the method are finished indoors, rust fungus summer spores are inoculated strictly and quantitatively, the disease incidence area is measured through a software system after disease is revealed, the experimental conditions are strictly controllable, the experimental operation is objective, accurate and repeatable, the research, development, evaluation and utilization of wheat rust fungus on indoor pesticide resistance bioassay of the bactericide are ensured, the foundation is laid for carrying out large-scale wheat rust fungus bactericide pesticide resistance monitoring work, and further, the theoretical basis is provided for chemical control of wheat rust and reasonable use of the bactericide.
2. The invention establishes a technical system for indoor biological measurement of the control effect of the bactericide on the wheat rust, and has stable and accurate result and good repeatability. The system can be used for the control effect detection of the bactericide on one hand and can also be used for monitoring the sensitivity of the rust fungi to the bactericide on the other hand, aiming at guiding the accurate medicine selection and scientific medicine application in production and furthest playing the effect of the bactericide on the basis of fully considering the ecological economic cost.
The present invention will be described in further detail with reference to the accompanying drawings and examples.
Drawings
FIG. 1 is a graph showing an indoor bioassay of wheat leaf rust in example 1 of the present invention.
FIG. 2 is a graph of the indoor bioassay of wheat leaf rust in example 2 of the present invention.
Detailed Description
Example 1
The indoor bioassay method for the control effect of the bactericide on the wheat rust comprises the following steps:
s1, seedling culture:
selecting and planting large and full wheat seeds in wheat seeds of which the variety is mingxian 169(MX169) in flowerpots with the specification of 10cm multiplied by 10cm, sowing 15 seeds in each flowerpot, culturing for 10 days under the greenhouse condition of alternately performing illumination and darkness, and selecting 10 strong seedlings and reserving the seedlings when the first leaves of the wheat are completely unfolded to obtain the wheat seedlings to be tested; the greenhouse conditions are as follows: culturing for 17h under the illumination condition at the temperature of 16 ℃, then culturing for 7h under the dark condition at the temperature of 13 ℃, and alternately performing illumination and darkness;
s2, quantitatively inoculating wheat rust:
adding wheat rust fungus summer spores into an electronic fluoridation solution (3M Novec TM 7100), and uniformly mixing to prepare a spore suspension with the concentration of 2 g/L; sucking 2.5 mu L of spore suspension by using a liquid transfer gun, and quantitatively inoculating the spore suspension to the middle 4cm leaf segment region of the first leaf of the tested wheat seedling obtained in S1 to obtain an inoculated wheat seedling; marking the 4cm leaf segment; the wheat rust is Puccinia striiformis f.sp.tritici; marking a leaf segment of 4cm in the middle of the first leaf by using a marker pen in advance;
s3, preparation of a culture medium:
heating and dissolving 0.12g of 6-BA by using a NaOH solution with the concentration of 1mol/L, and adding distilled water to dilute to obtain a mother liquor a with the concentration of 1000mg/L of 6-BA;
0.5128g of bactericide raw drug (triazolone raw drug with the mass fraction of 97.5%) is dissolved by adding a proper amount of acetone, and then the mixture is diluted by distilled water to prepare a mother solution b with the concentration of the active ingredient of the bactericide of 100 mu g/mL;
adding the obtained mother liquor a and the mother liquor b with different masses into sterilized water agar solution with the mass fraction of 0.5% to obtain culture media with the 6-BA concentration of 50 mu g/mL and different concentrations of active ingredients of a bactericide, and naming the culture media as culture media a to culture media h, respectively placing the culture media a to culture media h into culture dishes to obtain 8 culture dishes with medicines, and naming the culture media as the culture dishes with medicines a to culture dishes h in sequence, wherein the concentrations of the active ingredients of the triazolone in the culture media a to culture media h are 0 mu g/mL, 0.03 mu g/mL, 0.09 mu g/mL, 0.27 mu g/mL, 0.81 mu g/mL, 2.43 mu g/mL, 7.29 mu g/mL and 21.87 mu g/mL in sequence;
s4, leaf section shearing and culturing:
culturing the wheat seedlings inoculated in the S2 for 5 days until wheat rust spots completely grow out, cutting off the marked leaf segments according to a marking line, parallelly placing the wheat seedlings in 8 culture dishes with medicines obtained in the S3 with the leaf surfaces facing upwards, attaching 5 leaf segments to each dish to obtain 8 groups of leaves, pressing two ends of each leaf segment by using a transparent plastic film to ensure that the leaf segments are fully contacted with a culture medium, carrying out in-vitro culture, culturing each culture dish with the leaf segments in an incubator alternately carrying out illumination and darkness for 6 days until the leaf segments in the culture dishes a with medicines serving as a control group are fully attacked, and obtaining 8 groups of leaf segments to be photographed; the culture conditions in the incubator are as follows: culturing for 17h under the illumination condition at the temperature of 16 ℃, then culturing for 7h under the dark condition at the temperature of 13 ℃, and alternately performing illumination and darkness;
s5, photographing the 8 groups of leaf segments to be photographed obtained in the S4, respectively measuring the pixel values of the leaf segments to be photographed and the summer spores by using Photoshop CS6 software, calculating the proportion of the disease area, and then calculating a toxicity regression equation by using DPS7.05 software to obtain the EC of the strain to be photographed50Value using said EC50Classifying the resistance of the strain to be tested to the bactericide;
the EC50Values classify the resistance of the test strain to the fungicide as:
when the ratio of 0 mu g/mL to EC is less than or equal to50When the concentration is less than 0.75 mu g/mL, the resistance coefficient RI (resistance index) which is less than 5 times and is a strain sensitive to the bactericide by the rust fungi is more than or equal to 0;
when the concentration of the active carbon is less than or equal to 0.75 mu g/mL50When the concentration is less than 1.50 mu g/mL, the resistance coefficient RI (resistance index) is less than 10 times and is less than 5 times, and the resistance coefficient RI is a low-resistance strain of the rust bacteria to the bactericide;
when the ratio of 1.50 mu g/mL is less than or equal to EC50When the concentration is less than 6.00 mu g/mL, the resistance coefficient RI (resistance index) which is less than 40 times and is more than or equal to 10 times is used as the strain resistant to the bactericide by the rust bacteria;
when EC50When the resistance coefficient RI (resistance index) is more than or equal to 40 times when the concentration is more than or equal to 6.00 mu g/mL, the resistance coefficient RI (resistance index) is more than or equal to 40 times, and is a high-resistance strain of rust bacteria to bactericides.
As shown in FIG. 1, the leaf segments to be photographed in 8 groups are shown as followsThe concentration of the active ingredients of the bactericide in the culture dish is 0 mug/mL, 0.03 mug/mL, 0.09 mug/mL, 0.27 mug/mL, 0.81 mug/mL, 2.43 mug/mL, 7.29 mug/mL and 21.87 mug/mL in sequence from left to right in the first row and from left to right in the second row. Respectively measuring the pixel values of the leaf segment to be photographed and the summer spore by using Photoshop CS6 software, calculating the proportion of the disease incidence area, and then calculating a toxicity regression equation by using DPS7.05 software to obtain the EC of the strain to be measured50Indoor activity assays were completed.
The wheat stripe rust fungus group in Qinghai province is subjected to bioassay on indoor drug resistance of triadimefon, and the determination results are shown in table 1.
TABLE 1 indoor biological assay results of Puccinia striiformis in Qinghai province
All operations of this embodiment are all accomplished indoor, and the strict quantitative inoculation of rust fungus summer spore, shows that the back passes through software system survey the area of developing a disease, and experimental conditions is strict controllable, ensures that experimental operation is objective, accurate, can repeat, utilizes through the research and development and the evaluation of wheat rust fungus to the indoor drug resistance bioassay of germicide, lays the foundation for carrying out large-scale wheat rust fungus germicide drug resistance monitoring work, and then provides the theoretical foundation for wheat rust chemical control, the rational use of germicide.
Example 2
The indoor bioassay method for the control effect of the bactericide on the wheat rust comprises the following steps:
s1, seedling culture:
selecting and planting large and full wheat seeds in wheat seeds of which the variety is mingxian 169(MX169) in flowerpots with the specification of 10cm multiplied by 10cm, sowing 15 seeds in each flowerpot, culturing for 12 days under the greenhouse condition of alternately performing illumination and darkness, and selecting 10 strong seedlings and reserving the seedlings when the first leaves of the wheat are completely unfolded to obtain the wheat seedlings to be tested; the greenhouse conditions are as follows: culturing for 17h under the illumination condition at the temperature of 16 ℃, then culturing for 7h under the dark condition at the temperature of 13 ℃, and alternately performing illumination and darkness;
s2, quantitatively inoculating wheat rust:
adding wheat rust fungus summer spores into an electronic fluoridation solution (3M Novec TM 7100), and uniformly mixing to prepare a spore suspension with the concentration of 2 g/L; sucking 2.5 mu L of spore suspension by using a liquid transfer gun, and quantitatively inoculating the spore suspension to the middle 4cm leaf segment region of the first leaf of the tested wheat seedling obtained in S1 to obtain an inoculated wheat seedling; marking the 4cm leaf segment; the wheat rust is Puccinia recondita f.sp.tritici; marking a leaf segment of 4cm in the middle of the first leaf by using a marker pen in advance;
s3, preparation of a culture medium:
heating and dissolving 0.12g of 6-BA by using a NaOH solution with the concentration of 1mol/L, and adding distilled water to dilute to obtain a mother liquor a with the concentration of 1000mg/L of 6-BA;
0.5128g of bactericide raw drug (triazolone raw drug with the mass fraction of 97.5%) is dissolved by adding a proper amount of acetone, and then the mixture is diluted by distilled water to prepare a mother solution b with the concentration of the active ingredient of the bactericide of 100 mu g/mL;
adding the obtained mother liquor a and the mother liquor b with different masses into sterilized water agar solution with the mass fraction of 0.5% to obtain culture media with the 6-BA concentration of 50 mu g/mL and different concentrations of active ingredients of a bactericide, and naming the culture media as culture media a to culture media h, respectively placing the culture media a to culture media h into culture dishes to obtain 8 culture dishes with medicines, and naming the culture media as the culture dishes with medicines a to culture dishes h in sequence, wherein the concentrations of the active ingredients of the triazolone in the culture media a to culture media h are 0 mu g/mL, 0.03 mu g/mL, 0.09 mu g/mL, 0.27 mu g/mL, 0.81 mu g/mL, 2.43 mu g/mL, 7.29 mu g/mL and 21.87 mu g/mL in sequence;
s4, leaf section shearing and culturing:
culturing the wheat seedlings inoculated in the S2 for 6 days until wheat rust spots completely grow out, cutting off the marked leaf segments according to a marking line, parallelly placing the wheat seedlings in 8 culture dishes with medicines obtained in the S3 with the leaf surfaces facing upwards, attaching 5 leaf segments to each dish to obtain 8 groups of leaves, pressing two ends of each leaf segment by using a transparent plastic film to ensure that the leaf segments are fully contacted with a culture medium, carrying out in-vitro culture, culturing each culture dish with the leaf segments in an incubator alternately carrying out illumination and darkness for 7 days until the leaf segments in the culture dishes a with medicines serving as a control group are fully attacked, and obtaining 8 groups of leaf segments to be photographed; the culture conditions in the incubator are as follows: culturing for 17h under the illumination condition at the temperature of 16 ℃, then culturing for 7h under the dark condition at the temperature of 13 ℃, and alternately performing illumination and darkness;
s5, photographing the 8 groups of leaf segments to be photographed obtained in the S4, respectively measuring the pixel values of the leaf segments to be photographed and the summer spores by using Photoshop CS6 software, calculating the proportion of the disease area, and then calculating a toxicity regression equation by using DPS7.05 software to obtain the EC of the strain to be photographed50Value using said EC50Classifying the resistance of the strain to be tested to the bactericide;
the EC50Values classify the resistance of the test strain to the fungicide as:
when the ratio of 0 mu g/mL to EC is less than or equal to50When the concentration is less than 0.75 mu g/mL, the resistance coefficient RI is more than or equal to 0 and less than 5 times, and the resistance coefficient RI is a bacterial strain sensitive to the bactericide by the rust fungi;
when the concentration of the active carbon is less than or equal to 0.75 mu g/mL50When the concentration is less than 1.50 mu g/mL, the resistance coefficient RI is less than 10 times and is not more than 5 times, and the strain is a low-resistance strain of rust bacteria to the bactericide;
when the ratio of 1.50 mu g/mL is less than or equal to EC50When the concentration is less than 6.00 mu g/mL, the resistance coefficient RI is less than 40 times and is equal to or more than 10 times, and the obtained product is an anti-bacterial strain of rust bacteria to a bactericide;
when EC50When the concentration is more than or equal to 6.00 mu g/mL, the resistance coefficient RI is more than or equal to 40 times, which is a high-resistance strain of rust fungi to the bactericide.
As shown in FIG. 2, the concentration of the effective components of the bactericide in the culture dish is 0.03. mu.g/mL, 0.09. mu.g/mL and 0.09. mu.g/mL in the sequence from the left to the right along the first row and from the left to the right along the second row in the 8 groups of leaf segments to be photographed27. mu.g/mL, 0.81. mu.g/mL, 2.43. mu.g/mL, 7.29. mu.g/mL, 21.87. mu.g/mL, and 0. mu.g/mL. Respectively measuring the pixel values of the leaf segment to be photographed and the summer spore by using Photoshop CS6 software, calculating the proportion of the disease incidence area, and then calculating a toxicity regression equation by using DPS7.05 software to obtain the EC of the strain to be measured50Indoor activity assays were completed.
The above description is only for the preferred embodiment of the present invention, and is not intended to limit the present invention in any way. Any simple modification, change and equivalent changes of the above embodiments according to the technical essence of the invention are still within the protection scope of the technical solution of the invention.
Claims (7)
1. An indoor bioassay method for the control effect of a bactericide on wheat rust is characterized by comprising the following steps:
s1, seedling culture:
selecting and selecting large and full wheat seeds, planting the wheat seeds in a flowerpot, culturing the wheat seeds for 10 to 12 days under the greenhouse condition of alternating illumination and darkness, and selecting strong seedlings and keeping the strong seedlings when the first leaves of the wheat are completely unfolded to obtain wheat seedlings to be tested;
s2, quantitatively inoculating wheat rust:
adding the wheat rust fungus summer spores into the electronic fluoridation liquid, and uniformly mixing to prepare a spore suspension with the concentration of 2 g/L; quantitatively inoculating the spore suspension to the middle 4cm leaf segment region of the first leaf of the tested wheat seedling obtained in S1 to obtain an inoculated wheat seedling; marking the middle 4cm leaf segment of the first leaf in advance;
s3, preparation of a culture medium:
dissolving 6-BA by using NaOH solution with the concentration of 1mol/L, and adding distilled water to dilute to obtain mother liquor a with the concentration of 6-BA of 1000 mg/L;
dissolving the original pesticide of the bactericide by adding acetone, and diluting with distilled water to prepare a mother solution b with the concentration of the active ingredient of the bactericide being 100 mug/mL;
adding the obtained mother liquor a and the mother liquor b with different masses into sterilized water agar solution with the mass fraction of 0.5% to obtain culture media with the 6-BA concentration of 50 mu g/mL and different concentrations of active ingredients of the bactericide, and naming the culture media as culture media a to culture media h, respectively placing the culture media a to culture media h into culture dishes to obtain 8 culture dishes with medicines, and naming the culture media as the culture dishes with medicines a to culture dishes h in sequence, wherein the concentrations of the active ingredients of the bactericide in the culture media a to culture media h are 0 mu g/mL, 0.03 mu g/mL, 0.09 mu g/mL, 0.27 mu g/mL, 0.81 mu g/mL, 2.43 mu g/mL, 7.29 mu g/mL and 21.87 mu g/mL in sequence;
s4, leaf section shearing and culturing:
culturing the wheat seedlings inoculated in the S2 for 5-6 days until wheat rust spots completely grow out, cutting off the marked leaf sections according to a marking line, parallelly placing the wheat seedlings in 8 culture dishes with medicines obtained in S3 with the leaf surfaces facing upwards, attaching 5 leaf sections to each dish to obtain 8 groups of leaves, pressing two ends of each leaf section by using a transparent plastic film to ensure that the leaf sections are fully contacted with a culture medium, carrying out in-vitro culture, culturing each culture dish with the leaf sections in an incubator alternately carrying out illumination and darkness for 6-7 days until the leaf sections in the culture dish a with the medicines serving as a control group are fully attacked, and obtaining 8 groups of leaf sections to be photographed;
s5, photographing the 8 groups of leaf segments to be photographed obtained in the S4, respectively measuring the pixel values of the leaf segments to be photographed and the summer spores by using Photoshop CS6 software, calculating the proportion of the disease area, and then calculating a toxicity regression equation by using DPS7.05 software to obtain the EC of the strain to be photographed50Value using said EC50The resistance of the test strain to the bactericide is classified.
2. The indoor bioassay method for the control effect of a bactericide on wheat rust according to claim 1, characterized in that the greenhouse conditions in S1 are: culturing at 16 deg.C for 17h in light, and culturing at 13 deg.C for 7h in dark, with light and dark being alternated.
3. The indoor bioassay method for the control effect of a bactericide on wheat rust as claimed in claim 1, wherein said wheat rust in S2 is Puccinia striiformis f.sp.tritici or Puccinia recondita f.sp.tritici.
4. The indoor bioassay method for the control effect of a bactericide on wheat rust as claimed in claim 1, wherein the inoculation amount of the spore suspension on each leaf blade in S2 is 2.5 μ L.
5. The indoor bioassay method for the control effect of the bactericide on the wheat rust according to claim 1, characterized in that the bactericide raw drug in S3 is triazolone raw drug with a mass fraction of 97.5%.
6. The indoor bioassay method for the wheat rust control effect of a bactericide as claimed in claim 1, wherein the conditions for cultivation in the incubator of S4 are as follows: culturing at 16 deg.C for 17h in light, and culturing at 13 deg.C for 7h in dark, with light and dark being alternated.
7. The indoor bioassay method for the control effect of a bactericide on wheat rust as claimed in claim 1, wherein said EC in S550Values classify the resistance of the test strain to the fungicide as:
when the ratio of 0 mu g/mL to EC is less than or equal to50When the concentration is less than 0.75 mu g/mL, the resistance coefficient RI is more than or equal to 0 and less than 5 times, and the resistance coefficient RI is a bacterial strain sensitive to the bactericide by the rust fungi;
when the concentration of the active carbon is less than or equal to 0.75 mu g/mL50When the concentration is less than 1.50 mu g/mL, the resistance coefficient RI is less than 10 times and is not more than 5 times, and the strain is a low-resistance strain of rust bacteria to the bactericide;
when the ratio of 1.50 mu g/mL is less than or equal to EC50When the concentration is less than 6.00 mu g/mL, the resistance coefficient RI is less than 40 times and is equal to or more than 10 times, and the obtained product is an anti-bacterial strain of rust bacteria to a bactericide;
when EC50When the concentration is more than or equal to 6.00 mu g/mL, the resistance coefficient RI is more than or equal to 40 times, which is a high-resistance strain of rust fungi to the bactericide.
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Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107372492A (en) * | 2017-07-28 | 2017-11-24 | 中国科学院合肥物质科学研究院 | A kind of wheat rust inhibitor and preparation method thereof |
| CN110146668A (en) * | 2019-05-21 | 2019-08-20 | 西南大学 | A method of measurement fungicide is to wheat stripe rust virulence |
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Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107372492A (en) * | 2017-07-28 | 2017-11-24 | 中国科学院合肥物质科学研究院 | A kind of wheat rust inhibitor and preparation method thereof |
| CN110146668A (en) * | 2019-05-21 | 2019-08-20 | 西南大学 | A method of measurement fungicide is to wheat stripe rust virulence |
Non-Patent Citations (3)
| Title |
|---|
| FURONG PENG 等: "Rapid Quantification of Fungicide Effectivenesson Inhibiting Wheat Stripe Rust Pathogen(Puccinia striiformis f.sp. tritici)", 《PLANT DIS》 * |
| YUAN TIAN 等: "Trade-Off Between Triadimefon Sensitivity and Pathogenicity in a Selfed Sexual Population of Puccinia striiformis f. sp. Tritici", 《FRONTIERS IN MICROBIOLOGY》 * |
| 纪凡 等: "基于基因混池测序技术的小麦条锈菌对三唑酮抗性位点相关分析", 《植物保护学报》 * |
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